RAS FAMILY GTPASES INVOLVED IN BREAST CANCER
Ariella B. Hanker
A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Curriculum of Genetics and Molecular Biology.
Chapel Hill 2009
Approved by:
Channing J. Der (Advisor)
Christopher Counter
Adrienne Cox
H. Shelton Earp
Charles Perou
ABSTRACT
ARIELLA HANKER: Ras Family GTPases Involved in Breast Cancer (Under the direction of Channing J. Der)
The Ras branch of the Ras superfamily of small GTPases consists of over thirty-six
proteins that regulate a wide array of cellular processes. Mutations in the RAS oncogene that
cause aberrant activation of Ras signaling drive 30% of all cancers, but these mutations are
infrequent in breast cancer. Instead, other Ras family proteins may play more significant
roles in the development and progression of breast cancer. Here, we focus on two such
proteins, Rerg and Rheb. Hyperactive Rheb activity may enhance breast cancer
tumorigenesis, whereas Rerg has been proposed to negatively regulate breast cancer growth.
Expression of Rerg is controlled by estrogen; hence, Rerg is not expressed in ERα-negative
breast cancers. Whether loss of Rerg expression in these tumors contributes to breast cancer
progression is not known. We found that silencing Rerg expression did not significantly
enhance the growth or invasive properties of breast cancer cells, and likewise, inducing Rerg
expression in Rerg-negative breast cancer cell lines did not diminish their growth. Thus,
these studies argue against a role for Rerg as a tumor suppressor in breast cancer. More
studies are needed to determine whether Rerg is involved in breast cancer in vivo.
Rheb and its effector mTOR have been strongly implicated in the development of many cancers, including breast cancer. Upstream regulators of Rheb are commonly activated in the majority of breast cancers, leading to increased Rheb activity in breast cancer.
ii However, approaches to inhibit Rheb function in breast cancer have not been
comprehensively explored. Here, we investigated two strategies to inhibit Rheb-mTOR signaling. First, we asked whether CAAX-mediated posttranslational processing of Rheb is
required for its function. We found that, while farnesylation is absolutely required for Rheb
activation of mTOR, post-prenyl processing by the enzymes Rce1 and Icmt is dispensable for
Rheb signaling. Our studies further suggested that endomembrane localization of Rheb is not
required for its activation of mTOR. Finally, we found that the farnesylcysteine mimetic S-
trans, trans-farnesylthiosalicylic acid (FTS; salirasib) did not block Rheb localization, but potently inhibited mTOR-induced p70 S6 kinase (S6K) activation, suggesting that FTS is a direct inhibitor of mTOR. Therefore, FTS may be a promising therapy for the treatment of
Rheb- and mTOR-dependent cancers, including breast cancer. Together, these studies have
increased our understanding of the functions of Rerg and Rheb in breast cancer and have
suggested a potential therapeutic strategy to reverse aberrant Rheb signaling in breast cancer.
iii
ACKNOWLEDGEMENTS
I am indebted to many people for enriching and enhancing my graduate school experience. First, I would like to thank all members of the Der Lab from 2005-2009 for their advice and technical assistance. In particular, I must thank Kevin Healy for helping to ease my transition into the lab, as well as for his advice, Natalia Mitin for helpful discussions and generous technical assistance, and Cercina Onesto for her supportive friendship and technical advice. In addition, I thank my collaborators, including Rhonda Wilder, Adrienne Cox,
Gretchen Repasky, Fuyuhiko Tamanoi, and Elizabeth Petri-Henske for providing valuable advice and reagents. I also thank my committee members, Adrienne Cox, Christopher
Counter, H. Shelton Earp, and Charles Perou, for insightful comments and guidance. I am especially grateful to my advisor Channing Der for his mentorship, guidance, and incredible support. Finally, I thank my family, particularly my parents, Edward Hanker and Patricia
Redifer, for their extraordinary support and encouragement, and my husband, Lior Klirs, not just for editing this document, but for his unwavering belief in my abilities and all of the care and support he has provided for me. Without their support, this would not have been possible.
iv
TABLE OF CONTENTS
LIST OF FIGURES…………………………………………………...…………………….viii
LIST OF ABBREVIATIONS…………………………………………………………………x
Chapter
I. INTRODUCTION…………………………………………………………….1
Targeted therapies for breast cancer…………………………………………..1
Estrogen signaling in breast cancer……………………………………………3
The Ras family of small GTPases……………………………………………..4
Ras posttranslational processing and localization……………………………..7
Ras is an oncoprotein………………………………………………………...10
Ras in breast cancer………………………………………………………….12
Therapeutic targeting of Ras…………………………………………………14
Rheb………………………………………………………………...………..20
Inhibitors of the Rheb-mTOR pathway in breast cancer…………………….27
ARHI/Di-Ras3/Noey2………………………………………………………..28
Rerg………………….……………………………………………………….30
Other Ras family proteins in breast cancer…………………………………..33
Rationale for studies…………………………………………………………36
II. TOOLS TO STUDY THE FUNCTION OF THE RAS- RELATED, ESTROGEN-REGULATED GROWTH
v INHIBITOR IN BREAST CANCER………………………………………..38
Abstract………………………………………………………………………38
Introduction…………………………………………………………………..40
Materials and Methods……………………………………………………….42
Results and Discussion…………………………….………………………...48
Summary……………………………………………………………………..61
Acknowledgements…………………………………………………………..62
III. REQUIREMENT OF CAAX-MEDIATED POSTTRANSLATIONAL PROCESSING FOR RHEB LOCALIZATION AND SIGNALING………………………………………63
Abstract……………………………………………………………………....63
Introduction…………………………………………………………………..65
Materials and methods……………………………………………………….68
Results…………………………………………………………………..……71
Discussion……………………………………………………………………88
Acknowledgements…………………………………………………………..91
IV. CONCLUDING REMARKS AND FUTURE DIRECTIONS………………92
Is Rerg a growth inhibitor?...... 92
What is the role of Rerg in normal cells and in development?...... 94
What properties of Rerg are required for its function?...... 96
Does loss of Rerg expression contribute to the growth of other types of cancer?...... 98
Therapeutic reexpression of ARHI or Rerg in breast cancer………………...98
Which tumors are dependent on Rheb activity?...... 101
Is Rheb the FTI target responsible for its anti-tumor activity?...... 104
vi
Is Rheb localization important for its function?...... 107
Can more selective Rheb inhibitors be developed?...... 108
Rheb versus mTOR: which is a better therapeutic target?...... 110
Does FTS inhibit mTOR signaling in vivo?...... 112
Comparison of FTS to other mTOR inhibitors……………………………..114
What are the targets of FTS required for its anti-tumor activity?...... 116
Summary……………………………………………………………………118
REFERENCES……………………………………………………………………..119
vii LIST OF FIGURES
Figure
1-1. Targeted therapies for breast cancer currently in clinical use…………………2
1-2. The Ras branch of the human Ras superfamily of small GTPases……………5
1-3. Ras signal transduction………………………………………………………..6
1-4. Domain organization of Ras family proteins………………………………….8
1-5. Ras posttranslational processing and membrane association………………...11
1-6. Therapeutic strategies to inhibit Ras signaling………………………………15
1-7. Chemical structures of farnesyl-containing small molecules in clinical development………………………………………………………18
1-8. Rheb signal transduction……………………………………………………..21
1-9. Estrogen regulation of Rheb signaling……………………………………….23
1-10. Oncogenic properties of Rheb……………………………………………….25
1-11. Estrogen regulation of Rerg expression……………………………………...31
1-12. Unknown mediators of Rerg signal transduction……………………………32
2-1. Antibody detection of endogenous Rerg expression in breast cancer cell lines and in tumor tissue…………………………………………49
2-2. Transient and stable expression of wild-type and mutant Rerg proteins………………………...……………………………………….53
2-3. Tamoxifen-inducible expression of an estrogen receptor (ERα)-Rerg fusion protein…………………………………………………...56
2-4. Effects of induction of ERα-Rerg expression on growth and invasive properties in ERα-negative breast cancer cells…………………….58
2-5. Repression of Rerg expression by shRNA…………………………………...60
3-1. Rheb1 and Rheb2 are localized primarily to the endoplasmic reticulum and Golgi………………………………………………………….72
viii 3-2. CAAX-signaled modifications alone are sufficient for Rheb1 but not Rheb2 localization to the ER and Golgi……………………………..74
3-3. Rheb subcellular localization is dependent on Rce1- and Icmt- catalyzed modifications……………………………………………………...76
3-4. Rce1- and Icmt-catalyzed modifications are not required for Rheb1 activation of S6 kinase………………………………………………..80
3-5. FTS does not disrupt Rheb or H-Ras subcellular localization…………….…83
3-6. FTS inhibits mTOR-induced S6K activation by a mechanism independent of prenylated proteins…………………………………………..85
3-7. FTS blocks endogenous S6K activation in MCF-7 cells…………………….87
4-1. Epigenetic silencing of ARHI and Rerg and strategies to restore their expression………………………………………………………99
4-2. Knocking down Rheb1 expression does not significantly reduce estrogen-induced MCF-7 cell proliferation or anchorage- independent growth…………………………………………………………103
4-3. GG-Rheb1 does not reduce FTI sensitivity of MCF-7 cells………………..106
4-4. Inhibition of mTOR signaling by farnesyl-containing small molecules………………………………………………………………...…113
4-5. Inhibitors of the Rheb-mTOR pathway in clinical trials…………………...115
4-6. Feedback regulation of mTOR signaling…………………………………...117
ix LIST OF ABBREVIATIONS
4E-BP1 Eukaryotic initiation factor 4E-binding protein 1
4-OHT 4-hydroxytamoxifen
5-aza-dC 5-aza-2’-deoxycytidine
AdoHcy S-adenosylhomocysteine
AFC N-acetyl-S-farnesyl-L-cysteine
AMPK 5' adenosine monophosphate-activated protein kinase
ARHI Ras homolog member I
BCAR3 Breast cancer antiestrogen resistance 3
Bnip3 BCL2/adenovirus E1B 19 kDa interacting protein 3
BODIPY Boron-dipyrromethene
BSA Bovine serum albumin
C-terminus Carboxy-terminus
CAAX C=cysteine; A = aliphatic amino acid; X = terminal amino acid cDNA complementary deoxyribonucleic acid
DMEM Dulbecco’s modified Eagle’s medium
DMSO Dimethyl sulfoxide
DNA Deoxyribonucleic acid
DNase Deoxyribonuclease
DNMT DNA methyltransferase
E. coli Escherichia coli
E2 17β-estradiol (estrogen)
EDC 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide
x EDTA Ethylenediaminetetraacetic acid
EEA Early endosomal antigen 1
EGFR Epidermal Growth Factor Receptor eIF4E Eukaryotic initiation factor 4E
ER Endoplasmic reticulum
ERα-Rerg Estrogen receptor-Rerg fusion protein
ERα Estrogen receptor-alpha
ERE Estrogen response element
ERK Extracellular signal-regulated kinase
F-Rheb Farnesylated Rheb
FCS Fetal calf serum
FDA Food and drug administration
FKBP38 FK506 binding protein 8, 38 kDa
FL Full-length
FTase Farnesyltransferase
FTI Farnesyltransferase inhibitor
FTS S-trans, trans-farnesylthiosalicylic acid
GAP GTPase activating protein
GAPDH Glyceraldehyde 3-phosphate dehydrogenase
GDP Guanine nucleotide diphosphate
GEF Guanine nucleotide exchange factor
GFP Green fluorescent protein
GG-Rheb Geranylgeranylated Rheb
xi GGTase Geranylgeranyltransferase
GGTI Geranylgeranyltransferase-I inhibitor
Grb2 Growth receptor-bound protein 2
GTP Guanine nucleotide triphosphate
GTPase Guanine triphosphatase
HA Hemagglutinin
HDAC Histone deacetylase
HDACi Histone deacetylase inhibitor
HER1 Human epidermal growth factor receptor 1
HER2 Human epidermal growth factor receptor 2
Hsp90 Heat shock protein 90
Icmt Isoprenylcysteine-O-carboxyl methyltransferase
IGF1R Insulin-like growth factor 1 receptor
IHC Immunohistochemistry
IRS1 Insulin receptor substrate 1 kDa Kilodalton
KLH Keyhole limpet hemocyanin
LAM Lymphangioleiomyomatosis
LAMP2 Lysosomal-associated membrane protein 2
MAPK Mitogen-activated protein kinase
MEF Mouse embryonic fibroblast
MEK Mitogen-activated/extracellular signal-regulated protein kinase mLST8 Mammalian lethal with sec18 protein 8
xii MMTV Mouse mammary tumor virus mRNA Messenger ribonucleic acid mTOR Mammalian target of rapamycin mTORC1 Mammalian target of rapamycin complex 1 mTORC2 Mammalian target of rapamycin complex 2
MTT 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
N-terminus Amino-terminus
NF1 Neurofibromatosis type 1
OD Optical density
PBR Peripheral benzodiazepine receptor
PBS Phosphate-buffered saline
PCR Polymerase chain reaction
PDK1 Phosphoinositide-dependent kinase 1
PI3K Phosphatidylinositol 3-kinase
PIP Phosphatidylinositol phosphate
PLD1 Phospholipase D1
PM Plasma membrane
PTEN Phosphatase and tensin homolog
Rag Ras-related GTP-binding protein
RalGDS Ral guanine nucleotide dissociation stimulator
RasGRF Ras protein-specific guanine nucleotide-releasing factor
RasGRP Ras guanyl releasing protein
RASSF1A Ras association domain family 1A
xiii Rce1 Ras converting enzyme 1
REDD1 Regulated in development and DNA damage responses 1
Rerg Ras-related, estrogen-regulated growth inhibitor
Rheb Ras homolog enriched in brain
RIN1 Ras and Rab interactor 1
RNA ribonucleic acid
RNAi RNA interference
RSK p90 Ribosomal S6 Kinase
RT-PCR Reverse transcription-polymerase chain reaction
RTK Receptor tyrosine kinase
S6K p70 ribosomal protein S6 kinase
SAP Shrimp alkaline phosphatase
SCID Severe combined immunodeficiency
SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shRNA Short hairpin RNA
SOS Son of sevenless
STAT3 Signal transducer and activator of transcription 3
TBST Tris-buffered saline Tween-20
TCTP Translationally controlled tumor protein
TSA Trichostatin A
TSC Tuberous sclerosis complex
WT Wild-type
YFP Yellow fluorescent protein
xiv
CHAPTER 1
INTRODUCTION
Targeted therapies for breast cancer. Approximately 180,000 new cases of breast
cancer were diagnosed in the United States in 2008 (www.cancer.gov), and despite
significant advances in understanding the molecular pathways driving this disease, breast
cancer remains the second leading cause of cancer-related deaths among American women,
with more than 40,000 deaths per year. One complication in the treatment of breast cancer is that this disease is not simply one disease that exhibits a common genetic basis, clinical progression, and outcome; hence, not all breast cancers will respond to one therapeutic approach. Instead, breast cancer is comprised of at least five subtypes that are distinguished by distinct genetic and molecular profiles, and that exhibit differing prognoses and sensitivities to therapies (1-3).
Nearly 70% of breast cancers express the estrogen receptor alpha (ERα) and depend
on estrogen for growth. Current therapies for ERα-positive breast cancers exploit this
dependency on estrogen (Figure 1-1). These therapies include the antiestrogen tamoxifen
(Nolvadex®), which competes with estrogen for binding to the ERα, and aromatase
inhibitors (anastrazole [Arimidex®], exemestane [Aromasin®], and letrozole [Femara®]),
which prevent the synthesis of estrogen (4, 5). However, many tumors develop resistance to
these antiestrogen therapies (4-6). ERα-negative breast cancers tend to be more aggressive
and metastatic than ERα-positive tumors and therapeutic options for many of these patients
Aromatase EGFR HER2 inhibitors E2 Trastuzumab P ERα PI3K Fulvestrant P P Lapatinib P P P AKT Grb2 Ras plasma membrane GTP SOS TSC2P Raf P MEK nucleus Rheb
2 GTP ERKP E2 Raptor P P Tamoxifen ERα mTOR E2-responsive mLST8 ERE genes P P ie, Rerg S6K* 4E-BP1
Figure 1-1. Targeted therapies for breast cancer currently in clinical use. The estrogen receptor (ERα) drives proliferation of two-thirds of breast cancers. Current anti-estrogen therapies include aromatase inhibitors (anastrazole, exemestane, and letrozole), which prevent the synthesis of estrogen, the pure ERα antagonist fulvestrant, which causes degradation of the ERα, and the selective ERα modulator tamoxifen, which competes with estrogen binding to the ERα and prevents ERα-mediated gene transcription. HER2 is overexpressed in a subset of breast cancers and is inhibited by the anti-HER2 monoclonal antibody trastuzumab and the HER1/HER2 tyrosine kinase inhibitor lapatinib. Extensive cross-talk exists between estrogen signaling and growth factor-induced signaling, as described in the text. are much more limited (7). A subset of ERα-positive and ERα-negative breast cancers
overexpress the human epidermal growth factor receptor 2 (HER2/ErbB2/Neu), and while
the advent of HER2 inhibitors, including the HER2 monoclonal antibody trastuzumab
(Herceptin®) and the dual epidermal growth factor receptor (EGFR/HER1/ErbB1)-HER2
kinase inhibitor lapatinib (Tykerb®), have improved the survival of some of these patients,
again the development of resistance to this targeted therapy is a major problem (8-11). While
a number of other targeted therapies are in clinical trials either alone or in combination with
anti-estrogen or HER2 therapies, none has proven to be the “magic bullet” that selectively
targets the breast cancer cell. Therefore, new targeted therapies that block other molecules
driving breast tumor growth are required for breast cancer treatment.
Estrogen signaling in breast cancer. Estrogen stimulates breast cancer growth via genomic and non-genomic signaling mechanisms. Canonical, “genomic” estrogen signaling
involves binding of 17β-estradiol (E2) to nuclear ERα, causing a conformational change in the ERα. The ERα then dimerizes and acts as a transcription factor by binding directly to
DNA via estrogen response elements (EREs), recruiting coactivators, and activating
transcription (Figure 1-1). It is now appreciated that estrogen can also induce rapid, non-
genomic signaling. This signaling is initiated by E2 binding to the ERα in the cytoplasm or
plasma membrane. Ligand-associated ERα can interact with various signaling proteins,
including the EGFR tyrosine kinase, insulin-like growth factor receptor (IGF1R) tyrosine
kinase, and c-Src tyrosine kinase, leading to downstream activation of signaling through
ERK mitogen-activated protein kinase (MAPK; a serine/threonine kinase) and AKT
serine/threonine kinases (Figure 1-1) [reviewed in (4, 6, 12, 13)]. In turn, ERK, AKT, and
3 p70 S6 kinase (S6K) phosphorylate ERα, enhancing its transcriptional activity (14-17).
Membrane-bound ERα can also associate with the p85 regulatory subunit of the
phosphatidyl-inositol-3-OH kinase (PI3K), a lipid kinase that promotes formation of
phosphotidylinositol 3,4,5-phosphate (PIP3), which then facilitates AKT activation (18).
These signaling events occur within minutes and also stimulate cell proliferation. Non-
genomic estrogen signaling is not suppressed by tamoxifen and may contribute to tamoxifen
resistance (4, 6, 19, 20). Hence, components of non-genomic estrogen signaling pathways
may serve as targets for anti-cancer drug discovery.
The Ras family of small GTPases. Members of the Ras branch (36 genes encoding
39 proteins) of the Ras superfamily of small GTPases (156 members) regulate a multitude of
cellular processes, including cell adhesion, differentiation, survival, and proliferation (21)
(Figure 1-2). The three Ras isoforms (H-, K-, and N-Ras; the KRAS gene encodes two highly identical proteins that differ at their C-terminal sequences due to alternative exon 4 utilization, termed K-Ras4A and K-Ras4B) relay extracellular stimulus-mediated cell surface signals to the cytoplasm and the nucleus (Figure 1-3). Ras proteins cycle between a GTP- bound active state and an inactive, GDP-bound conformation. Ras-specific guanine nucleotide exchange factors (RasGEFs; e.g., Sos, RasGRF, RasGRP) promote the exchange of GDP for GTP and thereby promote formation of activated Ras-GTP, whereas GTPase
activating proteins (RasGAPs; e.g., NF1/neurofibromin) negatively regulate Ras GTPases by
catalyzing the hydrolysis of bound GTP to GDP, thereby forming the inactive Ras-GDP. The
conformation of Ras differs in these two nucleotide-bound states in amino acid residues
referred to as switch 1 (30-38) and switch 2 (60-76) (22). When bound to GTP, Ras proteins
4 RalB RalA Rit1 Rit2 Rap1A TC21 Rap1B R-Ras Rap2BRap2A M-Ras Rap2C Rheb1
H-Ras K-Ras Rheb2 N-Ras
E-Ras ARHI
Rad Di-Ras1
Gem Di-Ras2
Rem1 NKIRas1
Rem2 Rerg NKIRas2
RasL12 RasD1 RasD2 RasL11B RasL11A RasL10B RasL10A
Figure 1-2. The Ras branch of the human Ras superfamily of small GTPases. ClustalX was used to generate a dendrogram of the GTP-binding domains of human Ras subfamily members. The K-Ras2B isoform of K-Ras was used. Bubbles indicate proteins with established or putative roles in breast cancer, discussed in the text. Pink indicates proteins that suppress breast cancer growth (tumor suppressors), whereas green indicates proteins that promote tumor growth. While Rad (yellow) has also been implicated in breast cancer, whether it promotes or suppresses tumor growth is unclear. Adapted from Hanker and Der, Handbook of Cell Signaling, in press.
5 Extracellular stimuli (eg, growth factors)
RTKs* Other signals
GTP GEFs GDP
Ras Ras* On Off GDP GTP
GAPs* Pi
Raf PI3K RalGEF RASSF1A RIN1 Effectors*
Gene expression, cell cycle progression, differentiation, apoptosis, actin cytoskeletal remodeling, etc.
Figure 1-3. Ras signal transduction. Ras GTPases cycle between a GTP-bound “on” state and a GDP-bound “off” state. Ras proteins are positively regulated by GEFs and negatively regulated by GAPs. Ras is activated by growth-factor-stimulated receptor tyrosine kinases (RTKs), such as EGFR and HER2. When bound to GTP, Ras interacts with a variety of downstream effectors to control cellular processes. Asterisks denote proteins that are aberrantly expressed or mutated in breast cancer. The RTKs EGFR and HER2 are frequently overexpressed in breast cancer and loss of the RasGAP NF1 leads to an increased risk of breast cancer. While Ras itself is infrequently mutated in breast cancer, mutations in some of its effectors, such as PI3K (PIK3CA), are more common. In addition, loss of the Ras effector RIN1 has been observed in breast cancer. Adapted from Hanker and Der, Handbook of Cell Signaling, in press.
6 preferentially bind and regulate multiple, functionally distinct effectors (e.g., the Raf serine/threonine kinases) to initiate downstream cytoplasmic signaling networks that induce a
plethora of cellular responses.
The domain organization of Ras family GTPases is summarized in Figure 1-4A. The
amino acids G12 and Q61 of Ras are conserved in the majority of Ras superfamily proteins
and are essential for intrinsic and GAP-stimulated GTP hydrolysis. Missense mutations of
these residues are found in 33% of human cancers
(http://www.sanger.ac.uk/genetics/CGP/cosmic/) (23). These mutations render Ras
insensitive to GAPs and thus constitutively GTP-bound and active. Ras-GTP preferentially
binds to and activates its downstream effectors via its core effector domain (residues 32-40)
and flanking switch 1 and 2 sequences. Although this core effector binding domain is conserved in Ras family members, many members of this family signal through distinct effectors. Finally, most Ras family members terminate in a CAAX (C = cysteine, A = aliphatic amino acid, X = terminal amino acid) tetrapeptide sequence, a substrate for C15
farnesyl or C20 geranylgeranyl lipid modification responsible for membrane localization and
critical for Ras biological activity (Figure 1-5) (24).
Ras posttranslational processing and localization. Proper subcellular localization
is critical for activation and effector binding of many small GTPases. In order for Ras to
function properly, it must be targeted to the plasma membrane. This localization is
accomplished via posttranslational addition of a farnesyl isoprenoid lipid to the C-terminus of
Ras by the enzyme farnesyltransferase (FTase) (24). Next, Ras undergoes two additional
CAAX-signaled post-prenylation modifications (Figure 1-5). The Ras converting enzyme 1
7 A. 1 189 H-Ras G12 YDPTIEDSY Q61 CVLS
1 184 Rheb R15 YDPTIENTF Q64 CSVM
1 229 ARHI A46 YLPTIENTY G95 CIIM
1 199 Rerg A12 YDPTLESTY Q64 KISS
GTP Effector GTP HV CAAX Hydrolysis Binding Hydrolysis Domain
Figure 1-4A. Domain organization of Ras family proteins. ARHI contains a 35-residue N- terminal extension (N-extension) that may be important for its tumor suppressor activity. The amino acid residues G12 and Q61 in Ras are important for GAP stimulation of GTP hydrolysis; mutation of these residues in cancer leads to GAP insensitivity and therefore Ras is constitutively GTP-bound and active. Since these residues are not conserved in ARHI, ARHI may be constitutively GTP-bound. Rheb is regulated by the GAP TSC2, but whether the glutamine at position 64 is required for this regulation is unclear. Whether Rerg is regulated by GAPs is not known. Ras residues 32-40 comprise the core sequences important for effector binding. Although this core effector sequence is conserved between most Ras subfamily members, many Ras- related GTPases, such as Rheb, use distinct effectors. The residues in Rheb, ARHI, and Rerg that are not conserved in Ras are shown in bold. The hypervariable (HV) domain in Ras dictates subcellular localization, as does the C-terminal CAAX box, required for lipid modification and membrane targeting. Ras, Rheb and ARHI are farnesylated. Rerg is not farnesylated and does not localize to the membrane. Adapted from Hanker and Der, Handbook of Cell Signaling, in press.
8 B.
H-Ras: M---TEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILD Rerg: MAKSAEVKLAIFGRAGVGKSALVVRFLTKRFIWEYDPTLESTYRHQATIDDEVVSMEILD Rheb1: MPQSKSRKIAILGYRSVGKSSLTIQFVEGQFVDSYDPTIENTFTKLITVNGQEYHLQLVD Rheb2: MPLVRYRKVVILGYRCVGKTSLAHQFVEGEFSEGYDPTVENTYSKIVTLGKDEFHLHLVD * *:.:.* ***::*. ::: .* ****:*.:: : .:. : :.::*
TAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHQYREQIKRVKDSDDVPMVLVGNK TAGQED-TIQREGHMRWGEGFVLVYDITDRGSFEEVLPLKNILDEIKKPKNVTLILVGNK TAGQDEYSIFPQTYSIDINGYILVYSVTSIKSFEVIKVIHGKLLDMVGKVQIPIMLVGNK TAGQDEYSILPYSFIIGVHGYVLVYSVTSLHSFQVIESLYQKLHEGHGKTRVPVVLVGNK ****:: : . .*:: *: :.. **: : : :.::*****
CDLA-ARTVESRQAQDLARSYGIPYIETSAKTRQG-VEDAFYTLVREIR-----QHKLRK ADLDHSRQVSTEEGEKLATELACAFYECSACTGEGNITEIFYELCREVRRRRMVQGKTRR KDLHMERVISYEEGKALAESWNAAFLESSAKENQ-TAVDVFRRIILEAE------KM ADLSPEREVQAVEGKKLAESWGATFMESSARENQ-LTQGIFTKVIQEIA------RV ** * :. :.: ** . .: * ** : * : * :
LNPPDES-GPGCMSCKCVLS RSSTTHVKQAINKMLTKISS DGAASQGKSS------CSVM ENSYGQERR------CHLM .. . * - single, fully conserved residue : - conservation of strong groups . - conservation of weak groups - no consensus
Figure 1-4B. Clustal W was used to generate a complete sequence alignment of H-Ras, Rerg, Rheb1, and Rheb2. The core effector domain is highlighted in yellow. The CAAX tetrapeptide motif of H-Ras, Rheb1, and Rheb2 is shown in red. Rerg lacks a CAAX motif.
9 (Rce1) endoprotease cleaves the AAX residues and isoprenylcysteine methyltransferase
(Icmt) then methylates the C-terminal farnesylated cysteine residue (25, 26). These
modifications increase the hydrophobicity of Ras proteins and thus promote association with
lipids in the membrane. While farnesylation and post-prenyl processing are necessary for
proper Ras localization, they are not sufficient (24, 27). Rather, Ras requires a “second
signal” to reach the plasma membrane. For H-Ras and N-Ras, this second signal consists of
two palmitoylated cysteines in the C-terminal hypervariable domain upstream of the CAAX
motif. The second signal of K-Ras4B consists of polybasic residues that facilitate interaction
with negatively charged phospholipids in the plasma membrane. Ras proteins that lack these
second targeting elements show greatly impaired plasma membrane association and
transforming activities. As discussed below, inhibition of Ras posttranslational processing
and membrane association represents an attractive therapeutic strategy to disrupt aberrant
Ras activity in cancer.
Ras is an oncoprotein. Ras itself is an oncoprotein and is mutationally activated in
33% of all cancers, with the highest frequency seen in pancreatic (90%), colorectal (50%)
and lung (30%) cancers (23, 28). Extensive studies have implicated Ras in numerous aspects
of tumor formation and progression (23). However, Ras is only mutated in 5% of breast
cancers. Despite the fact that Ras is rarely mutated in breast cancer, Ras is activated in many
breast cancers by various upstream regulators, including the epidermal growth factor receptor family, in particular EGFR/HER1/ErbB1 and HER2/ErbB2/Neu (29, 30). Therefore, blocking Ras signaling in breast cancer may be therapeutically beneficial.
10 Plasma Membrane
FTS -C-OMe TLN-4601 Cytoplasm Ras Membrane-associated, Cysmethynil Icmt active Ras -C Rce1 Ras -CAAX
FTI FTase Cytosolic, -CAAX Ras inactive
CAAX = Cys-Aliphatic-Aliphatic-Ser/Met
= Farnesyl Isoprenoid Lipid
Figure 1-5. Ras posttranslational processing and membrane association. Most members of the Ras subfamily, including Ras, Rheb, and ARHI/Noey2, are processed by a series of posttranslational modifications that allow insertion into the plasma membrane or endomembranes. The first step in this process is the addition of a farnesyl isoprenoid lipid to the cysteine residue of the CAAX motif by FTase; FTIs block this step. Next, the endonuclease Rce1 cleaves the –AAX tripeptide and the methyltransferase Icmt methylates the now terminal farnesylated cysteine (O- Me). Complete processing is required for proper Ras localization and function. While FTIs have shown promise in breast cancer clinical trials, they do not inhibit K-Ras4B prenylation, and the FTase substrates responsible FTI inhibition of breast cancer growth are not known. Adapted from Hanker and Der, Handbook of Cell Signaling, in press.
11 Recently, several Ras family proteins have been implicated in breast cancer and may
play significant roles in the initiation and progression of this disease. These proteins include
Rheb (Ras homolog enriched in brain), an activator of the mTOR signaling pathway involved in estrogen-regulated breast cancer, and Rerg (Ras-related estrogen-regulated growth inhibitor) and ARHI (Ras homologue member I, also known as Di-Ras3 or Noey2), two Ras family proteins that, in contrast to Ras and Rheb, negatively regulate breast cancer growth and may function as tumor suppressors rather than oncogenes (Figure 1-2). Here, I focus on these Ras family proteins and their involvement in breast cancer. In particular, my thesis has explored the roles of two of these small GTPases, Rerg and Rheb, in breast cancer progression, with an emphasis on therapeutic strategies to reverse aberrant Rerg or Rheb activity in cancer.
Ras in breast cancer. Ectopic expression of mutationally activated Ras is capable of
transforming immortalized human mammary epithelial cells and transgenic Ras expression
drives mammary tumor development in mice (31). Furthermore, ectopic expression of Ras in
the estrogen-dependent MCF-7 cell line promotes estrogen-independent growth (32-34).
However, evidence for activation of endogenous Ras in human breast tumors is more limited.
The growth factor receptors EGFR and HER2 are aberrantly activated by overexpression in
breast cancers and can cause upstream activation of Ras (Figure 1-1). Studies have found that
the levels of activated, GTP-bound Ras are increased in breast tumors and cell lines
overexpressing EGFR or HER2 (29, 30). Therefore, Ras may be activated in breast tumors
in the absence of direct mutational activation of Ras. Ras proteins are also overexpressed in
20-50% of breast cancers. Another mechanism by which Ras may be activated in breast
12 cancer could be decreased expression of a negative regulator of Ras, such as a RasGAP.
Women with the genetic disease neurofibromatosis, caused by inactivating mutations of the
NF1 gene, which encodes the neurofibromin RasGAP, have an increased risk of developing breast cancer (35, 36), but whether the NF1 tumor suppressor is downregulated in spontaneous breast cancers is not known.
Recent work by Song and colleagues has revealed a mechanism by which H-Ras
expression is elevated in breast tumor-initiating cells, which may represent breast cancer
“stem cells” (37). They showed that expression of the microRNA let-7, a negative regulator
of H-Ras protein expression, is reduced in breast tumor-initiating cells (cancer stem cells)
and in clinical samples. They further showed that restoring let-7 expression reduced H-Ras
expression, cell proliferation, mammosphere formation, tumorigenicity, and metastasis, and
that silencing H-Ras alone was sufficient to reduce mammosphere formation, tumor
formation, and metastasis (37). Thus, H-Ras inhibition may represent a promising therapeutic approach for targeting breast cancer stem cells. Further work is needed to determine whether H-Ras levels are increased in independently-derived populations of breast cancer stem cells.
GTP-bound Ras associates with a variety of effectors to elicit a diverse array of
cellular responses. The best characterized Ras effectors implicated in oncogenesis include
the Raf serine/threonine kinases, the PI3Ks, and GEFs for the Ral GTPases (RalGEFs) (38).
Mutations in the genes encoding B-Raf and the p110α catalytic subunit of PI3K (PIK3CA)
have been identified in many cancers, supporting a role for these Ras effectors in
oncogenesis. While BRAF mutations are rare in breast cancer, PIK3CA is mutated in 20-40%
of human breast cancers (39) and its occurrence is mutually exclusive with Ras mutations in
13 breast cancer cell lines (40). However, Ras also activates some effectors that negatively
regulate cancer growth; silencing of these effectors, such as the RASSF1 tumor suppressor and its family members, is a common event in cancer (41, 42). Hypermethylation of the
RASSF1A promoter has been observed in 80-90% of breast cancers (43, 44), but a definitive role for RASSF1A in breast cancer has not yet been established. The Ras effector RIN1 was
recently implicated as a tumor suppressor in breast cancer. RIN1 regulates epithelial cell
properties such as cytoskeletal remodeling through Abl tyrosine kinases (45). Colicelli and
colleagues found that RIN1 levels are reduced in primary breast tumors and in breast cancer
cell lines, and that restoring RIN1 expression suppressed tumor growth (46). Whether loss of
RIN1 correlates with Ras activation, and whether its loss is necessary for Ras-induced tumor
growth, is not known.
Therapeutic targeting of Ras. Due to the unequivocal importance of Ras in human
cancer development and progression, there have been considerable efforts to develop
pharmacologic agents that block Ras function for cancer therapy. One major approach has
been the development of inhibitors of effector signaling, in particular inhibitors of the Raf-
MEK-ERK and PI3K-AKT-mTOR pathways (Figure 1-6) (47, 48), although combining inhibitors of these two pathways may be required for the treatment of tumors bearing Ras mutations (49). A second major focus has been inhibition of Ras membrane association
(Figure 1-5 and 1-6). FTase inhibitors (FTIs) were originally developed to block Ras localization and thereby block Ras function in cancer. However, it is now appreciated that
FTIs do not in fact block localization of the most commonly mutated Ras isoforms in cancer,
K-Ras4B and N-Ras. These Ras isoforms escape FTI inhibition by a process known as
14 Salirasib (FTS) Ras TLN-4601 Raf inhibitors, MEK inhibitors, PI3K Effectors inhibitors, mTOR Ras inhibitors 15 Lonafarnib FTase Tipifarnib Ras
Ras membrane association = Farnesyl isoprenoid Farnesyltransferase inhibitors Protein kinase inhibitors Figure 1-6. Therapeutic strategies to inhibit Ras signaling. Efforts in developing “anti-Ras” drugs have largely focused on inhibition of downstream effector signaling and inhibition of membrane association. In particular, protein kinase inhibitors targeting the Raf-MEK-ERK and PI3K-AKT-mTOR pathways are being tested in clinical trials. FTIs such as lonafarnib and tipifarnib are also in clinical trials but have failed to inhibit Ras oncogenic signaling. Farnesyl-containing small molecules such as FTS and TLN-4601 are being developed in the hopes of impairing Ras membrane association and function. Adapted from Yeh and Der, Expert Opin Ther Targets 2007;5:673-94. alternative prenylation, whereby they are modified by addition of another isoprenoid lipid,
geranylgeranol; geranylgeranylated Ras proteins remain functional and transforming in the presence of FTIs (50). Despite an inability to effectively block Ras, FTIs have shown some anti-tumor activity, particularly in breast cancer (51). Since other substrates of FTase do
have known roles in cell proliferation (e.g., Rheb) (52), the anti-tumor activity of FTIs may be due to inhibiting the function of these proteins. FTIs have also demonstrated clinical activity in combination with hormonal therapies in breast cancer (53, 54). Elucidating the
FTase substrates responsible for the anti-tumor activity of FTIs in breast cancer could be very beneficial in selecting patients that may respond to this targeted therapy.
Due to the failure of FTIs to successfully block K- and N-Ras activity, attention has
turned to other methods of disrupting Ras membrane association. Initial studies suggested that blocking Rce1- or Icmt-mediated modifications may not impair Ras transforming activity, since Ras mutants that were not modified by Rce1 or Icmt lost only 50% of their membrane localization and transforming activity (55). More recent studies using genetic ablation of Rce1 and Icmt have suggested that inhibition of these enzymes may indeed block
Ras-induced oncogenesis. For example, mice lacking Icmt displayed reduced lung tumor formation and reduced myeloproliferation phenotypes driven by mutant KRAS (56), but whether this was directly due to Ras inhibition is not known. Although Rce1 deficiency reduced Ras membrane association and transformation (57), Rce1 deficiency actually accelerated Ras-induced myoproliferative disease in mice (58), perhaps because Rce1 is required for proteolytic processing of many other CAAX-terminating proteins besides Ras.
Whether Rce1 deficiency blocks the growth of other Ras-dependent tumors is not known.
16 Therefore, recent efforts have focused on developing pharmacological inhibitors of Icmt to
block Ras-driven cancer.
Several inhibitors of Icmt have been reported, including S-adenosylhomocysteine
(AdoHcy), which inhibits many methyltransferases, and prenylcysteine analogs such as N-
acetyl-S-farnesyl-L-cysteine (AFC) (25, 26). However, these compounds lack specificity and
have considerable off-target effects unrelated to Icmt inhibition (26). More recently, Casey
and colleagues used a chemical library screen to identify a small molecule inhibitor of Icmt,
cysmethynil (59). Cysmethynil was shown to mislocalize Ras, block the anchorage-
independent growth of colon cancer cells in an Icmt-dependent fashion, and reduce tumor
xenograft growth of PC3 prostate cancer cells (59, 60). Cysmethynil was also shown to
induce autophagy and disrupt mTOR signaling (60). Whether the anti-tumor effects of
cysmethynil are due specifically to Ras inhibition is not known.
Farnesyl isoprenoid-containing small molecules represent another pharmacological approach to interfere with Ras membrane localization and function (Figure 1-6 and 1-7).
Because these compounds contain a farnesylated isprenoid, they are proposed to compete with farnesylated Ras for membrane-associated Ras binding partners (e.g., galectins) and dislodge Ras from the membrane, thereby antagonizing Ras signaling and function (61).
There are currently two such molecules undergoing clinical evaluation: FTS (salirasib) and
TLN-4601. FTS has been shown to dislodge Ras from the membrane, prevent Ras from binding to galectin-1 in the membrane, promote degradation of Ras protein, decrease levels of Ras-GTP, and inhibit Ras activation of the Raf-MEK-ERK pathway (62-64). In addition,
FTS reversed Ras-induced transformation, but not transformation driven by Raf (65, 66), indicating selectivity for Ras-transformed cells. Furthermore, FTS inhibited the growth of
17 Farnesylated Ras
Ras NH NH S
O O
Salirasib (S-trans-trans-farnesylthiosalicylic acid; FTS)
COOH
S 18
TLN-4601 O
N
HO HN OH HO
Dibenzodiazepinone Farnesyl isoprenoid
Figure 1-7. Chemical structures of farnesyl-containing small molecules in clinical development. FTS and TLN-4601 are farnesylcysteine mimetics that contain the C15 farnesyl isoprenoid lipid (shown in red) and are thought to displace farnesylated Ras from membrane binding sites. TLN-4601 also contains a dibenzodiazepinone moiety that binds to the peripheral benzodiazepine receptor, which is overexpressed in some tumor cells. Both small molecules are currently in Phase I/II clinical trials. many human cancer cell lines in vitro and in vivo (67-74), prompting clinical investigation.
FTS is currently in Phase I/II clinical trials (www.clinicaltrials.gov) and is well tolerated
(75). The results of a recent phase II trial of FTS in combination with gemcitabine for the
treatment of pancreatic cancer are particularly encouraging. The combination resulted in a
median survival period of greater than 10.8 months and a one-year survival rate of 50% (76).
Importantly, tumor biopsies showed that FTS treatment reduced the levels of Ras protein
(76). Interestingly, FTS has also been shown to inhibit the growth of breast cancer cells and
block mTOR signaling (77-79). Whether the anti-tumor effects of FTS are due to Ras
inhibition or inhibition of the mTOR pathway is not known. More potent FTS derivatives are
currently being developed (80).
Another farnesyl-containing small molecule is TLN-4601 (formerly ECO-4601), a
farnesylated dibenzodiazepinone natural product isolated from Micromonospora bacteria
(81). In addition to being a farnesylcysteine mimetic like FTS (Figure 1-7), TLN-4601 also
binds selectively to the peripheral benzodiazepine receptor (PBR) (81). The PBR is
overexpressed in some tumors and may promote the preferential uptake of TLN-4601 in
tumors (82). Micromolar concentrations of TLN-4601 have broad cytotoxic activity. TLN-
4601 was also shown to have significant antitumor activity in xenografts of several human
cell lines that overexpress the PBR (81). A phase I/II clinical trial found that TLN-4601 is
safe and well-tolerated in patients. TLN-4601 is now in a phase II clinical trial as a second
line therapy for glioblastoma multiforme (www.clinicaltrials.gov). Whether, like FTS, TLN-
4601 also inhibits Ras activity has not been thoroughly evaluated.
19 Rheb. Rheb1 and Rheb2 (51% amino acid identity; Figure 1-4B) are members of the
Ras family of small GTPases (83) that may have increased activity in breast cancer. Rheb is
a critical component of the AKT-TSC-mTOR pathway which responds to a plethora of
cellular signals to regulate protein translation, cell size, and cell cycle progression (Figure 1-
8) (84). The tumor suppressor TSC2 (tuberin), in complex with TSC1 (hamartin), functions
as a GAP towards Rheb and thereby suppresses Rheb-GTP formation and signaling (85, 86).
The role of TSC2 in Rheb signaling is analogous to that of the RasGAP NF1—loss of NF1 in
neurofibromatosis type I leads to hyperactivation of Ras. Similarly, loss-of-function
mutations in the TSC1 or TSC2 genes cause the Mendelian disorder tuberous sclerosis, which
is characterized by the formation of benign hamartomas in a variety of organs (87, 88). In this disease, loss of TSC2 or TSC1 results in hyperactivation of Rheb and promotes Rheb- mediated activation of the mTOR (mammalian Target of Rapamycin) complex 1 (mTORC1), which consists of mTOR, raptor, and GβL (mLST8). Rheb does not activate a second mTOR complex, mTORC2 (consisting of mTOR, rictor, GβL, and Sin1), which is regulated
by other mechanisms (89). Although Rheb binds to mTOR irrespective of GDP or GTP
association, Rheb activates mTOR only when bound to GTP (90). The mechanism by which
Rheb-GTP activates mTOR is incompletely understood, but may involve association with
FKBP38 and phospholipase D1 (PLD1). Sun et al. recently showed that Rheb binds and
activates PLD1 in a GTP-specific manner and that PLD1 is required for Rheb activation of
the mTOR pathway (91). Bai et al. suggested that FKBP38 negatively regulates mTORC1
signaling, and that Rheb-GTP association with FKBP38 relieves this negative regulation,
leading to mTORC1 activation (92-94). However, this model has recently been challenged
by multiple studies (95-97).
20 Growth factors/ Low energy Hypoxia insulin REDD1 LKB1 RTKs P P AMPK AKT Amino P p53 P ERK acids Pi P P P P Tsc1 Tsc2 P DNA RSK Low glucose Damage GAPDH RalGDS Rag Hypoxia GTP Rheb Rheb Bnip3 21 GDP GTP RalA GTP PLD1? GEF? FKBP38? P GDP GTP Raptor P mTOR Autophagy P B-Raf mLST8 Aggresome formation Protein translation, Cell growth, Cell cycle progression, Survival Figure 1-8. Rheb signal transduction. The TSC2-Rheb-mTOR pathway is tightly regulated by growth factor signaling, hypoxia, and amino acid, glucose, and energy availability. Many cellular signals converge on the TSC1/TSC2 RhebGAP complex to either enhance or disrupt its GAP activity. Rheb is also regulated independently of TSC2 by binding to GAPDH and Bnip3. mTORC1 is the primary Rheb effector, but the mechanism by which GTP-bound Rheb activates mTORC1 is incompletely understood. Amino acids promote Rheb activation of mTOR through Rag and Ral GTPases. Rheb also exhibits mTOR-independent activities: it inhibits aggresome formation and B-Raf activity. Rheb activation of mTOR may also involve enhanced recruitment of the mTORC1
substrates eIF4E binding protein 1 (4E-BP1) and p70 S6 kinase (S6K) (93, 96). mTOR-
mediated phosphorylation of these two substrates promotes protein translation (Figure 1-9).
Hypophosphorylated 4E-BP1 binds and sequesters the translation initiation factor eIF4E.
Phosphorylation of 4E-BP1 by mTOR allows the release if eIF4E, which promotes cap-
dependent mRNA translation and tumorigenesis (98). S6K phosphorylates the S6 ribosome subunit, also promoting increased protein translation, ribosome biogenesis, and the development of benign tumors.
Recent studies have found that Rheb can be regulated independently of TSC2 and that
Rheb also exhibits mTOR-independent functions (Figure 1-8). Although the Translationally
Controlled Tumor Protein (TCTP) was reported originally to be a GEF for Rheb in
Drosophila melanogaster (99), subsequent studies have failed to confirm it as a GEF for mammalian Rheb (95, 100). Thus, a bona fide GEF for Rheb in mammalian cells has yet to be identified. Several proteins have been shown to negatively regulate Rheb by binding and sequestering it away from mTOR. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) binds directly to Rheb and inhibits Rheb/mTOR signaling under low glucose conditions
(101). The hypoxia-inducible protein Bnip3 binds Rheb and blocks Rheb-induced mTOR activation during hypoxia (102). Rheb-mTOR signaling is also regulated by amino acid availability independently of TSC2. The Rag GTPases have been suggested to mediate amino acid-induced mTOR activation by promoting subcellular localization of mTOR to a compartment containing Rheb (103-105). RalGDS and RalA have also been shown to promote Rheb activation of mTOR in response to amino acids (106). This tight regulation of
22 E2 P ERα
PTEN* PI3K*
P AKT* AKT
P Tsc1*Tsc2* Tsc1*Tsc2*
FTI Rheb Rheb GTP GDP
PLD1? FKBP38? P Raptor P mTOR FKBP12 mLST8 rapamycin
P P S6K* 4E-BP1
P S6 eIF4E Protein Translation
Figure 1-9. Estrogen regulation of Rheb signaling. When bound to estrogen (E2), the ERα can initiate rapid, “non-genomic” signaling through PI3K. PI3K is a lipid kinase that converts PIP2 into PIP3. The reverse reaction is catalyzed by the lipid phosphatase PTEN, a negative regulator of the pathway. PIP3 leads to activation of Akt, which then phosphorylates a variety of substrates involved in cell proliferation and survival, including TSC2. Akt-mediated phosphorylation of TSC2 inactivates its RhebGAP activity, leading to increased levels of Rheb-GTP. The mechanism by which Rheb activates mTOR is not entirely understood, but may involve displacement of the mTOR inhibitor FKBP38 and/or activation of PLD1. The mTORC1 complex (mTOR, raptor, and mLST8) phosphorylates substrates involved in protein translation, including S6K and 4E-BP1. S6K can also phosphorylate the ERα, stimulating its transcriptional activity. Rheb is inhibited by FTIs and mTOR is inhibited by rapamycin. Green indicates positive regulators of the pathway; red indicates negative regulators. Asterisks denote proteins that are known to be mutated or aberrantly expressed in breast cancer. Adapted from Hanker and Der, Handbook of Cell Signaling, in press.
23 Rheb and mTOR activation ensures that the cell will not waste energy and nutrients by
engaging in protein synthesis under suboptimal conditions.
Although mTOR is the best studied Rheb effector, Rheb has been shown to possess
mTOR-independent functions (Figure 1-8). Multiple studies have found that Rheb binds to
Raf-1 and B-Raf and inhibits their activity (107-111). Whether tumors containing hyperactive Rheb exhibit reduced Raf activity is not known. Rheb has recently been shown
to inhibit dynein-dependent transport of misfolded proteins and thereby block aggresome
formation (112). The direct effector of Rheb responsible for this process has not yet been identified. While mTOR has been shown to be the primary Rheb effector involved in
tumorigenesis (113, 114), whether these mTOR-independent Rheb functions also contribute
to tumor formation, or perhaps restrict tumor growth in tuberous sclerosis patients, is not
known.
Recent studies have strengthened the case for the involvement of Rheb in sporadic
tumor development (Figure 1-10). Rheb1 and Rheb2 are overexpressed in a variety of tumors, including breast, prostate, lung, colon, ovary, glioma, and oral squamous cell
carcinoma (114-118). In addition, a Rheb mutation has been found in colon cancer (119),
and the RHEB gene is amplified in some prostate cancers (118). Furthermore, an activated
Rheb mutant was found to transform chick embryo fibroblasts (114) and Rheb
overexpression promoted tumor formation in mouse models of lymphoma and prostate
cancer (113, 118). Others have found that Rheb is involved in the survival of dormant tumor
cells (120) and in resistance to chemotherapeutics (113, 115). Therefore, it is likely that
Rheb inhibition will be therapeutically beneficial both for the treatment of TSC and for a
variety of cancers, including breast cancer.
24 Transformation of chick Tumor formation in embryo fibroblasts mouse models of Jiang and Vogt, Oncogene 2008 lymphoma and prostate Survival of dormant cancer tumor cells Mavrakis et al, Genes & Dev. 2008; Schewe and Aguirre-Ghiso, PNAS Nardella et al, Genes & Dev. 2008 2008 25 Rheb
Chemoresistance Senescence Mavrakis et al, Genes & Dev. 2008; Mavrakis et al, Genes & Dev. 2008; Basso et al, JBC 2005 Nardella et al, Genes & Dev. 2008 Figure 1-10. Oncogenic properties of Rheb. Not only does Rheb activation promote tumorigenesis in TSC patients, but Rheb has also been implicated in many types of sporadic cancers. Overexpression of activated Rheb promotes transformation of chick embryo fibroblasts and tumor formation in mouse models of lymphoma and prostate cancer. Rheb has also been implicated in the survival of dormant tumor cells and resistance to chemotherapeutics. Finally, like other oncogenes, overexpression of Rheb has been shown to induce senescence both in mouse fibroblasts and in mouse prostate tissue, suggesting that further molecular aberrations are required in order to bypass Rheb-induced senescence and promote tumorigenesis. Although mutations of TSC1 and TSC2 are not found in breast cancer, other
mechanisms may activate Rheb in breast cancer. Studies have found reduced expression of
TSC1 and TSC2 in breast cancer (121). The mRNA levels of both Rheb1 and Rheb2 are
elevated in breast cancer cells (115). The TSC2-Rheb-mTOR pathway can also be activated
by genetic alterations that cause activation of PI3K and its target, the serine-threonine kinase
AKT (Figure 1-9). PI3K promotes formation of PIP3, which facilitates AKT membrane association and activation. AKT phosphorylates and inactivates TSC2, leading to an increase in GTP-bound Rheb. The PI3K-AKT pathway is one of the most commonly activated pathways in breast cancer (39). In particular, mutational activation of the gene encoding p110α, the catalytic subunit of PI3K, is is one of the most frequent gene mutations found in breast cancer (119). Loss of the PTEN tumor suppressor, a negative regulator of PI3K, is alos prevalent in breast cancer (122). PTEN encodes a lipid phosphatase that converts PIP3 back to PIP2.
Like Ras, Rheb can be hyperactivated in breast cancer by upstream signals. HER2
and EGFR overexpression can cause activation of PI3K and thus Rheb (Figure 1-8). Other
growth factor-regulated kinases, such as ERK and RSK, also phosphorylate and inactivate
TSC2, thereby promoting Rheb-GTP formation (Figure 1-8) (123, 124). Furthermore,
estrogen can trigger Rheb signaling in the MCF-7 ERα-positive breast cancer cell line.
Estrogen activates PI3K and AKT through non-genomic mechanisms (Figure 1-9) and
estrogen treatment was recently found to increase Rheb-GTP levels and to stimulate
downstream signaling through mTOR (125, 126). Rheb was also shown to be required for
estrogen-induced DNA synthesis and cell cycle progression in MCF-7 cells (126). In
addition, mTOR is required for estrogen-induced MCF-7 cell proliferation (127). Rheb and
26 mTOR have also been implicated in tamoxifen resistance: Rheb inhibition increased
sensitivity to tamoxifen (115) and mTOR inhibition prevented AKT-induced tamoxifen
resistance in MCF-7 cells (128, 129). Therefore, inhibiting Rheb signaling may be
beneficial in ERα-positive and tamoxifen-resistant breast cancers. Further research is needed
to determine the role of Rheb in tamoxifen resistance and to validate Rheb as a therapeutic
target for the treatment of breast cancer.
Inhibitors of the Rheb-mTOR pathway in breast cancer. Like Ras, Rheb terminates in a CAAX motif and is farnesylated. Farnesylation is essential for its proper localization and function (86, 107, 114, 115, 130-132). FTIs block Rheb signaling and function and unlike Ras, Rheb does not undergo FTI-induced alternative prenylation (107,
115, 131). Therefore, Rheb inhibition may be partially responsible for the anti-breast cancer effects of FTIs. Basso et al. showed that the FTI lonafarnib (Sarasar®) enhanced tamoxifen- induced apoptosis, and this enhancement was prevented by a lab-generated FTI-resistant, geranylgeranylated mutant of Rheb (GG-Rheb) (115). GG-Rheb was also shown to rescue the anti-tumor effects of FTIs in lymphoma cells (113). Determining whether this FTI- resistant GG-Rheb variant prevents FTIs from decreasing breast cancer growth may validate
Rheb activation status as a biomarker for the selection of patients who may benefit from FTI therapy. My studies in Chapter 3 focus on blocking CAAX-mediated posttranslational processing as a means to inhibit Rheb-mTOR signaling in breast and other cancers.
FTIs are currently in clinical trials and have showed promise for the treatment of
advanced breast cancers. Preclinical studies have shown synergistic activity when FTIs were
given in combination with tamoxifen (53, 133). In a phase II clinical trial, 24% of patients
27 with hormone-refractory breast cancers showed clinical benefit when treated with the FTI
tipifarnib (Zarnestra®) (54). Tipifarnib has shown promising results in a phase II trial in combination with tamoxifen, achieving stable disease in 55% of patients (53). In contrast, a phase II study found that the addition of tipifarnib to letrozole did not improve the objective response rate (134), suggesting that tipifarnib may not be beneficial in combination with aromatase inhibitors.
mTOR has been well validated as a target for anti-cancer therapeutics and several
mTOR inhibitors are currently under clinical evaluation (135, 136). In addition, the
rapamycin analogs temsirolimus (Torisel®) and everolimus (Afinitor®) have recently been
approved for the treatment of advanced renal cell carcinoma (136). Rapamycin analogs have
also shown promise in combination with either endocrine therapies or HER2 inhibitors in
clinical trials for breast cancer (53, 137, 138). As mentioned above, FTS has also been
shown to inhibit mTOR (79), and FTS reduced the growth of estrogen-dependent and long-
term estrogen-deprived MCF-7 cells (77, 78, 139). Therefore, mechanistically distinct
inhibitors of the Rheb-mTOR pathway may help to overcome the development of resistance
to current targeted therapies in breast cancer.
ARHI/Di-Ras3/Noey2. Whereas RAS is an oncogene, and many genes encoding Ras
subfamily members, such as Rheb, promote growth, there are also several Ras-related genes that have been implicated as tumor suppressor genes in breast cancer. One such gene, ARHI, was identified originally by Bast and colleagues by using differential display PCR in ovarian cancer cells to identify genes whose expression is lost in tumor cells (140). ARHI is maternally imprinted and expressed monoallelically. Loss of heterozygosity, in which the
28 non-imprinted allele was deleted, was reported in 41% of breast and ovarian cancers (140).
ARHI expression is also negatively regulated by promoter hypermethylation, decreased
mRNA stability, and transcriptional regulation by histone deacetylase (HDAC)-E2F
complexes (141-143). ARHI expression was found to be reduced in 70% of invasive breast
carcinoma tissues and decreased expression was associated with breast cancer progression
and lymph node metastases (144, 145). Recently, mutations in ARHI were found in 42% of
breast cancer tissues as well as 34% of breast hyperplasias (146), suggesting that disruption
of ARHI is an early event in breast tumorigenesis. ARHI expression is also downregulated in
several other cancers, including pancreatic cancer, thyroid cancer, and non-small cell lung
cancer, suggesting that loss of ARHI may be an important step in oncogenesis (147).
Ectopic reexpression of the ARHI protein was found to reduce the clonogenic growth
of breast and ovarian cancer cells, reduce cyclin D1 promoter activity, and increase p21CIP1 expression (140), supporting a tumor suppressor and cell cycle inhibitory function. Bast and colleagues further showed that ARHI re-expression inhibited breast cancer cell proliferation and tumor xenograft formation and induced calpain-dependent apoptosis in breast and ovarian cancer cells (148). Since ARHI is divergent at two key residues important for the intrinsic GTPase activity of Ras and Ras-related proteins (residues A46 and G95), ARHI may be locked in a constitutively active conformation and may not be regulated by GDP/GTP cycling (Figure 1-4A). The mechanism of ARHI-induced tumor suppression was shown to depend on its unique N-terminal extension (149), absent in other Ras family members
(Figure 1-4A), and to involve inhibition of the signal transducer and activator of transcription
STAT3 (150). Strategies to cause reexpression of ARHI may represent an attractive therapy for ARHI-negative breast cancer patients.
29 Like Ras and Rheb, ARHI terminates in a CAAX motif, is farnesylated, and does not
undergo alternatively prenylation. The farnesylation of ARHI is essential for its membrane
association and growth inhibitory function (149). Therefore, it may be an important “off-
target” for FTIs that results in promotion, rather than inhibition, of growth.
Rerg. Like ARHI, Rerg is another Ras-related small GTPase that is downregulated
in breast cancers and is thought to play a growth-inhibitory role. Rerg was initially identified in a microarray gene expression screen where its expression was decreased in the most aggressive, ERα-negative subtypes (2, 151). Rerg was found to be a transcriptional target of the ERα, which functions as a transcription factor when bound to estrogen (Figure 1-11).
Rerg expression was stimulated by estrogen treatment and reduced by the anti-estrogen
tamoxifen (151). Other microarray studies have found decreased Rerg expression in several
different types of cancer and in metastatic cancers relative to their non-metastatic
counterparts (152), suggesting that loss of Rerg expression may play a role in tumor
progression and metastasis.
Unlike Ras and Rheb, which are evolutionarily conserved from yeast to humans, Rerg
has evolved more recently. Rerg is conserved in mammals, chickens (gallus gallus),
zebrafish (danio rerio), and frogs (xenopus tropicalis), but is not found in invertebrates or
outside of the animal kingdom. While Rerg shares substantial sequence identity with Ras
(42%), particularly in the core effector domain (Figure 1-4), Rerg does not bind to canonical
Ras effectors and no Rerg effectors have been identified to date (Figure 1-12). Rerg was shown to bind and hydrolyze GTP (151), but whether GTP binding is required for Rerg activity is not known. Additionally, no GEF(s) or GAP(s) for Rerg have been identified.
30 Tamoxifen E2 ERα nucleus
transcriptional E2 coactivators
ERα
ERE Rerg
Figure 1-11. Estrogen regulation of Rerg expression. When bound to estrogen (E2), the ERα can function as a transcription factor in the nucleus. The RERG promoter contains an upstream estrogen response element (ERE). When the ERα binds to EREs, it recruits transcriptional coactivators to activate transcription of estrogen-responsive genes such as Rerg. Rerg expression correlates with ERα expression in breast cancer; ERα-negative breast cancers lack Rerg expression. Adapted from Hanker and Der, Handbook of Cell Signaling, in press.
31 Upstream signals?
GTP GEF? GDP
Off? Rerg Rerg On? GDP GTP
GAP? Pi
Effectors?
Growth inhibition? Other functions? Gene expression?
Figure 1-12. Unknown mediators of Rerg signal transduction. Like Ras and Rheb, Rerg cycles between GDP- and GTP-bound states. Whether Rerg is only “active” when GTP-bound is not known. Furthermore, regulators of Rerg GTPase cycling and upstream signals that promote or inhibit Rerg GTP binding have not yet been identified. Finally, while the Rerg effector domain is similar to that of Ras, Rerg does not bind to canonical Ras effectors, and Rerg effectors that mediate growth inhibition or other functions of Rerg have yet to be identified. Adapted from Key et al., Methods Enzymol 2005;407:513-27.
32 Furthermore, whether Rerg is activated by growth factor signaling, like Ras and Rheb, is not known. Unlike most Ras family members, Rerg lacks the C-terminal CAAX motif and is localized primarily in the cytosol (151, 153). Overexpression of Rerg in MCF-7 cells decreased cell proliferation and xenograft formation (151), suggesting that Rerg is a growth inhibitor in breast cancer. However, ectopic reexpression of Rerg in ERα-negative, Rerg- negative breast cancer cell lines did not affect anchorage-dependent or –independent growth or invasion (152), casting doubt on the role of Rerg as a tumor suppressor in breast cancer.
My studies further evaluating the role of Rerg as a tumor suppressor in breast cancer will be presented in Chapter 2. More studies are needed to confirm the role of Rerg as a growth inhibitor in breast cancer in vivo.
Other Ras Family Proteins in Breast Cancer. There are several other members of
the Ras subfamily that have been implicated in breast cancer, but more studies are needed to
validate the importance of these proteins in breast cancer. Rap1, a Ras-related protein
involved in integrin and cadherin signaling, was shown to be involved in breast cancer lumen
formation (154). Rap1 activity was elevated in a malignant breast cancer cell line compared
to its nonmalignant counterpart when cultured in three-dimensional laminin-rich extracellular
matrix (154). Dominant-negative Rap1, which blocks GEF activation of endogenous Rap1,
restored tissue polarity and induced lumen formation in breast cancer cells. Dominant-
negative Rap1 also reduced tumor incidence in mice, whereas constitutively active Rap1
increased invasiveness and tumorigenicity (154). Determining whether GTP-bound Rap1 is
increased in primary breast cancer tissue and whether RNAi-mediated silencing of Rap1 in
33 multiple breast cancer cell lines reduces tumorigenicity and invasion will strengthen the
evidence for the importance of Rap1 in breast cancer tumorigenesis.
Rad (R-Rad/Rem3) is a Ras-related GTPase that is normally expressed in heart,
skeletal muscle, and lung tissues (155). Multiple groups have reported reduced expression of
Rad in breast cancers (156, 157), suggesting a role for Rad as a tumor suppressor. However,
ectopic expression of Rad in MDA-MB 435 breast cancer cells was shown to increase
anchorage-independent growth and tumor formation in mice, implicating Rad as an oncogene instead of a tumor suppressor (156). Coexpression of the metastasis suppressor nm23, which
interacts with Rad, inhibited Rad-induced breast cancer cell growth (156). More work is needed to resolve whether Rad is a tumor suppressor or an oncogene in breast cancer, and whether these discrepancies reflect cell context differences in function.
The Ral GTPases (RalA and RalB) have recently garnered attention for their roles in
promoting oncogenesis, tumor growth, and metastasis (158). Ral GTPases function downstream of the Ras effector, RalGEF, and the RalGEF-Ral pathway is required for Ras- induced transformation in human cells (159). However, whether RalA and RalB are involved in breast cancer is not known. Ral was shown to be required for EGFR-induced estrogen-independent growth in MCF-7 cells, but constitutively active RalA was not sufficient to promote estrogen-independent growth (160). Missense mutations in the RalGEF
Rgl1 have been found in breast cancer (161), but whether these mutations activate Ral and drive tumorigenesis has not been determined. Clearly, further research is warranted in order to determine whether targeting Ral or Rgl1 would be beneficial in breast cancer.
The GEF BCAR3 was found in a screen to identify proteins that promote tamoxifen
resistance (162). While BCAR3 overexpression does indeed promote antiestrogen
34 resistance, the question of which downstream GTPase it utilizes to do so is controversial.
Studies originally implicated the mouse homolog of BCAR3, AND-34, as a GEF for RalA,
Rap1A, and R-Ras (163). However, overexpression of AND-34 failed to activate RalA or R-
Ras in breast cancer cells (160, 164), and AND-34-induced antiestrogen resistance was found
to be mediated by PI3K and Rac1 (164, 165). Nevertheless, constitutively active R-Ras was
sufficient to induce estrogen-independent MCF-7 cell proliferation (160). Future studies to address whether R-Ras activity is elevated in tamoxifen-resistant breast cancer tissues will help to evaluate its role in breast cancer.
In summary, members of the Ras subfamily of small GTPases may represent
attractive targets for the treatment of breast cancer, but more research is needed to fully
validate the causal roles of these proteins in breast cancer. In addition, other small GTPases,
including members of the Rho and Rab subfamilies, have been implicated in breast cancer
and have been reviewed elsewhere (166-168). Unlike protein kinases or cell surface receptors, GTPases have not been classically considered promising druggable targets. The successful development of ATP-competitive inhibitors of protein kinases as anti-cancer drugs prompted the consideration of similar approaches for blocking Ras family small GTPase function. However, in contrast to the micromolar binding affinity of protein kinases for
ATP, GTPases bind GTP with picomolar affinities; therefore, small molecule approaches to block GTP binding are not feasible. As strategies to therapeutically inhibit small GTPase signaling improve, it will be imperative to understand the contributions of each of these proteins to breast cancer development and progression.
35 Rationale for studies. Several members of the Ras family of small GTPases have
been implicated in breast cancer and may represent therapeutic targets for the treatment of
either ERα-positive or ERα-negative breast cancers. I have chosen to focus on the roles of
Rerg and Rheb, two estrogen-regulated Ras family small GTPases, in breast cancer. Rerg is
regulated by classical, genomic estrogen signaling and is not expressed in ERα-negative
breast cancers. ERα-negative breast cancers are the most advanced, poorly differentiated,
invasive, and fatal types of breast cancer. There are currently no therapies tailored
specifically to ERα-negative breast cancers, particularly those that do not overexpress HER2.
Thus, ERα-negative breast cancers are in dire need of directed therapies that limit their
invasiveness and malignancy. Restoring expression of the ERα in ERα-negative breast
cancer cell lines reduces their invasiveness (169), but the key transcriptional targets of the
ERα responsible for inhibiting invasion are currently unknown. I hypothesized that loss of
Rerg might contribute to the enhaced aggressiveness of ERα-negative breast cancers, since
Rerg had been implicated as a growth inhibitor in breast cancer. As discussed above, many
facets of Rerg signaling are unknown, and thus ripe for discovery. In particular, I was
interested in determining whether reducing Rerg expression in ERα-positive breast cancer
cells might increase growth and invasion, or whether restoring Rerg expression in ERα-
negative breast cancer cells would decrease growth and invasion. Thus, I created tools to manipulate Rerg expression: I used short hairpin RNA (shRNA) targeting Rerg to knock down Rerg expression in two ERα-positive breast cancer cell lines and used a tamoxifen- inducible ER-Rerg fusion protein to restore Rerg expression in two ERα-negative breast
cancer cell lines. As discussed in Chapter 2, knocking down or restoring Rerg expression did
not significantly disrupt the growth or invasive properties of breast cancer cells. Therefore,
36 more studies are needed to determine the role of Rerg both in normal physiology and in
cancer development.
Rheb is also regulated by estrogen, but in a different manner: rather than being a transcriptional target of the ERα, Rheb is activated by non-genomic estrogen signaling in breast cancer. Rheb was also shown to be involved both in breast cancer and in many other cancers. Thus, Rheb represents an attractive therapeutic target for the treatment of breast and other cancers. Chapter 3 of my thesis explores strategies to inhibit Rheb signaling in cancer.
I hypothesized that inhibitors of CAAX-mediated posttranslational processing would block
Rheb localization, Rheb activation of mTOR, and, ultimately, Rheb-dependent tumor growth.
I found that the post-prenylation processing enzymes Rce1 and Icmt were required for proper
Rheb localization, but were not required for Rheb activation of mTOR, suggesting that inhibitors of these enzymes will not block Rheb activity. I also studied whether FTS blocks
Rheb farnesylation and function. Although FTS failed to inhibit Rheb localization, it potently inhibited both Rheb- and mTOR-dependent tumor growth, suggesting that it indeed inhibits mTOR and not Rheb. Therefore, FTS may be a beneficial treatment both for breast cancer and other Rheb- and mTOR-dependent tumors. Together, these studies have increased our understanding of the roles of Ras family small GTPases in breast cancer and have suggested potentially novel therapeutic strategies to treat breast cancer.
37
CHAPTER 2
TOOLS TO STUDY THE FUNCTION OF THE RAS-RELATED, ESTROGEN-
REGULATED GROWTH INHIBITOR IN BREAST CANCER1
Abstract
Rerg (Ras-related, Estrogen Regulated, Growth inhibitor) is a Ras-related small
GTPase and candidate tumor suppressor. Rerg gene expression is stimulated by the estrogen
receptor alpha (ERα) and Rerg gene expression is absent in ERα-negative breast cancers.
ERα-negative breast cancers are highly invasive and metastastic and are typically more
advanced than their ERα-positive counterparts. Like Ras, Rerg binds and hydrolyzes GTP,
but unlike Ras, Rerg has been shown to possess growth inhibitory activity in breast cancer
cells. The precise role that Rerg loss plays in breast cancer growth, and the mechanisms by
which it does so, are unknown. In this chapter, we describe tools to detect and manipulate
the expression of Rerg in breast cancer cells. We validated the use of an antibody to detect
Rerg expression by immunohistochemistry analyses of patient tumors and by western blot
analysis. We also generated mammalian expression vectors that encode wild-type and
mutants of Rerg that are altered in GDP/GTP regulation. Finally, we applied an inducible
Rerg expression system and utilized a retrovirus-based RNA interference approach to repress
1 Authors: Ariella B. Hanker, Staeci Morita, Gretchen A. Repasky, Douglas T. Ross, Robert S. Seitz, and Channing J. Der. All figures, except for Figure 2-1B, represent the work of Ariella B. Hanker. Rerg expression. These tools provide invaluable reagents for evaluating the biological function of Rerg in breast cancer.
39 Introduction
The Rerg (Ras-related, Estrogen Regulated, Growth inhibitor) small GTPase was initially identified in a microarray screen that grouped breast tumor samples into clinically relevant subtypes (2, 151). Rerg gene expression was found to be decreased in the most aggressive, ERα-negative subtypes (151). In fact, Rerg gene expression is directly regulated by estrogen. Recent microarray analyses by Perou and colleagues found that Rerg expression levels are a statistically significant predictor of patient outcome, even within ERα-positive breast cancers, indicating that low levels of Rerg expression correlate with breast cancer progression (personal communication, unpublished observations). Furthermore, other microarray studies have found that Rerg expression is decreased in some metastatic cancers compared to their non-metastatic counterparts, including prostate and lung cancer (170-172).
Rerg expression is also decreased or lost in kidney, ovary, and colon tumor tissues (153).
These studies suggest that loss of Rerg may be an important step in tumor progression and metastasis.
Rerg belongs to the Ras branch of the Ras superfamily of small GTPases (21). Rerg shares ~42% amino acid sequence identity with the Ras proto-oncogene proteins. Ras functions as a GTP/GDP regulated switch that relays extracellular ligand-stimulated signals; these signals regulate cell proliferation, differentiation, and survival. Ras is mutationally activated in 30% of human cancers, but only 5% of breast cancers. Like Ras, Rerg can bind to and hydrolyze GTP and cycles between active GTP-bound and inactive GDP-bound states
(151). Whether, like Ras, the GTP-bound form of Rerg is the “active” form is not yet known. Ras binds to and activates its many downstream effectors via its core effector domain (Ras residues 32-40). Key effectors implicated in Ras-mediated oncogenesis include
40 the Raf serine/threonine kinases, the phosphatidylinositol 3-kinases, and guanine nucleotide
exchange factors for the Ral Ras-like small GTPases (RalGEFs) (38). Although Rerg shares
significant sequence identity with the Ras effector domain, no known Ras effectors have
been shown to bind to Rerg. Additionally, no Rerg-specific effectors have been identified to date. Finally, while the intrinsic biochemical properties of Rerg suggest that, like Ras, Rerg
GDP/GTP cycling will be regulated by guanine nucleotide exchange factors (GEFs) and
GTPase activating proteins (GAPs), Ras GEFs and GAPs are not known to regulate Rerg and no Rerg-specific GAPs or GEFs have been identified.
A major difference between the sequences of Ras and Rerg is in their carboxyl-
terminal sequences. All Ras proteins terminate in a CAAX (C = cysteine, A = aliphatic
amino acid, X = terminal amino acid) tetrapeptide sequence, a substrate for farnesyl lipid
modification responsible for membrane localization and critical for Ras biological activity.
Rerg lacks this motif, and unlike Ras, Rerg is localized primarily to the cytosol (151).
Despite their sequence similarity, Rerg and Ras play opposing roles in human
oncogenesis. While Ras functions as an oncogene and drives growth transformation and
tumorigenesis, Rerg exhibits characteristics of a tumor suppressor and impedes tumor
growth. Previous studies have shown that ectopic overexpression of Rerg decreases cell
proliferation and tumorigenesis of Rerg-expressing, ERα-positive MCF-7 breast cancer cells
(151, 153), further supporting a tumor suppressor function for Rerg in breast cancer.
However, whether the absence of Rerg expression directly contributes to breast cancer advancement, and whether restoring Rerg expression in ERα-negative breast cancers limits their tumorigenicity and aggressiveness, has not yet been determined. Here, we describe our development and validation of tools to detect and manipulate the expression of the unique
41 GTPase Rerg in breast cancer cells. We have developed and validated antibodies that detect
ectopic and endogenous Rerg expression by western blot and immunohistochemical staining.
We have constructed mammalian expression vectors for expression of wild type and mutant
Rerg proteins. We have generated a tamoxifen-inducible ERα-Rerg fusion protein to inducibly express Rerg in ERα-negative breast cancers and have used this system to study the
effects of Rerg on cell growth and invasion. Finally, we have generated retrovirus vectors
for interfering short hairpin RNA (shRNA) to silence endogenous Rerg expression in ERα-
positive breast cancers. These reagents will undoubtedly prove useful in identifying and studying the precise biological function of Rerg in normal cells as well as in breast and other human cancers.
Materials and Methods
Molecular Constructs
cDNA sequences encoding Rerg wild-type (wt), Rerg S20N, and Rerg Q64L cDNA
sequences in the pKH3 expression vector plasmids (generous gifts of D. Andres, University
of Kentucky) were used as templates for polymerase chain reaction (PCR)-mediated DNA
amplification to generate open reading frame cassettes for subcloning into the pCGN-hygro
mammalian cell expression vector. Expression of the inserted cDNA is controlled from the cytomegalovirus promoter and the vector encodes for hygromycin resistance for selection of stably-transfected cells. The cassettes contained 5’ and 3’ BamHI sites to allow subcloning downstream of and in frame with sequences encoding the hemaglutinin (HA)-epitope tag.
Briefly, PCR-generated products and pCGN-hygro plasmid were digested with BamHI to generate sticky ends. BamHI-digested pCGN-hygro was then dephosphorylated with shrimp
42 alkaline phosphatase (SAP) to prevent religation of the plasmid. The BamHI-digested PCR
product was then incubated with BamHI-digested and SAP-treated pCGN-hygro using the
Rapid DNA Ligation kit (Roche Applied Science, Indianapolis, IN). Competent DH5α E.
coli were then transformed with the ligation products and positive clones were identified by
restriction digest analyses. The Rerg cDNA sequences were verified for accuracy by DNA
sequencing.
For the generation of retrovirus-base expression vectors the Rerg wild-type (WT),
Rerg Q64L, and Rerg S20N cDNA sequences were subcloned into the pBabe HAII retrovirus
vector. This vector is a variant of the pBabe-puro retrovirus expression vector (173) that has been modified to include coding sequences for the HA epitope sequence that are added to the amino terminus of the inserted cDNA sequence (174). Expression of the inserted cDNA
sequence is controlled by the Moloney leukemia virus long terminal repeat promoter and the
vector encodes for puromycin resistance for selection of stably-transfected or -infected cells.
pCGN Rerg WT, Q64L, or S20N plasmid DNAs were digested with BamHI and the resulting
Rerg cDNA fragments were ligated in-frame into linearized pBabe HAII vector (as described above).
pBabe ER expression vectors encoding wild-type or Q64L and S20N mutants of Rerg
were generated for tamoxifen-inducible expression of wild-type and mutant ER-Rerg fusion
proteins. In these constructs, the cDNA sequence for the ligand binding domain of the ERα
is placed 5’ to the Rerg cDNA coding sequence. pCGN Rerg plasmid DNAs were used as
templates for PCR-mediated DNA amplification to generate open reading frame cassettes
containing 5’ and 3’ EcoRI restriction sites. The primers used were: 5’
TTTTGAATTCATGGCTAAAAGTGCGG 3’ and 5’
43 TTTTGAATTCTTTCTAACTACTGATTTTG 3’. PCR products were digested with EcoR1
and Rerg fragments were ligated into the Zero Blunt Topo Kit (Invitrogen, Carlsbad, CA).
Rerg was then excised from the Zero Blunt Topo vector and ligated into the EcoR1 site of the
linearized pBabe ER vector (derived from the pBabe ER-H-Ras vector).
Rerg shRNA Constructs
For generation of short hairpin RNA (shRNA) against Rerg, we selected target
sequences using the web server at http://jura.wi.mit.edu/bioc/siRNA. We chose three target
sequences for generation of shRNA Rerg expression vectors. However, our subsequent
analyses found that target 2 showed no knockdown activity against Rerg and will not be
discussed here. The target sequence for Rerg shRNA 1 corresponds to nucleotide residues
2115-2137 in the 3’ untranslated region of Rerg cDNA, whereas the Rerg shRNA 3 target sequence corresponds to nucleotide residues 427-449 in the coding sequence. The following oligonucleotides were used for insertion into the BglII and HindIII sites of the pSUPER.retro.puro vector: 5’-
GATCCCCAGCGTTAGCGGCATTAATTTTCAAGAGAAATTAATGCCGCTAACGCTT
TTTTGGAAA-3’ and 5’-
AGCTTTTCCAAAAAAGCGTTAGCGGCATTAATTTCTCTTGAAAATTAATGCCGCT
AACGCTGGG-3’ for Rerg shRNA 1 and 5’-
GATCCCCGCAACCATCGATGATGAAGTTCAAGAGACTTCATCATCGATGGTTGCT
TTTTGGAAA-3’ and 5’-
AGCTTTTCCAAAAAGCAACCATCGATGATGAAGTCTCTTGAACTTCATCATCGAT
GGTTGCGGG-3’ for Rerg shRNA 3. Oligonucleotides were phosphorylated and annealed prior to ligation into the BglII and HindIII sites of the pSUPER.retro.puro vector
44 (Oligoengine, Seattle, WA), as described previously (175). The negative control
pSUPERIOR.retro.puro GFP shRNA (176) was a gift from Natalia Mitin (UNC-Chapel Hill).
All inserted oligonucleotide sequences in these molecular constructs were verified by sequencing.
Cell Culture and Expression Vector Transfection and Infection
293T human embryonic kidney epithelial cells were maintained in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 100
units/mL penicillin and 100 μg/mL streptomycin in a 10% CO2 humidified incubator at 37ºC.
All human breast carcinoma cell lines, with the exception of the SUM149 cells, were obtained from the American Type Culture Collection and cultured as described previously
(29). The SUM149 basal-like breast carcinoma cell line (obtained originally from Stephen
Ethier, Karmanos Cancer Institute), and provided by Carolyn Sartor, UNC-Chapel Hill) was grown in Ham’s F12 medium supplemented with 5% FCS, 5 μg/mL insulin, 1 μg/mL
hydrocortisone, 0.5 μg/mL fungizone, and 5 μg/mL gentamycin. All breast cancer cells were grown in a 5% CO2 incubator at 37ºC.
Sub-confluent cultures of each cell line were transfected with the various Rerg or
shRNA expression plasmid DNAs using Lipofectamine and Plus reagents (Invitrogen)
according to the manufacturer’s instructions. Cells were harvested 24 h following
transfection.
All retroviral infections were performed as described previously (175). Briefly, 293T
host cells were transfected by calcium chloride precipitation with 10 μg of the pCL10A1
virus packaging vector plasmid DNA along with 10 μg of the appropriate Rerg or shRNA
45 expression plasmid DNA. The medium was replaced 16 h prior to infection of target cells.
Two days following 293T transfection, host cells were infected with filtered virus-containing medium from the 293T cells supplemented with 8 μg/mL polybrene to increase infection efficiency. Twenty-four to forty-eight h following infection, growth medium supplemented with puromycin was used for the selection of stably-infected T-47D (2 μg/mL), MDA-MB-
231 and Hs578T (1 μg/mL), and SUM149 (0.5 μg/mL) cells. After selection, multiple drug- resistant colonies (>100) were pooled together to establish mass populations of stably- infected cells. All stable cell lines were maintained in medium supplemented with puromycin.
Cell Growth and Invasion Assays
For the MTT cell viability assay, MDA-MB-231 cells stably expressing ER-Rerg fusion proteins were seeded at 2 x 103 cells per well in growth medium. Cells were seeded in replicates of eight in 96-well plates and allowed to attach overnight. The growth medium was then removed and replaced with fresh growth medium supplemented with 1 μM 4- hydroxytamoxifen (4-OHT) to induce ER-Rerg expression. For each time point, 3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich, St. Louis,
MO), which stains only viable cells, was added to a final concentration of 0.5 mg/mL in the media of each well and plates were incubated for 4 h. The formazan (MTT metabolic product) was then resuspended in 100 μL dimethyl sulfoxide (DMSO) and the optical density was measured at 560 nM using an ELx100 Universal Microplate Reader (Bio-Tek
Instruments, Inc., Winooski, VT). Optical density directly correlates with cell quantity.
46 The transwell Matrigel invasion assay using the Biocoat Matrigel Invasion Chamber
(BD Biosciences, Bedford, MA) was performed according to the manufacturer’s instructions.
Briefly, MDA-MB-231 stable cell lines were seeded at 5 x 103 cells per chamber in triplicate
in serum-free DMEM supplemented with 1% fatty acid-free bovine serum albumin (BSA).
Medium containing 3% FCS was added to each bottom well of a 24-well plate to serve as a
chemoattractant. After two h, growth medium supplemented with ethanol (vehicle) or 1 μM
4-OHT was added to each chamber and bottom well. After an additional 22 h incubation at
37ºC, non-invading cells were removed with a cotton swab and invading cells were fixed and stained using Diff-Quick Stain Set (Dade Behring). Invading cells were quantified by counting 5 fields of view using the 20x objective lens under an inverted phase contrast microscope.
The soft agar assay to evaluate anchorage-independent growth potential was
performed as described previously (177). Briefly, 104 SUM149 cells stably expressing ER-
Rerg or H-Ras fusion proteins were seeded in triplicate in 60 mm plates into growth medium
supplemented with 0.4% bacto-agar and overlaid onto a bottom layer containing growth
medium supplemented with 0.6% agar. Agar layers contained SUM149 growth medium
supplemented with 10% FCS and penicillin/streptomycin. Either ethanol (vehicle) or 1 μM
4-OHT final concentration was added to both top and bottom layers. Media containing
ethanol or 1 μM 4-OHT was replaced twice weekly for three weeks. After 21 days, viable
colonies were stained in 2 mg/mL MTT and plates were scanned. Visible colonies were
counted from scanned images on Adobe Photoshop.
Generation of Anti-Rerg Antibodies
47 The S0222 anti-Rerg antiserum was raised in rabbits to bacterially-expressed His6-
thioredoxin-Rerg fusion protein from the pET32a-Rerg E. coli expression vector (151). The
S0068 antiserum was raised against a 21-mer peptide corresponding the carboxyl-terminus of
RERG (residues 179-199; RRSSTTHVKQAINKMLTKISS) conjugated to keyhole limpet
hemocyanin (KLH) by EDC coupling. The protein or KLH conjugated peptide was mixed
with Freund's adjuvant, injected and serially boosted into two New Zealand white rabbits
over a 14 week period. The boosts were made approximately three weeks apart. Antisera
were collected and affinity purified by attaching the recombinant protein or peptide to
Sepharose 4B. Antibody was eluted by a pH gradient of glycine-HCL pH 4 decreasing to pH
2 in borate buffer.
Results and Discussion
Validation of S0222 Anti-Rerg Antibody for Western Blot Analyses
In order to determine whether the S0222 anti-Rerg antibody can detect endogenous
human Rerg protein expression, we first tested it against ectopically expressed Rerg. We
determined that the optimum dilution for western blot analyses was 1:500 (final
concentration of 740 ng/mL) in 5% non-fat dry milk/TBST, incubated overnight. S0222
effectively detected expression of HA-Rerg (Figures 2-1A and 2-2B). Furthermore, S0222
detected expression of the ER-Rerg fusion protein (Figure 2-3). However, S0222 was not as sensitive as the HA.11 antibody (Covance, Berkeley, CA) for detection of HA-Rerg (Figure
2B) and typically required longer exposure.
Next, we asked whether S0222 could detect endogenous Rerg expression. To test
this, we generated lysates from ERα-positive and ERα-negative breast cancer cells treated
48 A. + HA-Rerg E2: -+ -+ -+ -+ - + - BT-474 MCF-7 T-47D Hs578T MDA-MB-231
α-Rerg (S0222) *
α-β-actin
ERα-positive ERα-negative B. Tumor #1 Tumor #2 Tumor #3 Tumor #4
α-Rerg (S0068)
α-ERα
Figure 2-1. Antibody detection of endogenous Rerg expression in breast cancer cell lines and tumor tissue. (A) Detection of endogenous Rerg expression in breast carcinoma cell lines. Endogenous Rerg and ectopically-expressed HA-tagged Rerg were detected in breast cancer cells by western blot analysis using the S0222 anti-Rerg antibody. The indicated cell lines were incubated in complete growth media, either with or without 10 nM 17β-estradiol (E2, Sigma), for 24 h. A strong band at ~26 kDa (asterisk) was seen in all Rerg mRNA-positive cell lines (BT-474, MCF-7 and T-47D), whereas a band at ~30 kDa (arrow) was detected in MDA-MB-231 cells stably-infected with the pBabe-puro HAII Rerg plasmid. Blot analysis for β-actin (Sigma) was used as a loading control. (B) Rerg expression levels correlate with ERα expression in breast cancer tissue. Rerg and ERα protein expression in primary invasive breast tissues were detected by IHC analyses. Parallel staining is shown for Rerg (S0068) and ERα from four representative samples. Adapted from Hanker et al., Methods Enzymol 2008;439:53-72. Panel B is the work of Douglas Ross and Robert Seitz (Applied Genomics Inc.).
49 with 10 nM 17β-estradiol (Sigma-Aldrich) for western blot analysis with S0222. We
previously showed that Rerg mRNA was detected in ERα-positive, but not ERα-negative,
breast cancer cells (151). In the ERα-positive cell lines, S0222 detected a band at the proper
molecular weight (Figure 2-1A). However, a weaker band at a similar weight was also seen
in Hs578T and MDA-MB-231 cells (Figure 2-1A), two cell lines that were negative for Rerg
mRNA by northern blot analyses and reverse transcription (RT)-PCR (data not shown). It is
possible that the S0222 antibody is cross-reacting with another protein of a similar size in
these samples. We are fairly certain that the band seen in the ERα-positive samples is indeed
endogenous Rerg, as knocking down Rerg gene expression by shRNA decreased the intensity
of this band (Figure 2-5C). We did not find that treatment with estradiol stimulated an
increase Rerg expression (Figure 2-1A), perhaps because the cells were maintained in
phenol-red containing medium and were not estrogen-starved. Since phenol red can
stimulate the ERα, Rerg may have been expressed at maximal levels even in cells not directly
stimulated with estrogen. Taken together, these results suggest that the S0222 antibody can
effectively detect endogenous Rerg expression by western blot analysis.
Use of S0068 Anti-Rerg Antibody for Immunohistochemical Staining of Breast Tumor Tissue
The tissue source and our protocols for immuohistochemical (IHC) staining of
paraffin-embedded primary invasive breast tumor tissues array blocks have been described in
detail elsewhere (178). Briefly, prior to staining, tissue arrays were de-paraffinized and dehydrated by submerging in xylene three times for 10 min each. Tissue arrays were then rinsed three times in 100% ethanol and twice in 95% ethanol and treated by microwaving
boiling for 11 min in 10 μM buffered citrate (pH 6.0). Slides were allowed to cool to room
50 temperature and rinsed in distilled water followed by phosphate-buffered saline (PBS).
Slides were dipped in 0.03% hydrogen peroxide, rinsed with PBS, and then stained using
dilutions of antibodies in DAKO Diluent (DAKO Cytomation Inc.) for 1 h at room
temperature. Secondary antibody was applied for 1 h and staining was visualized using the
DAKO Cytomation Envision staining kit according to the manufacturer’s instructions.
Staining with an antibody directed against ERα was performed by a commercial service (US
Labs Inc.).
Anti-Rerg (S0068) and anti-ERα staining was executed in parallel using slides
comprised of tissue from primary invasive breast cancers. Consistent with array analyses in
breast tumors and western blot analyses of breast tumor cell lines (151), the intensity of anti-
Rerg staining correlated with that of ERα. Additionally, whereas ERα staining is nuclear,
Rerg staining is cytosolic, as we have seen in cell lines. These results provide validation for
the use of S0068 anti-Rerg antibody for IHC analyses.
Transient and Stable Expression of Wild-Type and Mutant Rerg Proteins
Like Ras, Rerg cycles between GTP- and GDP-bound states (151), but whether
GDP/GTP cycling is important for Rerg function is not known. In order to determine
whether Rerg function depends on its GTP-bound state, we introduced missense mutations
that resulted in amino acid substitutions in Rerg, based on analogous mutants of Ras proteins,
that render Rerg either constitutively GTP-bound (Rerg Q64L) or preferentially bound to
GDP (Rerg S20N). The Rerg Q64L mutation is analogous to the GAP-insensitive Q61L
mutation in Ras and has been shown previously to possess decreased intrinsic GTPase
activity in vitro (151). Since no GAP specific for Rerg has been identified, whether this
51 mutant is also impaired in GAP-stimulated hydrolysis is not known. However, in vitro biochemical analyses support the existence of a GAP (151). The Rerg S20N mutant has not been described previously but is based on the preferentially GDP-bound S17N mutation in
Ras (179). Introduction of the analogous mutation has generated dominant negative variants
of a wide variety of Ras superfamily proteins. Such dominant negatives function by forming
nonproductive complexes with GEFs that stimulate the activation of the wild-type GTPase.
While a Rerg-specific GEF remains to be identified, an indirect evaluation of such mutants
can be performed by expressing them in cells. For example, the growth suppressive activity
of the Ras S17N mutant is consistent with the requirement for Ras in normal cell
proliferation. Therefore, if Rerg functions as a tumor suppressor, then dominant negative
Rerg would be expected to stimulate growth in cells in which Rerg is expressed.
In order to test the expression of these mutants in the pCGN-hygro vector, we
transiently transfected 293T cells with pCGN-hygro expression vectors encoding Rerg wild- type, Q64L, or S20N. As shown in Figure 2A, wild-type Rerg and Rerg Q64L were expressed at high levels, but expression of Rerg S20N was substantially reduced. Similar
expression patterns were observed when these constructs were stably expressed in breast
cancer cells (data not shown). We also tested expression of pBabe HA-tagged Rerg
constructs stably expressed in Hs578T breast cancer cells. Wild-type Rerg and Rerg Q64L
were expressed at equal levels, but Rerg S20N expression was not detected (Figure 2-2B).
Furthermore, we consistently failed to detect transient expression of pBabe-Rerg S20N in
293T cells (data not shown) and ER-Rerg S20N expression was not efficiently induced
(Figure 2-3). Thus, we found that Rerg S20N protein was inefficiently expressed regardless
of the expression vector or cell line used, or whether we tried transient or stable expression.
52 A. Vector Rerg 20N Rerg 64L Rerg WT
α-HA
α-β-actin
B. Vector Rerg WT Rerg 64L Rerg 20N
α-HA
α-Rerg (S0222)
α-β-actin
Figure 2-2. Transient and stable expression of wild-type and mutant Rerg proteins. (A) Transient expression of HA-Rerg proteins in 293T cells. 293T cells were transfected with the empty pCGN-hygro plasmid (Vector), or encoding Rerg WT, Rerg Q64L, or Rerg S20N. HA-Rerg expression was detected using the HA.11 (Covance) anti-HA epitope antibody. (B) Stable expression of HA-Rerg proteins in ER-negative Hs578T cells. Western blot analysis to verify expression of ectopically-expressed Rerg proteins was performed for Hs578T cells stably-infected with the empty pBabe-puro HAII vector, or encoding Rerg WT, Rerg Q64L, or Rerg S20N. The lysates were blotted with either anti-HA epitope or S0222 anti-Rerg antibody. Blot analysis with anti-β-actin was used as a loading control to verify equivalent total protein. Adapted from Hanker et al., Methods Enzymol 2008;439:53-72.
53 We believe that the reduced expression level is most likely due to decreased protein stability.
The mutation in residue 20 may disrupt Rerg protein structure. Alternatively, the mutation
may decrease GDP- and GTP-binding, which may account for the reduced protein stability.
The future identification of a Rerg GEF will be required to address these issues. Future
directions may include the generation of additional Rerg mutants as candidate dominant
negatives.
Establishment of a Tamoxifen-inducible Rerg Protein Expression System
A major obstacle in the study of tumor suppressors and other growth inhibitory
proteins is that their stable expression is not well tolerated in cells. Either toxicity prevents
suitable long-term expression, or cells overcome growth inhibition by secondary,
compensatory mechanisms. In order to evade these scenarios and to obtain a more accurate
measure of Rerg growth inhibition, we generated an inducible expression system for Rerg.
We constructed a chimeric gene that encodes a tamoxifen-inducible ER-Rerg fusion protein.
This fusion protein is composed of a mutant form of the ERα ligand binding domain, that
responds only to 4-hydroxytamoxifen (4-OHT) and not to phenol red or estrogen, and is
fused to the N-terminus of full-length Rerg. In the absence of tamoxifen, the fusion protein
is inactive because it is bound by an Hsp90 complex and degraded. Upon ligand binding, the
Hsp90 complex dissociates, allowing stable expression of the fusion protein (180). The use
of ERα fusion proteins to generate inducible protein expression has been described for Ras and many other signaling proteins (180-184). While it is possible that addition of the ERα
sequences to the amino terminus of Rerg may disrupt Rerg function, previous observations
54 with Ras and other Ras superfamily proteins indicate that the addition of sizeable sequences
to the amino termini of small GTPases generally does not cause perturbations in function.
To determine whether ectopic expression of Rerg in ERα-negative breast cancer cells
decreases their tumorigenicity, we retrovirally infected two ERα-negative cell lines, MDA-
MB-231 and SUM149, with the empty pBabe-puro plasmid (Vector), or with plasmids
encoding ER-Rerg WT, Q64L, or S20N. To induce ER-Rerg protein expression, we treated
stably-infected cells with 1 μM 4-OHT for a minimum of 24 h. Figure 2-3 shows that in both
cell lines, tamoxifen treatment efficiently induced expression of wild-type Rerg and Rerg
Q64L in both cell lines. In contrast, Rerg 20N was not induced efficiently. Some leakiness
of expression was observed in untreated cells when the film was exposed for a longer time
period (Figure 2-3A). Despite this leakiness, it is clear that tamoxifen treatment greatly
enhanced ER-Rerg protein expression in this system. Furthermore, H-Ras 12V expression
was also efficiently induced by 1 μM 4-OHT in SUM149 cells (Figure 2-3B).
We next asked whether inducing Rerg expression decreases the growth or invasive
properties of the two ERα-negative breast cancer cell lines. First, we used an MTT assay to
measure the anchorage-dependent cell proliferation rate of MDA-MB-231 cells stably
expressing ER-Rerg wt, Q64L, and S20N. Figure 2-4A shows that inducing wild-type or mutant Rerg with 4-OHT did not significantly alter the viability or proliferation rate of
MDA-MB-231 cells. Next, we used the Matrigel invasion assay to determine whether inducing wild-type or mutant Rerg decreased the invasiveness of MDA-MB-231 cells in
vitro. Adding 4-OHT to cell lines stably-infected with the empty pBabe-ER plasmid
(Vector), or with plasmids encoding ER-Rerg WT, Q64L, or S20N, did not significantly and
reproducibly affect invasion through Matrigel in vitro (Figure 2-4B). In addition, we did not
55 A. Vector Rerg WT Rerg 64L Rerg 20N 4-OHT (3 days) - + -++ -+ - short exposure
-Rerg long α
(S0222) exposure
α-β-actin
B. Vector Rerg WT Rerg 64L Rerg 20N 4-OHT (1 day): - + -++ -+ - α-Rerg (S0222) α-β-actin
C. H-Ras 12V 4-OHT (1 day): - +
α-H-Ras
α-β-actin
Figure 2-3. Tamoxifen-inducible expression of an ER-Rerg fusion protein. The plasmids encoding ER-Rerg fusion proteins were retrovirally infected into (A) MDA-MB-231 cells and (B) SUM149 cells. Stable cell lines were treated with either vehicle (ethanol) or 1 μM4- hydroxytamoxifen (4-OHT) for 3 days (A) or 1 day (B). Western blot analysis using the S0222 anti- Rerg antibody detected a ~55 kDa band corresponding to the ER:Rerg fusion protein in samples treated with 4-OHT. Similar results were obtained when cells were treated with 4-OHT for 7 days (data not shown). As a control for 4-OHT induction, SUM149 cells stably-infected with an expression vector encoding the ER-H-Ras G12V protein (C) was included. Expression was induced by 1 day treatment with 4-OHT and detected by western blot analysis with an H-Ras antibody (#146; Quality Biotech, Camden, NJ). Blot analysis for β-actin in total cell lysates was used as a loading control to verify equivalent total protein. Adapted from Hanker et al., Methods Enzymol 2008;439:53-72.
56 observe significant differences in soft agar growth or invasion through collagen in these cell lines (data not shown). Together, these data show that Rerg expression does not significantly affect the growth or invasive properties of ERα-negative MDA-MB-231 breast cancer cells.
We also examined whether inducing Rerg expression affected the growth of SUM149 cells. We used a soft agar assay to measure the anchorage-independent growth of these cells stably expressing ER-Rerg and ER-H-Ras constructs. As expected, treating vector control cells with 4-OHT did not affect their ability to grow in soft agar. Furthermore, as we expected, inducing oncogenic H-Ras G12V with 4-OHT drastically increased the ability of these cells to form colonies in soft agar (Figure 2-4C). However, inducing wild-type Rerg or
Rerg Q64L did not significantly affect their anchorage-independent growth. Inducing Rerg
S20N may have slightly increased the anchorage-independent growth of these cells; this may be because Rerg S20N can act as a dominant negative and may inhibit Rerg exchange factors in these cells. Thus, consistent with results obtained with the MDA-MB-231 cell line, inducing Rerg expression did not significantly reduce the anchorage-independent growth of the ERα-negative SUM149 breast cancer cells.
We have shown previously that ectopic overexpression of HA-tagged Rerg decreases cell proliferation and soft agar growth of the Rerg-expressing MCF-7 breast cancer cell line
(151). However, Rerg did not show significant growth inhibition when ectopically expressed in the two ERα-negative cell lines we tested. This discrepancy may be due to overexpression artifacts—in our previous studies, Rerg was expressed from the CMV promoter, which drives expression much more strongly than the pBabe promoter described here. Another possibility is that the growth inhibitory function of Rerg may be cell-type specific; Rerg may act as a growth inhibitor in ERα-positive but not in ERα-negative cells. Alternatively, it remains
57 A. 1 Vector B. ER-Rerg WT 120 Vehicle 0.8 ER-Rerg 64L 4-OHT ER-Rerg 20N 0.6 80 0.4 40 OD (560 nM) 0.2 cells Invading 0 0 0246 Vector WT 64L 20N Day ER-Rerg
C. 400 Vehicle 300 4-OHT 200 100
Colonies per dish 0 Vector WT 64L 20N ER-H-Ras ER-Rerg 12V
Figure 2-4. Effects of induction of ER-Rerg expression on growth and invasive properties in ER-negative breast cancer cells. (A) Evaluation of growth rate upon induction of ER-Rerg expression in MDA-MB-231 cells. Cells were incubated in growth medium supplemented with 1 μM 4-OHT to induce Rerg expression. MTT was added and optical density was measured at the indicated time points. Results are an average of eight replicates per time point; error bars represent standard deviation. (B) Evaluation of Matrigel invasion upon induction of ER-Rerg in MDA-MB-231 cells. Stable cell lines were seeded in triplicate and allowed to invade towards growth medium supplemented with 3% FCS (chemoattractant). After two h, growth media supplemented with ethanol (Vehicle) or 1 μM 4-OHT was added to each top and bottom well to induce Rerg expression. After incubation for 22 h, non-invading cells were removed and invading cells were fixed and stained. Invading cells were quantified by counting five fields of view using the 20x objective lens under a phase contrast microscope. Error bars represent standard deviation of triplicate wells. (C) Evaluation of anchorage-independent growth upon induction of ER-Rerg in SUM149 cells. Cells were seeded in triplicate into 0.4% soft agar over a 0.6% bottom layer; ethanol (Vehicle) or 1 μM 4-OHT was added to the top and bottom agar layers. After 21 days, viable colonies were stained in 2 mg/mL MTT and visible colonies representing >50 cells were quantified. Results represent the average of triplicate plates; error bars represent standard deviation. Adapted from Hanker et al., Methods Enzymol 2008;439:53-72.
58 possible that the large ERα domain fused to Rerg is disrupting its function. However, we believe this is not the case for the following reasons: 1) placing an HA tag N-terminal to
Rerg does not disrupt its growth inhibitory function, and 2) placing the ERα ligand binding domain N-terminal to Ras does not disrupt Ras function (180, 184). Therefore, although we were unable to identify an assay for Rerg function, we believe that the ER-Rerg fusion proteins generated here will be very useful in the study of Rerg as other functions are identified in the future.
Suppression of Rerg Expression by RNA Interference
To further define the role of Rerg in breast cancer, we used interfering shRNA to repress Rerg in Rerg-expressing cells. First, we validated that our Rerg shRNA construct reduced expression of ectopically expressed Rerg protein in 293T cells. Of the two constructs described here, only pSUPER.retro Rerg shRNA 3 targets a sequence in the coding region of Rerg. This construct efficiently reduced expression of pBabe HAII Rerg
(Figure 2-5A), verifying that Rerg shRNA3 does indeed target Rerg mRNA.
Next we generated mass populations of the T-47D ERα-positive cell line stably- infected with either the empty pSUPER.retro.puro vector, or encoding Rerg shRNA 1, Rerg shRNA 3, or GFP shRNA. After selection in growth medium supplemented with puromycin, multiple drug-resistant colonies were pooled together to establish cell lines for biological analyses. To determine whether the Rerg shRNA constructs efficiently reduced endogenous
Rerg mRNA levels, we used quantitative real-time RT-PCR. RNA was isolated from each T-
47D cell line using the TRIzol reagent (Invitrogen) and genomic DNA was removed by treatment with RQ1 DNase (Promega, Madison, WI). mRNA was reverse-transcribed to
59 HA-Rerg A. Vector Vector shRerg 3 α-HA α-Rerg (S0222) α-β-actin B. 0.12
0.08
0.04
Ratio of Rerg to actin 0 Vector shRerg 1 shRerg 3 shGFP C. shRerg 3 HA-Rerg shRerg 1 shGFP Vector α-Rerg HA-Rerg (S0222) α-β-actin
Figure 2-5. Repression of Rerg expression by shRNA. (A) Reduction of ectopically expressed Rerg by shRNA. 293T cells were transiently co-transfected with either 2.5 μg of the empty pSUPER.retro.puro vector (lanes 1, 2) or encoding Rerg shRNA 3 (lane 3), together with 0.5 μg the empty pBabe-puro HAII vector (lane 1) or encoding HA-Rerg (lanes 2, 3). HA-Rerg expression was detected by immunoblotting with the anti-HA or anti-Rerg (S0222) antibodies. Blot analysis for β-actin was used to verify equivalent total protein. (B) shRNA-induced reduction of endogenous Rerg mRNA. cDNA from T-47D cells stably-infected with the empty pSUPER.retro.puro vector, Rerg shRNA 1, Rerg shRNA 3, or GFP shRNA was evaluated using quantitative real-time RT- PCR. Samples were run in duplicate. Rerg:actin ratio was determined using the equation 2ΔCT, where ΔCT = β-actin CT value – Rerg CT value. (C) shRNA-mediated reduction of endogenous Rerg protein. T-47D cells stably-infected with the empty pSUPER.retro.puro vector, or encoding Rerg shRNA 1, Rerg shRNA 3, or GFP shRNA, or the pBabe-puro HAII-Rerg expression vector, were lysed and analyzed by western blot. The S0222 anti-Rerg antibody was used to detect Rerg expression. Blot analysis for β-actin was used as a loading control. Adapted from Hanker et al., Methods Enzymol 2008;439:53-72.
60 cDNA using the SuperScript III First-Strand Synthesis System (Invitrogen). Rerg cDNA was
amplified using Absolute SYBR Green ROX Mix (ABGene House, Surrey, UK) and the
following primers specific to Rerg cDNA: 5’ GGCATCTTCACCTTGCTTT 3’ (sense) and
5’ GGCATGGTTAAGCTCCATT 3’ (antisense). Real-time RT-PCR was performed on the
ABI PRISM real-time PCR machine (Applied Biosystems, Foster City, CA) using the SYBR
green detector and data was analyzed using SDS 2.0 software. Both Rerg shRNA sequences
significantly reduced expression of Rerg mRNA (Figure 2-5B). Similar results were
obtained with semi-quantitative RT-PCR (data not shown)
We used western blot analysis with the S0222 anti-Rerg antibody to determine
whether the Rerg shRNA constructs could reduce the expression of endogenous Rerg protein.
As shown in Figure 2-5C, Rerg shRNA 3, but not Rerg shRNA 1, decreased expression of endogenous Rerg protein. We cannot explain why Rerg shRNA 1 reduced expression of
Rerg mRNA but not Rerg protein. Consistent with our results from inducing Rerg expression, knocking down endogenous Rerg expression did not significantly affect soft agar growth or Matrigel invasion of the T-47D breast cancer cells (data not shown). These studies do not support a critical role for Rerg in breast cancer growth or invasion.
Summary
In this chapter, we have described reagents for inducible, transient, or sustained
expression of ectopic Rerg or to reduce endogenous Rerg expression in breast cancer cells.
We have also described antibodies that detect Rerg expression by western blot or IHC analyses. We have validated expression of putative gain-of-function and loss-of-function
Rerg mutants. While inducing Rerg expression or knocking down Rerg by shRNA did not
61 significantly affect the in vitro growth or invasive properties of the breast cancer cell lines we
tested, it remains possible that Rerg expression will impair the tumorigenic, invasive or metastatic properties of breast carcinoma cells when evaluated in mouse models. The reagents described here will prove to be useful in identifying a function for Rerg and for delineating its role in breast cancer progression.
Acknowledgements
We thank Douglas Andres (U. Kentucky) for the pKH3 Rerg wild-type, Rerg Q64L,
and Rerg S20N plasmids; Christopher Counter (Duke University) for pBabe ER H-Ras
G12V; Kevin Healy (UNC-Chapel Hill) for the pBabe ER empty vector; Natalia Mitin
(UNC-Chapel Hill) for the pSUPERIOR.retro.puro GFP shRNA plasmid; Stephen Ethier (U.
Michigan) and Carolyn Sartor (UNC-Chapel Hill) for the SUM149 cells; and Brian Z. Ring
(Applied Genomics Inc.) for assistance in manuscript writing. This work was supported in
part by a Susan G. Komen Foundation grant to C.J.D. (BCTR0402537). A.B.H. is a recipient
of the UNC Cancer Cell Biology Training Grant and Department of Defense Breast Cancer
Research Program Predoctoral Fellowship (BC061107).
62
CHAPTER 3
REQUIREMENT OF CAAX-MEDIATED POSTTRANSLATIONAL PROCESSING FOR
RHEB LOCALIZATION AND SIGNALING2
Abstract
The Rheb1 and Rheb2 small GTPases and their effector mTOR are aberrantly
activated in human cancer and are attractive targets for anti-cancer drug discovery. Rheb is
targeted to endomembranes via its C-terminal CAAX (C = cysteine, A = aliphatic, X =
terminal amino acid) motif, a substrate for posttranslational modification by a farnesyl
isoprenoid. Following farnesylation, Rheb undergoes two additional CAAX-signaled
processing steps, Rce1-catalyzed cleavage of the AAX residues and Icmt-mediated
carboxylmethylation of the farnesylated cysteine. However, whether these post-prenylation processing steps are required for Rheb signaling through mTOR is not known. We found that Rheb1 and Rheb2 localize primarily to the endoplasmic reticulum and Golgi apparatus.
We determined that Icmt and Rce1 processing is required for Rheb localization, but is dispensable for Rheb-induced activation of the mTOR substrate p70 S6 kinase (S6K).
Finally, we evaluated whether farnesylthiosalicylic acid (FTS) blocks Rheb localization and function. Surprisingly, FTS prevented S6K activation induced by a constitutively active mTOR mutant, indicating that FTS inhibits mTOR at a level downstream of Rheb. We
2 Authors: Ariella B. Hanker, Rhonda S. Wilder, Elizabeth Petri Henske, Fuyuhiko Tamanoi, Adrienne D. Cox, and Channing J. Der. All data shown represent the work of Ariella B. Hanker. conclude that inhibitors of Icmt and Rce1 will not block Rheb function, but FTS could be a promising treatment for Rheb- and mTOR-dependent cancers.
64 Introduction
Rheb is a member of the Ras branch of the Ras superfamily of small GTPases and is evolutionarily conserved from yeast to humans (83). The two mammalian Rheb isoforms,
Rheb1 (Rheb) and Rheb2 (RhebL1; 51% identity), function as GTP/GDP regulated binary switches. Although both are expressed in many tissues and tumor types, current knowledge of Rheb comes largely from the study of Rheb1. Like Ras, Rheb proteins cycle between activated, GTP-bound and inactive GDP-bound states. Rheb is negatively regulated by the tumor suppressor TSC1 (hamartin)-TSC2 (tuberin) complex which functions as a GTPase activating protein (GAP) towards Rheb (185).
Rheb is a critical component of the phosphatidylinositol 3-kinase (PI3K)-Akt-TSC- mTOR pathway which is frequently hyperactivated in cancer. PI3K activates Akt, which phosphorylates and inactivates TSC2, leading to an increase in GTP-bound Rheb (185).
Rheb-GTP is an activator of the rapamycin-sensitive mTOR complex 1 (mTORC1) (90), which regulates the initiation of protein translation, nutrient sensing, and cell growth. mTOR controls protein synthesis by phosphorylating proteins involved in translation initiation, such as the p70 ribosomal S6 kinase (S6K).
Loss-of-function germline mutations in TSC1 or TSC2 cause Rheb hyperactivation and tuberous sclerosis complex disease which is characterized by the formation of hamartomas in a variety of organs (87, 88). Rheb and mTOR are also hyperactivated in many sporadic cancers due to mutations in PI3K and PTEN. Rheb1 and Rheb2 are overexpressed in a variety of tumors, including glioma, oral squamous cell carcinoma, and breast, prostate, lung, colon, and ovarian cancer (114-118). In addition, a Rheb mutation has
66 been found in colon cancer (119), and the RHEB gene is amplified in some prostate cancers
(118). Therefore, Rheb inhibition may be therapeutically beneficial for a variety of cancers.
Like Ras, Rheb terminates in a C-terminal CAAX motif (C = cysteine, A = aliphatic,
X = terminal amino acid), a substrate for farnesyltransferase (FTase)-catalyzed
posttranslational modification by a C15 farnesyl isoprenoid lipid (83). Two additional
CAAX-signaled posttranslational processing steps, proteolytic cleavage of the AAX residues
[catalyzed by Ras converting enzyme (Rce1)] and carboxylmethylation [(catalyzed by
isoprenylcysteine carboxyl methyltransferase (Icmt)], are required for Rheb localization
(186). However, whether these processing steps are also required for Rheb signaling through mTOR is not yet known.
The CAAX-signaled modifications are necessary but not sufficient for the proper
membrane association and subcellular localization of a majority of Ras and Rho family small
GTPases (27, 50). In addition, a second membrane-targeting signal positioned in sequences
immediately upstream of the CAAX motif is required. For example, in H-Ras, the second
signal is comprised of two palmitoylated cysteines upstream of the CAAX motif, whereas in
K-Ras4B and Rac1, it is comprised of polybasic-rich sequences. Rheb1 and Rheb2 lack
either of these, which may account for the absence of any plasma membrane-associated
Rheb. However, other unidentified sequence elements may provide a second signal (187), so
it remains possible that Rheb subcellular localization is not dictated solely by CAAX-
signaled modifications.
FTase inhibitors (FTIs) are a class of anti-cancer agents that were originally
developed to inhibit Ras farnesylation and membrane association. While FTIs have shown anti-tumor activity, this activity is not due to inhibition of Ras, but rather to inhibition of
65 other FTase substrates (27, 50), possibly including Rheb (86, 113, 115). Rce1 and Icmt
inhibitors, such as cysmethynil, are being developed as potential inhibitors of Ras membrane
association and function (25, 26), but whether they block Rheb signaling and function is not known. Additionally, farnesyl-containing small molecules, such as S-trans, trans- farnesylthiosalicylic acid (FTS; salirasib), have been developed as potential inhibitors of Ras membrane association and are currently undergoing clinical evaluation (61). Intriguingly,
FTS has also exhibited properties of an mTOR inhibitor (77-79), but whether this is due to inhibition of farnesylated Rheb is not known.
In this study, we determined whether inhibition of CAAX-signaled modifications
could be used to block aberrant Rheb signaling in cancer. We found that Rheb1 and Rheb2 both localized to the endoplasmic reticulum (ER) and Golgi apparatus and that the CAAX motif of Rheb1, but not of Rheb2, was sufficient for proper localization. While Rce1- and
Icmt-catalyzed modifications were required for proper Rheb localization, surprisingly, they were dispensable for Rheb-mediated activation of mTOR. Finally, we found that FTS directly blocked mTOR-induced activation of S6K independently of inhibition of Rheb or other prenylated proteins, suggesting that FTS is a novel type of mTOR inhibitor.
67 Materials and methods
Molecular constructs
pEGFP rat Rheb1 wt, M184L, and C181S and pEGFP human Rheb2 wt, M183L, and
C180S were generated by polymerase chain reaction (PCR) amplification and ligation into the pEGFP-C1 vector. All primer sequences are available upon request.
For generation of pEGFP-C3 Rheb C-terminal constructs (Figure 3a), the following oligonucleotides were used: 5’-AATTCCCTGCTCGGTGATGTGAG-3’ and 5’-
GATCCTCACATCACCGAGCAGGG-3’ (Rheb1 C4); 5’-
AATTCCCCAAGGCAAGTCTTCATGCTCGGTGATGTGAG-3’ and 5’-
GATCCTCACATCACCGAGCATGAAGACTTGCCTTGGGG-3’ (Rheb1 C9); 5’-
AATTCCCAAAATGGACGGGGCAGCTTCACAAGGCAAGTCTTCATGCTCGGTGAT
GTGAG-3’ and 5’-
GATCCTCACATCACCGAGCATGAAGACTTGCCTTGTGAAGCTGCCCCGTCCATTT
TGGG-3’ (Rheb1 C16); 5’-AATTCCCTGCCATCTCATGTGAG-3’ and 5’-
GATCCTCACATGAGATGGCAGGG-3’ (Rheb2 C4); 5’-
AATTCCCCAAGAGCGTCGCTGCCATCTCATGTGAG-3’ and 5’-
GATCCTCACATGAGATGGCAGCGACGCTCTTGGGG-3’ (Rheb2 C8); 5’-
AATTCCCCGTGTGGAGAATTCCTATGGGCAAGAGCGTCGCTGCCATCTCATGTGA
G-3’ and 5’-
GATCCTCACATGAGATGGCAGCGACGCTCTTGCCCATAGGAATTCTCCACACGG
GG-3’ (Rheb2 C15). Oligonucleotides were ligated into the EcoRI and BamHI sites of the pEGFP-C3 vector and verified by DNA sequencing.
68 pcDNA3 HA-K-Ras G12V, pcDNA3 FLAG-Rheb1 N153T, and pcDNA3 AU1- mTOR E2419K were described previously (174, 188, 189). pEYFP H-Ras (wild-type) was a gift from Dr. Mark Philips (New York University) (190). pRK7 HA-S6K1 was obtained from Addgene (Cambridge, MA) (191).
Cell culture and transfections
NIH 3T3 mouse fibroblasts were maintained in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% calf serum. COS-7 monkey epithelial cells were maintained in DMEM supplemented with 10% fetal calf serum. Rce1 +/+, Rce1 -/-, Icmt
+/+, and Icmt -/- mouse embryo fibroblasts (MEFs) were from Dr. Stephen Young
(University of California at Los Angeles) and were cultured as described previously (192).
FTI-2153 and GGTI-2417 were obtained from Dr. Saïd Sebti (Moffit Cancer Center) and Dr. Andrew Hamilton (Yale University) (193, 194) and FTS was obtained from Dr.
Yoel Kloog (Tel Aviv University) (195). Lovastatin and rapamycin were purchased from
Sigma-Aldrich (St. Louis, MO).
Transient transfections were performed in serum-free Opti-MEM using
Lipofectamine and Plus reagents (Invitrogen, Carlsbad, CA). For live cell microscopy, the medium was replaced with phenol red-free complete growth medium, and cells were imaged
24 to 48 h following transfection. When indicated, cells were serum-starved overnight 24 h following transfection, and then amino acid-starved for 1 h in phosphate buffered saline
(PBS) containing magnesium and calcium.
Immunofluorescence and confocal microscopy
69 Live cells were stained with 5 μM BODIPY TR C5 Ceramide (Molecular Probes,
Carlsbad, CA), 500 nM MitoTracker Red (Molecular Probes), or 75 nM LysoTracker Red
(Molecular Probes).
For immunofluorescence, cells were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 in PBS, blocked in 2% BSA in PBS, and incubated with the following primary antibodies: GM130 (BD Biosciences, Bedford, MA), EEA1 (BD
Biosciences), ERp72 (Calbiochem, La Jolla, CA), or LAMP2 (Developmental Studies
Hybridoma Bank, University of Iowa, Iowa city, IA), followed by Alexa Fluor 594 goat anti- mouse antibody or goat anti-rabbit antibody (Molecular Probes). Images were captured from a Zeiss 510 Meta Inverted Laser Scanning Confocal microscope using LSM 510 Meta software.
Western blot analysis
Cells were lysed in buffer containing 1% NP40, 0.25% deoxycholic acid, 10% glycerol, 25 mM HEPES, 50 mM NaCl, and 10 mM MgCl2, supplemented with EDTA-free protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN) and phosphatase inhibitor I and II (Sigma-Aldrich). Equal amounts of protein were resolved by SDS-PAGE, transferred onto Immobilon P membranes (Millipore, Bedford, MA), and immunoblotted with primary antibodies: anti-phospho-p70 S6 kinase (Thr389), anti-p70 S6 kinase, anti- phospho-S6 ribosomal protein (Ser235/236), anti-S6 ribosomal protein, and anti-mTOR (Cell
Signaling Technology, Danvers, MA); anti-HA.11 (Covance, Berkeley, CA); anti-GFP
(Clontech, Mountain View, CA); anti-Rheb1 (Millipore); anti-K-Ras (CalBiochem); anti-
Rap1 (Santa Cruz Biotechnology, Santa Cruz, CA); and anti-β-actin (Sigma-Aldrich).
70 Results
Rheb1 and Rheb2 localize to the ER and Golgi.
Previous reports indicated that ectopically expressed Rheb1 localizes to the Golgi
apparatus (130), endoplasmic reticulum (ER) (114), and vesicular structures (103, 130, 196),
and that endogenous Rheb localizes to mitochondria (92). The precise subcellular localization of Rheb2 has not been described. Therefore, we first wanted to confirm and extend the previous disparate observations on Rheb1 and additionally to evaluation Rheb2 localization.
Because no Rheb2 antibody was available for evaluation of endogenous protein, we
utilized green fluorescent protein (GFP)-tagged Rheb proteins for our analyses. Previous studies with similar GFP-tagged Ras family small GTPases have validated this approach for accurate determination of endogenous protein subcellular distribution (197-199). We transiently transfected COS-7 epithelial cells with either GFP alone or with GFP-tagged
Rheb1 and Rheb2 and treated live cells with BODIPY TR C5 ceramide, which detects cis-
and trans-Golgi and associated vesicles, as well as with MitoTracker, a mitochondrial marker, and LysoTracker, a lysosome marker. In addition, we fixed cells and stained with markers of the following intracellular compartments: ERp72 (ER), GM130 (cis-Golgi),
EEA1 (early endosomes), and LAMP2 (late endosomes). GFP-Rheb1 and GFP-Rheb2 both exhibited strong colocalization with BODIPY TR C5-ceramide (live cells) and GM130 (fixed
cells), confirming that they localize to the Golgi apparatus (Figures 3-1A and 3-1B). We also
observed colocalization with ERp72, indicating ER localization (Figure 3-1C). We did not
observe colocalization of either Rheb1 or Rheb2 with EEA1, LAMP2, LysoTracker, or
MitoTracker, suggesting that Rheb1 and Rheb2 do not localize to early endosomes, late
71 A. B.
C.
Figure 3-1. Rheb1 and Rheb2 are localized primarily to the ER and Golgi. COS-7 cells were transiently transfected with pEGFP vector or with pEGFP encoding GFP-tagged Rheb1 or Rheb2. (A) Rheb localization in cis- and trans-Golgi and associated endomembranes. Live cells were imaged after staining with BODIPY TR C5 ceramide. (B) Rheb localization in cis-Golgi. Cells were fixed and immunostained with mouse anti-GM130 antibody followed by Alexa Fluor 594- conjugated anti-mouse secondary antibody. (C) Rheb localization in the ER. Cells were fixed and immunostained with rabbit anti-ERp72 antibody followed by Alexa Fluor 594-conjugated anti-rabbit secondary antibody. All images were acquired on a Zeiss confocal microscope using LSM software. Data shown are representative of more than 10 images from at least two independent experiments.
72 endosomes, lysosomes, or mitochondria. We confirmed these results in NIH 3T3 mouse
fibroblasts (data not shown) and we observed similar localization of GFP-Rheb in fixed and
live cells.
The CAAX motif of Rheb1, but not Rheb2, is sufficient for proper localization.
Next, we determined the minimal C-terminal amino acid sequences sufficient for
proper Rheb localization. Since Rheb proteins lack either a cysteine or a polybasic amino
acid-rich stretch upstream of their CAAX motifs (Figure 3-2A), we hypothesized that they lack a “second signal” for localization and that the CAAX motif alone would dictate proper subcellular localization. To address this possibility, we engineered expression constructs encoding the last 4 amino acids of either Rheb1 (CSVM; Rheb1 C4) or Rheb2 (CHLM;
Rheb2 C4) fused to GFP (Figure 3-2B). We also created similar constructs with progressively longer C-terminal Rheb sequences to determine the minimum membrane targeting sequence of Rheb and transiently transfected them into NIH 3T3 cells. As controls, we used GFP-tagged full-length (FL) Rheb1 and Rheb2, as well as GFP-tagged Rheb1
C181S and Rheb2 C180S (“SAAX” mutants), which cannot be farnesylated, and hence display a nuclear and cytoplasmic localization identical to GFP alone (Figure 3-2C). Rheb1
C4, C9, and C16 all exhibited a subcellular localization indistinguishable from that of full length Rheb1, suggesting that the CAAX motif-signaled modifications alone are sufficient for proper Rheb1 localization (Figure 3-2C). Surprisingly, Rheb2 C4 did not localize to the distinct perinuclear region as seen with full length Rheb2, but instead exhibited a more diffuse cytoplasmic localization, and indicating that it was partially mislocalized, but still distinct from the localization of the unfarnesylated SAAX mutant. In contrast, Rheb2 C8
73 A.
B.
C.
Figure 3-2. CAAX-signaled modifications alone are sufficient for Rheb1 but not Rheb2 subcellular localization to the ER and Golgi. (A) Comparison of C-terminal membrane targeting sequence elements of H-Ras, K-Ras4B, Rnd3, Rheb1, and Rheb2. Palmitoylated cysteines (in H-Ras) and polybasic residues (in K-Ras 4B and Rnd3) are underlined. The C- terminal CAAX motifs are shaded. (B) GFP fusion proteins terminating in the Rheb1 or Rheb2 C- terminal sequences shown were encoded by cDNA sequences subcloned into the pEGFP vector. (C) NIH 3T3 cells were transiently transfected with pEGFP vector, or with pEGFP encoding the following proteins: GFP-tagged full-length Rheb1 C181S (Rheb1 SAAX), full-length Rheb2 C180S (Rheb2 SAAX), the indicated C-terminal Rheb1 or Rheb2 sequences, or full-length (FL) Rheb1 or Rheb2. Live cells were imaged by confocal microscopy. Data shown are representative of two independent experiments.
74 displayed a localization identical to the full length protein, suggesting that amino acid
residues immediately upstream of the CAAX motif are required for proper Rheb2
localization to the Golgi.
Localization of farnesylated Rheb is more dependent on Rce1- and Icmt-catalyzed
modifications than geranylgeranylated Rheb.
A previous study showed that Rheb1 localization is impaired in MEFs lacking Rce1
or Icmt, but whether Rheb1 function was impaired in these MEFs was not addressed (186).
To determine if Rheb1-mediated activation of mTORC1 was dependent on these
modifications, we first sought to confirm these localization findings, and to extend them to
Rheb2. We transiently transfected wild-type (Rce1 +/+ and Icmt +/+), Rce1-deficient (Rce1
-/-), and Icmt-deficient (Icmt -/-) MEFs with expression vectors encoding GFP-Rheb1 and
GFP-Rheb2. Consistent with previous results for Rheb1 (186), we found that the normal
localization of both Rheb1 and Rheb2 was completely impaired in Icmt -/- MEFs. Both
Rheb1 and Rheb2 showed significant nuclear accumulation and were indistinguishable from
the subcellular distribution of GFP alone (Figures 3-3A and 3-3B). Rce1 deficiency is expected to prevent the subsequent Icmt modification. However, Rheb1 and Rheb2 were
only partially mislocalized in Rce1 -/- MEFs: Rheb1 and Rheb2 accumulated in the nucleus
and cytosol, but Golgi localization was still visible (Figures 3-3A and 3-3B). Thus, Rheb1
and Rheb2 are more dependent on Icmt-mediated methylation than on Rce1-mediated AAX
cleavage, a pattern that we have observed with other farnesylated small GTPases (192).
The CAAX motifs of some GTPases, particularly Rho GTPases, are substrates for
geranylgeranyltransferase-I (GGTase-I)-catalyzed addition of a longer C20 geranylgeranyl
75 A. Rce1 +/+ Rce1 -/- Icmt +/+ Icmt -/- GFP F-Rheb1 GG-Rheb1 F-Rheb2 GG-Rheb2
Figure 3-3A. Rheb subcellular localization is dependent on Rce1- and Icmt-catalyzed modifications. Rce1 +/+, Rce1 -/-, Icmt +/+, and Icmt -/- MEFs were transiently transfected with pEGFP vector or with pEGFP encoding GFP-tagged Rheb1 (F-Rheb1), Rheb1 M184L (GG- Rheb1), Rheb2 (F-Rheb2), or Rheb2 M183L (GG-Rheb2). Live cells were imaged by confocal microscopy. Data shown are representative of three independent experiments.
76 B. completely mislocalized partially mislocalized properly localized Rce1 +/+ Rce1 -/- 100% 100%
80% 80%
60% 60%
40% 40%
20% 20% Percent of cells 0% 0% F-Rheb1 GG-Rheb1 F-Rheb2 GG-Rheb2 F-Rheb1 GG-Rheb1 F-Rheb2 GG-Rheb2 77
Icmt +/+ Icmt -/- 100% 100%
80% 80%
60% 60%
40% 40%
20% 20% Percent of cells 0% 0% F-Rheb1 GG-Rheb1 F-Rheb2 GG-Rheb2 F-Rheb1 GG-Rheb1 F-Rheb2 GG-Rheb2
Figure 3-3B. Rheb subcellular localization is dependent on Rce1- and Icmt-catalyzed modifications. Quantification of the data shown in (A). At least 30 cells in each condition were scored.. isoprenoid lipid. A previous study found that the localization of geranylgeranylated proteins
is less dependent on Rce1- and Icmt-catalyzed modifications than farnesylated protein
localization (200). Whether modification by the more hydrophobic geranylgeranyl group
also reduces Rheb sensitivity to Icmt and Rce1 loss was not known. To obtain
geranylgeranylated Rheb (GG-Rheb) mutants, we mutated the C-terminal amino acid of
Rheb1 and Rheb2 from methionine to leucine (Rheb1 M184L and Rheb2 M183L). Similar
CAAX mutants of S. pombe and human Rheb showed FTase-independent function (113, 115,
131, 201). We utilized pharmacologic inhibitors of FTase and GGTase-I (GGTI) and
confirmed that GG-Rheb was less sensitive to inhibition by FTI than wild-type farnesylated
Rheb (F-Rheb; data not shown).
To determine whether localization of GG-Rheb also depends on Rce1- and Icmt-
catalyzed modifications, we transiently transfected wild-type, Rce1 -/-, and Icmt -/- MEFs
with GFP-tagged GG-Rheb1 and GG-Rheb2. As expected, in wild-type MEFs, GG-Rheb2
displayed identical subcellular localization to F-Rheb. However, we were surprised to find
that GG-Rheb1 localization was similar to that seen with wild type F-Rheb1 in only a subset
of cells. Instead, a majority (74-77%) of GG-Rheb1 expressing cells showed partially (48-
66%) or completely (11-26%) mislocalized distributions (Figures 3-3A and 3-3B). Thus,
unlike most Ras and Rho small GTPases, where the type of isoprenoid modification did not
alter protein localization, geranylgeranyl modification did not substitute for farnesylation to
fully support Rheb1 subcellular localization.
In Rce1 -/- MEFs, F-Rheb1 and GG-Rheb1 were partially mislocalized (74-76%)
(Figure 3-3B). However, when expressed in Icmt -/- MEFs, a much lower percentage (48%)
of GG-Rheb1 was completely mislocalized when compared to F-Rheb1 (95%). Similarly, a
78 majority of GG-Rheb2 (>50%) retained proper localization in both Rce1 -/- and Icmt -/- cells,
although all of the F-Rheb2 was partially or completely mislocalized in these cells. Thus, modification by a geranylgeranyl group reduces the requirement of both Rheb1 and Rheb2 for the subsequent CAAX modifications, particularly by Icmt.
Rheb activation of mTOR is more dependent on farnesylation than on postprenylation CAAX
processing.
Since Rheb1 and Rheb2 are mislocalized in the absence of Rce1 and Icmt, we asked
if Rheb signaling was impaired in Rce1 -/- and Icmt -/- MEFs. First, we wanted to confirm in our assays that Rheb activation of mTOR is dependent on farnesylation (130). We transfected wild-type or knockout MEFs with constructs encoding GFP-tagged fusion proteins of an activated mutant of Rheb1 (Q64L) with an intact CAAX sequence or with two
CAAX missense mutants: a geranylgeranylated variant (X=L; M184L), designated GG-
Rheb1, and a nonfarnesylated mutant (X=S; M184S), designated Rheb1-SAAX. As expected, both F-Rheb and GG-Rheb efficiently induced S6K phosphorylation in wild-type
MEFs (Figure 3-4A). In agreement with previous observations (107, 115, 130), we found that the nonfarnesylated Rheb1-SAAX mutant was greatly impaired in stimulating S6K phosphorylation.
Since Rheb1-SAAX fails to undergo all three CAAX-signaled modifications, we next determined a role for the Rce1- and Icmt-catalyzed modifications in Rheb1 function.
Surprisingly, both F-Rheb and GG-Rheb retained the ability to stimulate S6K phosphorylation when expressed in either Rce1 -/- or Icmt -/- MEFs (Figure 3-4A).
79 A.
B.
Figure 3-4. Rce1- and Icmt-catalyzed modifications are not required for Rheb1 activation of S6K. (A) Wild-type (Icmt +/+) Rce1 -/-, and Icmt -/- mouse embryo fibroblasts were transiently transfected with pEGFP vector or with pEGFP encoding Rheb1 Q64L (F-Rheb1 64L), Rheb1 M184L/Q64L (GG-Rheb1 64L), or Rheb1 C181S/Q64L (Rheb1-SAAX 64L), along with pRK7 HA- S6K1, and serum- and amino-acid starved as described in Materials & Methods. Cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies to phospho-S6K, HA, GFP, or β-actin (loading control). (B) Wild-type (Icmt +/+), Rce1 -/-, and Icmt -/- MEFs were transiently transfected with pcDNA3 vector, or with pcDNA3 encoding HA-tagged K-Ras G12V, FLAG-tagged Rheb1 N153T, or AU1-tagged mTOR E2419K, along with pRK7 HA-tagged S6K1, and serum- and amino acid-starved as in (A). Cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies to phospho-S6K, HA, Rheb1, K-Ras, or β-actin (loading control). Data shown are representative of at least two independent experiments.
80 Since these observations were unexpected, we also compared the activity of Rheb1
with a different N-terminal tag (FLAG) and activating mutation (N153T) (188) with that of constitutively activated K-Ras4B(G12V), which is expected to require these modifications, and with that of constitutively activated mTOR (E2419K; a kinase-domain mutation) (189), which is not a substrate for either CAAX modifying enzyme. We transiently transfected wild-type, Rce1 -/-, and Icmt -/- MEFs with either pcDNA3 empty vector, or with pcDNA3 encoding constitutively activate mutants of K-Ras (HA-K-Ras G12V), Rheb1 (FLAG-Rheb
N153T), or mTOR (AU1-mTOR E2419K), along with pRK7 HA-S6K1, and used western
blot analysis to determine S6K phosphorylation. Consistent with previous studies (188, 189,
202), we observed that activated K-Ras4B, Rheb1, and mTOR all efficiently induced
phosphorylation of S6K in wild type MEFs. As expected, since mTOR is not a substrate for
Rce1 and Icmt, constitutively active mTOR also induced S6K phosphorylation in Rce1 -/-
and Icmt -/- cells (Figure 3-4B). Unexpectedly, K-Ras4B also did not show impaired S6K phosphorylation when expressed in Rce1 -/- or Icmt -/- MEFs. However, this result is consistent with our recent observation that K-Ras4B subcellular localization and plasma membrane association were not impaired to any significant degree in Rce1- or Icmt-null cells
(192). Consistent with GFP-tagged Rheb1, FLAG-tagged Rheb-induced S6K phosphorylation was also not impaired in Rce1 -/- or Icmt -/- MEFs. Thus, while these
CAAX modifications are necessary for proper subcellular localization, Rheb activation of mTOR is not impaired in the absence of Rce1 or Icmt function. Taken together, these data suggest that Rheb activation of mTOR is more dependent on farnesylation than on Rce1- and
Icmt-catalyzed modifications.
81 FTS does not disrupt the subcellular localization of Rheb.
FTS (also called salirasib) is a farnesylcysteine mimetic that is proposed to dislodge
Ras proteins from the plasma membrane and thus inhibit Ras activity (61, 203). However,
whether FTS also displaces and inhibits the function of other farnesylated proteins, such as
Rheb, is not known. FTS has also been shown to reduce phosphorylation of the mTOR
substrates S6K and 4E-BP1 (77, 78), but whether this is due to Rheb inhibition was not
investigated. To determine if FTS inhibits Rheb localization, we transiently expressed GFP-
tagged Rheb1 or YFP-tagged H-Ras in NIH 3T3 cells and treated them with FTS. As a
control, we treated cells with FTI-2153, since FTIs are known to impair Rheb and H-Ras localization (130, 186, 192). Indeed, GFP-Rheb and YFP-H-Ras were completely mislocalized in FTI-treated cells (Figure 3-5). Surprisingly, FTS did not cause mislocalization of either H-Ras or Rheb. These results suggest that FTS does not detectably disrupt the membrane association and subcellular localization of Rheb or Ras proteins under these conditions.
FTS inhibits mTOR-induced S6K activation and reduces mTOR protein levels.
Since it remained possible that FTS may disrupt Rheb interaction with mTOR, we
next asked if FTS inhibits Rheb-induced activation of mTOR. Activated K-Ras4B, Rheb1,
and mTOR all efficiently induced S6K phosphorylation in vehicle (DMSO)-treated cells
(Figure 3-6A). As expected, FTI treatment strongly inhibited Rheb1-induced S6K
phosphorylation and resulted in a shift in Rheb mobility as seen by SDS-PAGE, indicating
the accumulation of unprenylated Rheb1. We also observed an increase in Rheb protein
levels in FTI-treated cells, in agreement with previous studies (130). Importantly, FTI
82 Figure 3-5. FTS does not disrupt Rheb or H-Ras subcellular localization. NIH 3T3 cells were transiently transfected with pEGFP vector, pEGFP encoding GFP-tagged Rheb1, or pEYFP encoding YFP-tagged H-Ras and treated with DMSO (Vehicle), 5 μM FTI-2153, or 75 μM FTS overnight. Live cells were imaged by confocal microscopy.
83 treatment did not block mTOR-induced S6K phosphorylation, since mTOR E2419K is
constitutively active and is not dependent on Rheb. In contrast, FTS strongly inhibited K-
Ras, Rheb, and mTOR-induced S6K phosphorylation, suggesting that FTS inhibits S6K
activation at the level of mTOR, and not by inhibiting upstream activators.
Surprisingly, we found that FTS treatment of NIH 3T3 cells decreased levels of both
endogenous and ectopically expressed mTOR protein (Figure 3-6A), but did not decrease
levels of K-Ras, Rheb, or β-actin. Thus, FTS inhibition of mTOR may be due, in part, to promoting the loss of mTOR protein. However, FTS treatment did not decrease endogenous mTOR in MCF-7 human breast carcinoma cells, whereas it did decrease phospho-S6K in these cells (Figure 3-7, discussed below), suggesting that the FTS-induced reduction in
mTOR levels is not the primary cause of the reduction in S6K phosphorylation.
FTS inhibition of mTOR does not require protein prenylation.
One explanation for the above results could be that FTS is blocking the function of an
unidentified prenylated protein required for mTOR-induced S6K phosphorylation. For
example, Cdc42 and Rac1, which are geranylgeranylated, have previously been implicated in
S6K phosphorylation (204). In addition, RalA, another geranylgeranylated protein, was
recently shown to be required for Rheb-induced S6K phosphorylation (106). To determine
whether prenylated proteins are required for mTOR-induced S6K phosphorylation, we
transiently transfected NIH 3T3 cells as described above, and treated cells with FTI, a
GGTase-I inhibitor (GGTI), or both. We verified that the GGTI was functional and inhibited
modification of Rap1A (Figure 3-6B). As expected, FTI inhibited Rheb1-induced, but not
mTOR-induced, S6K phosphorylation (Figure 3-6B). Combined treatment with FTI and
84 A. B.
C. 85
Figure 3-6. FTS inhibits mTOR-induced S6K activation by a mechanism independent of prenylated proteins. (A) NIH 3T3 cells were transiently transfected with pcDNA3 vector, or with pcDNA3 encoding HA-tagged K-Ras G12V, FLAG-tagged Rheb1 N153T, or AU1-tagged mTOR E2419K, along with pRK7 HA-tagged S6K1. Three h following transfection, medium was replaced with complete medium containing either DMSO (Vehicle) or 5 μM FTI-2153. Twenty-four h following transfection, cells were serum-starved in DMEM supplemented with 0.1% BSA containing DMSO, 5 μM FTI-2153, or 75 μM FTS overnight, and then amino-acid starved in PBS containing the indicated compound for 1 h. Cell lysates were resolved by SDS-PAGE and immunoblotting was performed with antibodies to phospho-S6K, HA, Rheb1, K-Ras, or β-actin (loading control). Data shown are representative of three independent experiments. (B) NIH 3T3 cells were transiently transfected with the indicated plasmids, as described above. Three h following transfection, medium was replaced with complete medium supplemented with DMSO, 10 μM FTI-2153, 20 μM GGTI-2417, or 10 μM FTI-2153 + 20 μM GGTI-2417. Cells were serum- and amino-acid starved as in (A). Cell lysates were resolved by SDS-PAGE and immunoblotting was performed with antibodies to phospho-S6K, HA, Rheb1, K-Ras, Rap1, or β-actin (loading control). (C) NIH 3T3 cells were transiently transfected with the indicated plasmids. Three h following transfection, medium was replaced with complete medium supplemented with DMSO (Vehicle), 10 μM FTI-2153, 20 μM lovastatin, or 30 μM lovastatin. Serum- and amino acid-starved cell lysates were resolved by SDS-PAGE and immunoblotting was performed with antibodies to phospho-S6K, HA, Rheb1, K-Ras, H-Ras, or β-actin (loading control). GGTI efficiently inhibited K-Ras4B-induced S6K phosphorylation, presumably as a consequence of blocking alternative prenylation of K-Ras4B (205, 206). In contrast, FTI and
GGTI in combination failed to prevent mTOR-induced S6K phosphorylation, suggesting that
prenylated proteins modified by either farnesyltransferase or GGTase-I are not required for
mTOR activation of S6K.
To confirm these results and to exclude the possibility that prenylated GGTase-II
substrates (such as Rab GTPases) are required for mTOR activation of S6K, we treated cells
with lovastatin, which blocks all protein prenylation. As expected, lovastatin treatment
caused a mobility shift in Rheb, and reduced both Rheb- and K-Ras-induced S6K
phosphorylation (Figure 3-6C). However, lovastatin failed to inhibit mTOR-induced S6K
phosphorylation. Thus, FTS inhibition of mTOR does not involve inhibition of a prenylated
protein.
FTS inhibits endogenous S6K activity in breast cancer cells.
As described previously (77), we confirmed that FTS inhibited endogenous S6K activity in MCF-7 cells, but treatment did not affect mTOR or Rheb1 protein levels (Figure
3-7). These data are consistent with the hypothesis that FTS is an mTOR inhibitor.
86 Figure 3-7. FTS blocks endogenous S6K activation in MCF-7 cells. MCF-7 cells growing in complete medium were treated with 100 μM FTS in complete medium for 0, 2, 6, 16, or 24 h. Cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies to phospho-S6K (pS6K), total S6K, phospho-S6 (pS6), total S6, mTOR, and β-actin (loading control). Data shown are representative of two independent experiments.
87 Discussion
Rheb functions as a key intermediate in the PI3K-AKT-TSC-Rheb-mTOR network
that is aberrantly activated in a majority of human cancers (122). While pharmacologic
inhibitors of PI3K, AKT and mTOR are currently in Phase I/II clinical trials, Rheb may also
be a useful target. A key goal of our study was to determine if specific approaches can block
Rheb function by disrupting the membrane association and subcellular distribution of Rheb
essential for its biological activity. We assessed two different strategies for blocking Rheb function. The first strategy involves preventing the CAAX-mediated posttranslational modifications that promote Rheb membrane association. We found that both Rheb1 and
Rheb2 localization, but not mTOR activation, was disrupted in the absence of the post- prenylation processing enzymes Rce1 or Icmt, suggesting that inhibitors of these enzymes will not block Rheb signaling and oncogenic activity. The second strategy involves inhibition of Rheb endomembrane association by FTS. Unexpectedly, we found that FTS did
not effectively impair Rheb or Ras protein membrane association. Instead, we found that
FTS blocked Rheb activation of mTOR, but this effect was independent of inhibition of Rheb
or of any other prenylated protein. Rather, we suggest that a potent mechanism for FTS-
mediated growth inhibition may involve direct inhibition of mTOR.
Although the subcellular localization of Rheb proteins has been addressed in multiple
previous studies, conflicting observations were made (92, 103, 114, 130, 186). Furthermore,
the precise subcellular localization of Rheb2 had not been determined. We confirmed that
Rheb1 is primarily localized to the Golgi and ER endomembranes, in agreement with others
(114, 130). We also found that the localization of Rheb2 is indistinguishable from that of
Rheb1, and that Rheb localization overlaps with the observed localization of Rheb binding
88 partners (207-209). The essentially identical subcellular localization of Rheb1 and Rheb2
contrasts with the situation seen with closely related isoforms of other Ras family small
GTPases. For example, although RalA and RalB share 82% overall sequence identity, their
distinct subcellular distributions result in strikingly distinct biological roles (158).
We found that, unlike most Ras and Rho small GTPases, the CAAX motif alone of
Rheb1 was sufficient for proper localization. These data are consistent with Choy et al., who
showed that the CAAX motif of K-Ras and N-Ras alone promoted localization to the ER and
Golgi (197). In contrast, the CAAX motif alone was not sufficient for proper localization of
Rheb2, which required amino acids immediately upstream of the CAAX motif, suggesting
that the targeting motifs of Rheb1 and Rheb2 are not functionally identical.
We also found that geranylgeranylated mutants of Rheb1 and Rheb2 (Rheb1 M184L
and Rheb2 M183L), like their farnesylated wild-type counterparts, localized to the ER and
Golgi. However, the localization of GG-Rheb was less dependent on Icmt- and Rce-1 catalyzed modifications, in agreement with previous studies showing that the localization of geranygeranylated Ras mutants was less dependent on these modifications than farnesylated
Ras (200).
Farnesylation of Rheb was required for its ability to activate mTOR, as shown by the
inability of the nonfarnesylated SAAX mutant of Rheb1 to induce S6K phosphorylation.
These observations are consistent with several studies showing that FTI impairs S6K or S6
phosphorylation (113, 115, 131, 210). In contrast, neither of the post-prenyl processing steps
was required for Rheb-induced mTOR activation, which was not disrupted in either Rce1 -/-
or Icmt -/- MEFs. In addition, we found that the Icmt inhibitor cysmethynil did not block
Rheb activation of mTOR (data not shown). These data are consistent with those of Buerger
89 et al., who showed that the methyltransferase inhibitor AFC does not block Rheb-induced
S6K activation (130). Therefore, we suggest that inhibitors of Rce1 and Icmt will not inhibit
Rheb activity.
We found that FTS strongly inhibited both Rheb- and mTOR-induced S6K activation.
FTS is proposed to compete with farnesylated Ras for membrane binding sites and dislodge
Ras from the membrane, thereby disrupting its function (62, 65, 73, 195). However, we
failed to observe detectable disruption of H-Ras or Rheb membrane association. Instead, our
data strongly suggest that the FTS-mediated inhibition of mTOR-induced S6K activation is
not due to inhibition of a prenylated protein. Rather, our data are consistent with the
observation that FTS disrupts the association of mTOR with raptor (79). We failed to
observe a concomitant decrease in Akt phosphorylation on S473 in FTS-treated cells (Figure
4-4), suggesting that FTS inhibits mTORC1 but not mTOR complex 2 (mTORC2) activity.
In summary, we have found that inhibition of Rce1- and Icmt-catalyzed modifications
does not prevent Rheb activation of mTOR, and that FTS inhibits mTOR through a
mechanism that does not involve Rheb inhibition. Although FTS is not a direct Rheb
inhibitor, mTOR is essential for Rheb oncogenic activity (113, 114); hence, FTS would be
expected to disrupt both Rheb- and mTOR-induced transformation. Since FTS is currently in
clinical trials for cancer treatment, our results have significant implications for the selection
of who that may benefit from FTS therapy and suggest that S6K phosphorylation may be a
useful biomarker to monitor FTS activity.
90 Acknowledgements
We thank Yoel Kloog, Saïd Sebti, and Andrew Hamilton for inhibitors, Lawrence
Quilliam and Mark Philips for providing plasmid constructs, Stephen Young for MEF cell lines deficient in Rce1 and Icmt, and Natalia Mitin and the UNC Michael Hooker
Microscopy Facility for imaging assistance.
91
CHAPTER 4
CONCLUDING REMARKS AND FUTURE DIRECTIONS
Aberrant Ras signaling drives the growth and progression of many cancers. Although
Ras mutations are rare in breast cancers, other related Ras family members may be deregulated in breast cancer and may contribute to breast cancer progression. I have chosen to focus on the roles of two estrogen-regulated Ras family small GTPases in breast cancer.
The majority of breast cancers depend on estrogen for growth, and hence proteins regulated by estrogen may represent attractive therapeutic targets for the treatment of breast cancer.
The primary aims of my studies were to define the roles of Rerg and Rheb in breast cancer progression. Unexpectedly, I found that loss of Rerg expression did not significantly contribute to breast cancer progression. While I was unable to further examine the role of
Rheb in breast cancer due to technical limitations, my studies did identify strategies to block
Rheb-mTOR signaling that may be therapeutically beneficial for patients with breast and other cancers. My studies have also laid a foundation for future studies of Rerg and Rheb function in normal cells and in tumorigenesis.
Is Rerg a growth inhibitor? Previous studies have implicated Rerg as a growth inhibitor in ERα-positive MCF-7 breast cancer cells (151). In Chapter 2, I further investigated the effects of Rerg expression on breast cancer cell growth and invasion. However, I failed to observe a difference in the growth of these cells in vitro upon depletion of Rerg expression by shRNA. Furthermore, a tamoxifen-inducible ER-Rerg fusion protein did not reduce the aggressiveness of ERα-negative breast cancer cells in vitro. What accounts for these discrepancies? First, the growth inhibitory properties of Rerg may depend on cell context. It is possible that high expression of Rerg may restrain the growth of ERα- positive breast cancers, but may not function the same way in ERα-negative breast cancer cells. Indeed, high Rerg expression correlated with favorable outcome in ERα-positive breast cancer patients (Perou and colleagues, unpublished observations). Secondly, either my results or the previous studies may have been compromised by nonphysiologic experimental conditions. In the previous study, the growth inhibition observed upon transfection with
Rerg may be the result of ectopic Rerg expression at higher than physiological levels.
Conversely, in my studies, insufficient knockdown levels may have prevented an accurate assessment of the effects of loss of Rerg expression in ERα-positive breast cancer cells.
Other lentiviral shRNAs targeting Rerg should be tested for a more complete knockdown of
Rerg expression and may lead to more pronounced differences in the growth or invasive properties of ERα-positive breast cancer cells. It is also possible that the fusion of the ERα ligand binding domain to Rerg disrupted Rerg activity, as discussed in chapter 2. Thus, creation of a doxycycline-inducible Rerg expression system would circumvent this caveat; it will be important to test whether induction of untagged Rerg decreases the growth of breast cancer cells. Finally, it remains possible that Rerg may have a more pronounced role in breast cancer growth in vivo.
Although in vitro soft agar formation strongly correlates with tumorigenic potential in vivo, there are instances of proteins with no apparent effect on soft agar assays, yet these proteins
93 do affect xenograft growth or metastasis in vivo. As I have only studied the effects of
manipulating Rerg expression in vitro, my studies do not rule out a possibility that Rerg is
involved in tumor formation or metastasis in vivo. For example, Rerg may play a role in the
tumor microenvironment: it may mediate tumor/stromal interactions, immune response,
angiogenesis, etc. Therefore, it will be important to test whether knocking down or restoring
Rerg expression enhances or reduces orthotopic tumor xenograft growth (by injecting tumor
cells containing Rerg shRNA or ER-Rerg into the mammary fat pad) or affects the formation
of metastases in the lung (by injecting tumor cells into the tail veins of SCID mice).
The generation of a Rerg knockout mouse will also shed considerable light on
whether Rerg is involved in breast cancer initiation or progression. First, it will be important
to determine if Rerg is expressed in current transgenic mouse models of breast cancer, such
as the mouse mammary tumor virus (MMTV)-HER2 model. If so, then crossing this model
with a Rerg knockout mouse will determine if Rerg loss enhances tumor growth and
metastasis. If the current mouse models of breast cancer do not express Rerg, then a
transgenic mouse expressing Rerg under a mammary-specific promoter should be crossed to
these mice to determine if Rerg expression reduces tumor growth or metastasis. Assuming
that deleting the Rerg gene does not result in embryonic lethality, it will also be interesting to determine if mice lacking Rerg exhibit increased tumor incidence, suggesting that Rerg may suppress the formation of tumors.
What is the role of Rerg in normal cells and in development? Rerg mRNA is
expressed in many tissues, including heart, skeletal muscle, kidney, and ovary (153),
suggesting that it may play an important role in a variety of normal cellular processes.
94 Several approaches can be used to identify the function of Rerg in normal cells and in mammalian development. First, the identification of Rerg binding partners and effectors will shed considerable light on the signaling pathways and cellular processes in which Rerg participates. Although yeast 2-hybrid screens have not been successful in identifying Rerg binding partners, other approaches may be used. One potential method would be to express
GFP-tagged Rerg in cells, immunoprecipitate Rerg using an anti-GFP antibody, and separate interacting proteins by SDS-PAGE. Bands may then be removed and proteins identified by mass spectrometry analysis (211, 212). This proteomic approach has the advantage over the yeast 2-hybrid method because it allows expression and post-translational modifications to occur in mammalian cells and allows for the precipitation of multi-protein complexes to detect indirect interactions. Our laboratory has successfully used this approach to identify binding partners of other small GTPases, such as RhoBTB2 and RhoBTB3 (unpublished observations). To identify specific effectors of Rerg, this approach may be modified by expressing the GFP-tagged Rerg Q64L mutant, which exhibits increased GTP binding (151), along with GFP-Rerg S20N, predicted to be bound to GDP (153). Proteins that preferentially associate with Rerg Q64L and require an intact effector domain would be predicted to be bona fide Rerg effectors. This approach could also be performed in Rerg-positive cancer cell lines in order to identify effector proteins that may mediate the putative growth inhibitory properties of Rerg. Interacting proteins should be validated by co-immunopredicipation using antibodies to endogenous proteins.
Secondly, determining the genes that are regulated in response to Rerg expression may also give clues to the function of Rerg. The Rerg shRNA I generated may be used to reduce Rerg expression in cells that normally express high levels of Rerg. Next, microarrays
95 may be used to determine which genes are up- or down-regulated in response to Rerg repression. Gene ontology programs may then be used to determine enrichment for groups of genes involved in specific cellular processes. It is likely that Rerg would play a role in these processes.
A third, and perhaps the most direct, approach to elucidate the function of Rerg is, again, the generation of a Rerg knockout mouse. Rerg-deficient mice should be studied for apparent defects in development, immune regulation, etc. It is possible that Rerg deficiency will result in embryonic lethality, in which case it will be interesting to determine the function Rerg plays in embryonic development. Conversely, it is possible that mice lacking
Rerg will not exhibit an obvious phenotype in normal development. However, genetic or chemical carcinogen-induced oncogenesis in Rerg-deficient mice may reveal a function that occurs only under situations of stress. Finally, determining Rerg binding partners and Rerg- regulated genes may provide clues as to how to find a phenotype associated with Rerg loss.
What properties of Rerg are required for its function? Elucidating the biological function of Rerg will lay the groundwork for exciting studies to determine the mechanism by which Rerg acts. Ideally, a cellular assay would be developed in which shRNA-mediated knockdown of endogenous Rerg causes a biological phenotype. Ectopic expression of a shRNA-insensitive Rerg cDNA could then be used to rescue this effect and restore normal cellular function. Then, mutants of Rerg could be tested to determine whether specific properties of Rerg are required for its function. One major question is whether the GTP- bound form of Rerg is the “active” form. Therefore, the Rerg Q64L or S20N mutants could be re-expressed in Rerg shRNA-expressing cells. I would predict that the GTP-bound Rerg
96 Q64L mutant would rescue the phenotype, whereas the GDP-bound S20N mutant would not.
As mentioned above, discovering Rerg effectors will be a key breakthrough in elucidating a
Rerg signaling pathway. Effector domain mutants of Rerg could then be generated to determine which effectors mediate the biological functions of Rerg. shRNA targeting Rerg effectors could then be used to determine if loss of Rerg effectors phenocopies Rerg loss.
Alternatively, activated versions of Rerg effectors could be used to rescue the phenotype of
Rerg knockdown cells. Finally, it will be important to determine whether Rerg localization is
important for its function. As mentioned above, Rerg is unique from other Ras family
proteins and lacks a CAAX motif. My studies found that GFP-tagged Rerg is targeted to the
cytoplasm. The Rerg C-terminus could be replaced with that of Ras: will a membrane-
targeted version of Rerg still function properly? Clearly, once a validated biological function
is ascribed to Rerg, this will be an active and exciting field of study. The tools I developed to
manipulate and detect Rerg expression will be invaluable in these future studies.
It will also be interesting to determine the upstream regulators of Rerg GTP/GDP cycling. Rerg has been shown to bind, exchange, and hydrolyze GTP (151); are there GEFs and GAPs for Rerg that promote these processes? In addition, many small GTPases, such as
Ras and Rheb, are regulated by extracellular growth factors and other signals. Do extracellular signals similarly promote Rerg-GTP, or perhaps inhibit Rerg? To test this, one
could serum-starve cells overnight, then stimulate with serum or with other growth factors
for 15 mins, and use thin layer chromatography to detect Rerg binding to radiolabeled GTP
(151). These experiments will establish the cellular context of Rerg signaling.
97 Does loss of Rerg expression contribute to the growth of other types of cancer?
Although my studies did not find a role for Rerg in the growth of breast cancer cells, it
remains plausible that loss of Rerg expression promotes growth or metastasis of other types
of tumors. According to the oncomine database (www.oncomine.org), Rerg mRNA expression is frequently downregulated in breast and other types of tumors as well as in metastatic tumors compared to non-metastatic counterparts (170-172). In order to extend these findings, one could use the Rerg antibody for IHC analysis on tissues from metastatic and primary tumor samples. A tissue microarray would be particularly useful in determining whether Rerg protein is indeed downregulated in different types of tumors. If this is found to be the case, then the Rerg shRNA and tamoxifen-inducible ER-Rerg fusion protein I generated in Chapter 2 will be valuable tools in validating a role for Rerg in suppressing the growth or metastatic properties of these tumor cells. If Rerg is found to be a tumor or metastasis suppressor, then a primary goal will be to develop strategies that restore Rerg expression in tumors.
Therapeutic reexpression of ARHI or Rerg in breast cancer. Due to the putative
roles of ARHI and Rerg as tumor suppressors or growth inhibitors in breast (or perhaps
other) tumors, therapeutic reexpression of these proteins in tumors that have lost their
expression may be beneficial. Although a gene therapy approach to simply restore
expression may be the most direct strategy, this feat remains difficult to accomplish with
current technology. Instead, targeting the epigenetic regulation of ARHI and Rerg may be
more promising. Expression of both genes is negatively regulated by HDACs (Figure 4-1).
The HDAC inhibitor trichostatin A restored expression of ARHI and Rerg in breast cancer
98 HDACi (TSA) 5-aza-dC
E2F HDAC DNMT
ARHI
HDACi (TSA) MEK
Src E2 HDAC DNMT ERα ERE Rerg
= methylated CpG in DNA
= deacetylated histone tail
Figure 4-1. Epigenetic silencing of ARHI and Rerg and strategies to restore their expression. The ARHI gene can be epigenetically silenced by promoter hypermethylation, catalyzed by DNMTs, and by HDAC/E2F complexes. The DNMT inhibitor 5-aza-dC and the HDAC inhibitor (HDACi) TSA have been shown to restore ARHI expression in some breast cancer cells. TSA can also restore Rerg expression in an ERα-negative cell line. Other methods to restore Rerg expression in ERα-negative breast cancers could include strategies to upregulate or to stabilize the ERα, such as with Src inhibitors, MEK inhibitors, or DNMT inhibitors. Adapted from Hanker and Der, Handbook of Cell Signaling, in press.
99 cells (213, 214). Several HDAC inhibitors are currently in clinical trials to treat cancer,
including breast cancer, and the HDAC inhibitor vorinostat (Zolinza®) has been approved by
the FDA for the treatment of cutaneous T-cell lymphoma (215). It will be important to
determine whether these HDAC inhibitors restore expression of ARHI or Rerg in primary
breast cancers lacking expression of these proteins and whether restoration of ARHI or Rerg
expression correlates with patient response.
Many tumor suppressors, including ARHI, are epigenetically silenced by promoter
hypermethylation (Figure 4-1). DNA methyltransferases (DNMTs) have thus attracted
interest as therapeutic targets (216). The DNMT inhibitor 5-aza-2’-deoxycytidine (5-aza-dC)
reactivated ARHI expression in breast cancer cells (142, 214). Another way to restore ARHI
expression could be E2F inhibitors, since RNA interference-mediated suppression of E2F family member expression was shown to increase ARHI expression in SKBR3 breast cancer cells (141). Whether the Rerg promoter is hypermethylated or whether Rerg expression can be restored by 5-aza-dC treatment is not known, but 5-aza-dC can restore ERα expression in
ERα-negative breast cancer cells (217). It will be important to determine if ERα expression is sufficient to restore Rerg expression. If this is the case, then Rerg expression may also be restored by inhibitors of the Src tyrosine kinase or MEK1/2 dual specificity kinases, which have been shown to increase ERα levels in breast cancer cells (218, 219). If Rerg or ARHI are found to be upregulated by HDAC or other inhibitors, then it will be important to determine whether the anti-tumor effects of these pharmacologic agents may be due to restoring expression of these GTPases. To test this, shRNA targeting Rerg or ARHI could be used to prevent upregulation in response to treatment, and then tumor growth may be monitored. Since many of these therapies are in preclinical or clinical studies, understanding
100 the effects of ARHI or Rerg re-expression in breast cancer may help to predict patient
response to these therapies.
Which tumors are dependent on Rheb activity? Rheb overexpression and
activation of the Rheb-mTOR pathway have been implicated in many types of cancers,
including breast cancers, suggesting that Rheb may be a good therapeutic target for these
cancers. I initially began work on Rheb in the hopes of defining the role of Rheb in estrogen-
regulated breast cancers. However, my studies were inconclusive due to technical concerns,
so I began work on potential strategies to inhibit Rheb function in cancer. I focused on
blocking Rheb CAAX-mediated posttranslational processing and localization, mimicking
similar strategies aimed at blocking oncogenic Ras function in cancer. My results suggest
that targeting Rheb post-prenylation processing would not strongly inhibit Rheb function, but
that the farnesylcysteine mimetic FTS may be more promising in inhibiting Rheb/mTOR
signaling. Furthermore, my studies reinforced the concept that farnesylation is strongly
required for Rheb function, and hence FTIs would be predicted to block Rheb-induced
oncogenesis, in agreement with previous studies (113, 115). As more Rheb inhibitors are
identified, it will be essential to determine the types of tumors that depend on Rheb signaling
for growth and survival in order to identify an appropriate patient population in which to test
inhibitors of Rheb.
One approach to define Rheb-dependent tumor cells would be to screen panels of
tumor cell lines for sensitivity to Rheb shRNA. A high-throughput assay, such as the MTT cell viability assay, could be used to measure cell viability in response to knocking down
Rheb expression. A similar approach using K-Ras shRNA in K-Ras-mutant cells has
101 successfully identified cell lines “addicted” to mutant K-Ras activity (220). Alternatively, a
more accurate measure of tumorigenic potential may be the use of soft agar assays to
determine cell lines that require Rheb for anchorage-independent growth. Once sensitive cell
lines are identified, they should be analyzed for specific oncogenic mutations, such as
PIK3CA or PTEN mutations, as well as Rheb overexpression, to determine if any of these
molecular aberrations correlate with sensitivity. In addition, phosphorylation status of
mTOR downstream targets, such as S6K and 4E-BP1, should be measured in these cell lines
to determine if sensitivity to Rheb depletion correlates with phosphorylation status of mTOR targets. Phosphorylation of raptor on Ser 863 (221) and phosphorylation of mTOR on Ser
1261 (222) have also been suggested to correlate with Rheb activity and therefore should be tested. These studies may identify a molecular biomarker to predict patient response to Rheb inhibitors.
However, there are caveats to this approach. In my studies, I failed to observe a decrease in cell proliferation or anchorage-independent growth in breast cancer cells expressing Rheb shRNA (Figure 4-2). This may be due to the presence of Rheb2, which may be functionally redundant with Rheb1. Therefore, it may be necessary to deplete both Rheb1 and Rheb2 using shRNA targeting each isoform. Alternatively, overexpression of TSC2 would be predicted to block activation of both Rheb1 and Rheb2. Since I was unable to achieve stable expression of TSC2 in breast cancer cell lines, presumably due to its strong tumor suppressive activity, an inducible TSC2 expression system may be required.
An emerging theme of targeted anti-cancer therapy is that targeted therapies will most likely work better in combination than as single agents (223). Thus, future studies should determine appropriate combination therapy for Rheb inhibitors. In breast cancer, one could
102 A. shLuciferase shRheb (1) shRheb (2) shRheb (3) pSR vector shGFP
pS6
Rheb
β-actin
B. 1.6
1.2 vehicle 1 nM E2 0.8 1 nM E2 + 1 uM Tam 0.4 Optical Density
0 r o P F iferase Rvect shG c S p shLu shRheb (1)shRheb (2)shRheb (3) C. 500 400 300 200 100 0 r Colonies per Plate Colonies o P F Rvect shG iferase S c p shRheb (1) shRheb (2) shRheb (3) shLu Figure 4-2. Knocking down Rheb1 expression does not significantly reduce estrogen- induced MCF-7 cell proliferation or anchorage-independent growth. (A) MCF-7 cells were stably infected with empty pSuper.retro.puro vector, or encoding shRNA targeting green fluorescent protein (GFP) or luciferase (negative controls), or Rheb1. Western blots were probed with antibodies to phospho-S6 (pS6), Rheb1, or β-actin (loading control). (B) MCF-7 stable cell lines were seeded at equal numbers in 96-well plates and treated with Vehicle, 1 nM estrogen, or 1 nM estrogen + 1 μM tamoxifen for 5 days. Cell viability was measured using the MTT assay. Results represent the average of 8 replicate wells; error bars represent standard deviation. (C) MCF-7 stable cells were seeded in triplicate into 0.4% soft agar over a 0.6% bottom layer. After 27 days, viable colonies were stained in 2 mg/mL MTT. ImageJ software was used to quantify number of colonies from images of scanned plates. Results represent the average of triplicate plates; error bars represent standard deviation.
103 foresee combining Rheb inhibitors with endocrine therapies, such as tamoxifen. The AKT pathway is hyperactivated in tamoxifen-resistant breast cancer cell lines (224) and constitutively active AKT has been shown to promote tamoxifen resistance in MCF-7 cells
(14, 128, 225). This increased tamoxifen resistance was blocked by rapamycin, and is thus dependent on mTOR (128). Furthermore, Rheb inhibition was shown to increase sensitivity to tamoxifen-induced apoptosis (115). Thus, Rheb may play a role in tamoxifen resistance, and Rheb inhibition may delay or reduce development of resistance to endocrine therapies.
To test this, a constitutively active mutant of Rheb (Rheb N153T) could be stably expressed in MCF-7 cells, and tamoxifen sensitivity could be determined using the MTT or soft agar assay. Does activated Rheb, like activated AKT, impart resistance to tamoxifen? It will also be interesting to determine whether Rheb is required for tamoxifen resistance. Tamoxifen- resistant MCF-7 cells have been developed by culture in the presence of tamoxifen for six months; these cells have overcome tamoxifen-induced growth inhibition (226). Does knocking down Rheb using shRNA delay or prevent the development of tamoxifen resistance in these cells? Experiments addressing these questions will be critical to determine whether the combination of Rheb inhibitors with endocrine therapy is a rational strategy for breast cancer treatment.
Is Rheb the FTI target responsible for FTI anti-tumor activity? FTIs have exhibited some anti-tumor activity in clinical trials, particularly in breast cancer (54, 133,
227-229). However, the clinical development of these agents has been hampered by the inability to identify a molecular biomarker to predict response or to define a clear patient population that will benefit from these agents (50, 230). Considerable evidence points to a
104 role for Rheb in mediating the anti-tumor activity of FTIs (27, 231). First, FTIs were found
to decrease phosphorylation of S6K and 4E-BP1 in cancer cell lines and mammary tumors of
transgenic mice (115, 126, 210, 232). In support of these studies, treatment of melanoma cells with the FTI lonafarnib reduced levels of phospho-S6K but not phospho-ERK or
phospho-AKT (233). Further evidence of the importance of Rheb in FTI anti-tumor activity
comes from studies using the FTI-insensitive GG-Rheb variant. Basso et al. found that GG-
Rheb prevented lonafarnib enhancement of tamoxifen-induced apoptosis in MCF-7 cells
(115), which are exquisitely sensitive to FTIs (234), but whether GG-Rheb also rescued lonafarnib-induced inhibition MCF-7 cell growth was not tested. More recently, Mavrakis and colleagues found that GG-Rheb Q64L promoted FTI resistance in lymphoma cells (113).
However, in my studies, I failed to observe a change in FTI sensitivity (measured using the
MTT assay and soft agar assay) when I stably expressed GG-Rheb in MCF-7 cells (Figure 4-
3). This may be due to low expression levels of GG-Rheb. Alternatively, GG-modified
Rheb may not fully recapulate the activities of authentic Rheb. In addition, I did not use activated GG-Rheb, which may have given a stronger phenotype. I also failed to observe a difference in the FTI sensitivity of MCF-7 cells expressing Rheb shRNA. Again, this may be due to expression of Rheb2. Consistent with my results, a recent clinical trial of the FTI tipifarnib in breast cancer found that response to tipifarnib did not correlate with Rheb overexpression (229). However, S6K and 4E-BP1 phosphorylation levels were not examined. Thus, the role of Rheb in patient response to FTIs remains unclear.
More than seventy different cellular proteins are predicted to be farnesylated (52).
Therefore, it is likely that FTI inhibition of more than one farnesylated protein contributes to its anti-tumor effects. Furthermore, it is conceivable, and even probable, that the critical FTI
105 A. vector Rheb wt GG-Rheb [FTI-2153] (μM): 0 0.01 0.1 1 0 0.01 0.1 1 0 0.01 0.1 1 Flag-Rheb
β-actin B. vector 1.2 Rheb wt 1 0.8 GG-Rheb 0.6 0.4 0.2 OD (normalized) 0 DMSO 0.01 μM 0.1 μM 1 μM FTI 10 μM FTI FTI FTI C. 1200 vehicle 0.01 μM FTI 0.1 μM FTI 800 1 μM FTI 400
Number of colonies 0 vector Rheb wt GG-Rheb
Figure 4-3. GG-Rheb1 does not reduce FTI sensitivity of MCF-7 cells. (A) MCF-7 cells were stably transfected with the pcDNA3 empty vector or encoding wild-type Rheb1 or GG-Rheb1 (Rheb CVLL). Lysates of cells treated with the indicated concentrations of FTI-2153 were probed with antibodies to Rheb1 and β-actin (loading control). FTI induced a mobility shift in wild-type Rheb1, but not in GG-Rheb1. (B) MCF-7 stable cell lines were seeded in replicates of 8 in 96-well plates and treated with DMSO or the indicated concentration of FTI-2153 for 3 days. Cell viability was measured using the MTT assay. Data represent an average of 8 replicate wells; error bars represent standard deviation. (C) MCF-7 stable cells were seeded in triplicate into 0.4% soft agar over a 0.6% bottom layer. After 22 days, viable colonies were stained in 2 mg/mL MTT. ImageJ software was used to quantify number of colonies from images of scanned plates. Results represent the average of triplicate plates; error bars represent standard deviation.
106 targets for anti-tumor activity are different in different tumor types. So how can we identify
the targets of FTIs responsible for their anti-tumor activity in different types of tumors? One
approach would be to generate a shRNA library containing shRNA directed against every
known and predicted farnesylated protein (52). This library could then be screened to
identify shRNAs that sensitize cells to FTI treatment. It would be predicted that the targets
of these shRNAs would contribute to the efficacy of FTIs. Such studies would certainly aid
in identifying groups of patients who may benefit from FTI therapy. A limitation of this
approach is that, if FTI sensitivity is based on inhibiting multiple proteins, this will not be
mimicked by shRNA inhibition of only one substrate at a time.
Is Rheb localization important for its function? Because farnesylation is required
for Rheb localization and signaling [Chapter 3 and (86, 107, 114, 115, 130-132, 186)], and
proper localization of many small GTPases, such as Ras, is required for their function (21,
235, 236), we hypothesized that proper localization of Rheb will be required for it to function properly and activate mTOR. Surprisingly, my studies in Chapter 3 found that although
GFP-Rheb1 and GFP-Rheb2 were mislocalized in MEFs lacking Rce1 or Icmt, Rheb-induced
activation of mTOR signaling was not impaired in these cells. My results suggested that
farnesylation of Rheb may be more important than proper localization for Rheb activation of
mTOR. However, an important caveat of these studies is that they were done using
ectopically expressed protein. Studies in our laboratory are underway to determine whether
endogenous Rheb is mislocalized in MEFs lacking Rce1 or Icmt and whether endogenous
Rheb function is impaired in these cells. Specifically, we are planning to activate endogenous Rheb in these cells by using shRNA to deplete cells of the RhebGAP TSC2.
107 Knocking down TSC2 should increase endogenous S6K activation in wild-type MEFs, and this should be inhibited by FTI. Based on my studies in Chapter 3, I predict that knocking down TSC2 will also increase Rheb activity and S6K activation in Rce1- and Icmt-deficient cells, whereas FTI will prevent this increase. These results would be consistent with my studies, where I found that a farnesylation-defective Rheb mutant could not activate S6K in
Rce1- or Icmt-deficient cells, whereas farnesylated Rheb strongly induced S6K activation
(Figure 3-4A). If we indeed confirm that endogenous Rheb localization, but not activity, is impaired in the absence of Rce1 or Icmt, it would suggest that perhaps Rheb farnesylation is required for interaction with effectors such as PLD1 or mTOR (90, 91). For example, Ras farnesylation is required for its interaction with galectin-1 (63). Notably, Rheb farnesylation is not required for binding to and inhibition of B-Raf (109). It will be interesting to determine whether Rheb inhibition of aggresome formation is dependent on farnesylation or post-prenyl processing.
My studies suggest that Icmt and Rce1 inhibitors will not block Rheb activation of mTOR. However, it will be important to test this in vivo. Mice deficient in Icmt or Rce1 in specific tissues have been generated (56, 58); will crossing a transgenic mouse model overexpressing Rheb (113, 118) to these mice impair Rheb-induced tumor growth? These in vivo studies will more definitively determine whether Rheb post-prenyl processing is required for Rheb-induced oncogenesis.
Can more selective Rheb inhibitors be developed? As mentioned above, cells contain dozens of farnesylated proteins, and even more proteins are modified by Icmt and
Rce1. Therefore, inhibitors of CAAX-mediated posttranslational processing are not expected
108 to be specific for Rheb and may exhibit numerous off-target and potentially deleterious side
effects. For example, as discussed in Chapter 1, the tumor suppressor ARHI is farnesylated
(149). Hence, FTIs may inhibit the tumor suppressive function of ARHI. Thus, if Rheb is proven to be a good target for anti-cancer therapeutics, it will be critical to develop more specific inhibitors of Rheb that do not block other farnesylated proteins.
How can more specific Rheb inhibitors be designed? One possibility is to use the yeast 2-hybrid system to screen for small molecules that disrupt Rheb association with mTOR or PLD-1. Such a screen has been described to identify small molecule inhibitors of
Ras association with its effector Raf-1 (237). However, such small molecule inhibitors may not prevent Rheb association with effectors other than PLD-1 or mTOR. Once a GEF for
Rheb is identified, another possible approach is to find small molecules that inhibit the
exchange activity of this GEF (238, 239), or that prevent Rheb binding to its GEF, similar to the small molecule NSC23766, which inhibits the interaction of Rac with the GEFs Trio and
Tiam1 (240). A third approach may be to block Rheb GTP binding. Such an approach has
not been deemed feasible for most small GTPases due to extremely high affinities for GTP.
Nevertheless, studies from our lab have shown that the small molecule Rac inhibitor EHT
1864 displaces the guanine nucleotide from Rac and inhibits its downstream signaling (241).
Currently, small molecules that displace guanine nucleotides from other small GTPases have
not yet been identified.
Recently, hyperactive Rheb was shown to sensitize cells to apoptosis induced by
proteasome inhibitors due to the inability to form aggresomes (112). Therefore, proteasome
inhibitors or other agents that promote accumulation of misfolded proteins may represent an
indirect way to inhibit the growth of Rheb-dependent tumors. The proteasome inhibitor
109 bortezomib (Velcade®) is FDA-approved for the treatment of multiple myeloma and is
currently in clinical trials in solid tumors in combination with various chemotherapeutics
(www.clinicaltrials.gov). Indeed, bortezomib was shown to preferentially induce apoptosis
in the livers of TSC1-deficient mice (112), suggesting that it may be a useful therapy for
tuberous sclerosis and other tumors harboring hyperactive Rheb. More studies are needed to
determine whether bortezomib blocks Rheb-induced oncogenesis in vivo.
Rheb versus mTOR: which is a better therapeutic target? Since relatively
selective mTORC1 inhibitors already exist and exhibit some clinical benefit (136), is it
practical to pursue Rheb inhibitors for cancer treatment? Several lines of evidence suggest that Rheb inhibition may be clinically relevant and may even be superior to mTOR inhibition under certain circumstances. First, in tuberous sclerosis, Rheb activation is more directly linked to loss of TSC1 or TSC2 function; mTORC1 activation in these cells is most likely a result of Rheb activation. Rapamycin and rapamycin analogs are currently in clinical trials for tuberous sclerosis-associated malignancies, such as angiomyolipoma kidney tumors and the lung disease lymphangioleiomyomatosis (LAM) (242). While such therapy did reduce tumor volume, in some cases up to 50-75%, the tumors did not disappear, and regrowth occurred in the year of follow-up off therapy (242). Furthermore, in one clinical trial, rapamycin treatment did not significantly improve lung function in patients with LAM (242).
Therefore, these conditions are far from cured with mTOR inhibitors.
FTI has been shown to reduce S6 phosphorylation, cell viability, and soft agar growth in TSC1- and TSC2-deficient MEFs (131). Whether FTI also inhibits the growth of tuberous sclerosis patient-derived tumor cell lines, such as the TRI-101 renal angiomyolipoma cell
110 line harboring a loss-of-function mutation in TSC2 (243), should be determined.
Furthermore, to directly compare whether Rheb inhibition would be superior to mTORC1 inhibition in these cells, shRNA could be used to knock down either raptor (to exclusively inhibit mTORC1) or the combination of Rheb1 and Rheb2 in TRI-101 cells, and the tumorigenic potential of these cells could be tested. If Rheb inhibition is found to be superior to mTOR inhibition, and if FTIs inhibit the growth of TRI-101 cells, then perhaps FTIs should be considered for tuberous sclerosis clinical trials.
A second line of evidence suggesting that Rheb inhibitors may be advantageous under certain circumstances is that numerous studies have detected Rheb overexpression or amplification in a variety of cancers (114-118), while similar mTOR aberrations have not been reported. Thus, Rheb inhibitors may be more beneficial in these cancers.
Finally, Rheb may have mTOR-independent functions that contribute to tumorigenesis, such as inhibition of aggresome formation (112). Other as-yet-unidentified downstream effectors of Rheb may also contribute to its oncogenic activity, independently of mTOR. An analogy could be drawn to Ras-induced oncogenesis, where it is thought that multiple downstream effectors contribute to Ras-driven tumor formation (38). Whether Rheb functions in a similar manner has not been determined.
Inhibition of Rheb may also be therapeutically beneficial in diseases other than tuberous sclerosis and cancer. For example, since Rheb inhibits aggresome formation, it may be an attractive therapeutic target for diseases associated with misfolded proteins (112), such as cystic fibrosis and Parkinson’s disease (244). Since this function of Rheb is independent of mTOR, rapamycin analogs would not be expected to be as efficacious as Rheb inhibitors.
111 Does FTS inhibit mTOR-dependent tumor growth? My studies in Chapter 3
found that FTS potently blocked mTOR activation of S6K in cellulo. Future work should be
aimed at whether FTS also inhibits mTOR signaling and mTOR-dependent tumor growth in
vivo. Does FTS impair the growth of TSC2-deficient cells, such as TRI-101
angiomyolipoma cells, in vitro and in vivo? Does FTS, like rapamycin, inhibit Rheb-induced
transformation of chick embryo fibroblasts (114), or Rheb-induced oncogenesis in vivo (113,
118)? Does FTS promote autophagy, like other mTOR inhibitors (245)? It will also be
important to test whether FTS treatment reduces phospho-S6K and phospho-4EBP1 in vivo,
and whether S6K or 4E-BP1 phosphorylation should be used as biomarkers to predict patient
response. However, some tumors with high phospho-S6K levels may harbor S6K
amplification, which has been observed in a subset of breast cancers (246, 247), and would
be predicted to impart resistance to mTOR inhibitors; perhaps the combination of high S6K
and 4E-BP1 phosphorylation would be most predictive. Finally, my studies found that
another farnesyl-containing small molecule, TLN-4601, also blocks mTOR activation of S6K
(Figure 4-4). Thus, TLN-4601 should be included in the above studies to determine whether
it behaves similarly to FTS.
FTS may be particularly efficacious in breast cancer. Santen and colleagues found
that FTS inhibited the growth of hormone-dependent breast cancer cells and reduced antiestrogen resistance (77, 78, 248, 249). It will be important to determine whether, like rapamycin analogs, FTS synergizes with endocrine therapies and HER2 inhibitors in the clinic.
112 A. K-Ras Rheb mTOR Vector G12V N153T E2419K FTS FTS DMSO 4601 4601 DMSO FTS DMSO FTI 4601 4601 FTI FTS FTI DMSO FTI p85 S6K pS6K p70 S6K
β-actin
B. 100 μM FTS 30 μM TLN-4601
Treatment time (h): 0 3 6 16 24 0 3 6 16 24
pS6K
pAKT
pERK
β-actin
Figure 4-4. Inhibition of mTOR signaling by farnesyl-containing small molecules. (A) NIH 3T3 cells were transiently transfected with pcDNA3 vector, or with pcDNA3 encoding HA-tagged K- Ras G12V, FLAG-tagged Rheb1 N153T, or AU1-tagged mTOR E2419K, along with pRK7 HA- tagged S6K1, and serum- and amino acid-starved. Cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies to phospho-S6K or β-actin (loading control). (B) MCF-7 cells growing in complete medium were treated with 100 μM FTS or 30 μM TLN-4601 for 0, 3, 6, 16, or 24 h. Cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies to phospho- S6K (pS6K), phospho-AKT (pAKT), phospho-ERK (pERK), or β-actin (loading control).
113 Comparison of FTS to other mTOR inhibitors. In addition to rapamycin analogs
(“rapalogs”), mTOR kinase inhibitiors (“TORKinibs”) have also entered clinical studies.
Figure 4-5 summarizes the mTOR inhibitors currently under clinical evaluation for cancer
treatment. Unlike rapalogs, TORKinibs inhibit the kinase activity of both mTORC1 and the rapamycin-insensitive mTOR complex mTORC2, which phosphorylates AKT. Recent studies have suggested that mTORC2 may contribute to oncogenesis independently of mTORC1 (250, 251). TORKinibs were also shown to inhibit mTORC1 activity more completely than rapalogs (252). Since FTS did not inhibit AKT phosphorylation at Ser 473
(the site phosphorylated by mTORC2) (Figure 4-4), FTS is not expected to inhibit mTORC2.
Although two rapalogs have been FDA-approved for the treatment of renal cell
carcinoma, the success of rapalogs has been limited due to several factors. First, side effects
associated with treatment with rapalogs include immunosuppression, mucositis, fatigue, and thrombocytopenia (253). Second, the identification of an appropriate patient population that will benefit from mTOR inhibition has been daunting (136). A third problem with mTORC1 inhibition is the elimination of a negative feedback loop which dampens PI3K-AKT signaling (Figure 4-6). Normally, S6K phosphorylates and inactivates IRS-1, resulting in a decrease in AKT phosphorylation and AKT-mediated cell survival signaling (254-256).
Rapalogs prevent this feedback inhibition; hence, patients treated with rapalogs exhibit increased levels of phospho-AKT (255), which promotes tumor cell survival. In addition, treatment with rapalogs also results in increased levels of phospho-ERK by similar elimination of a negative feedback loop (257, 258). These events may promote resistance to rapamycin and its derivatives.
114 BGT226 PTEN Ras EGFR BEZ235 PX-866 XL147 PI3K GDC-0941 mTORC2 P P T308 S473 mTOR PDK1 AKT Rictor mLST8
PTSC2 TSC1 NVP- BEZ235 Lonafarnib Rheb XL765 Tipifarnib
Sirolimus Salirasib mTOR Deforolimus TLN-4601? mTORC1 Raptor mLST8 Everolimus* P Temsirolimus* P S473 4E-BP S6K *FDA-approved eIF4G P S6 eIF4E eIF4B
Translation initiation
Figure 4-5. Inhibitors of the Rheb-mTOR pathway in clinical trials. The primary classes of mTOR inhibitors are rapamycin analogs, or “rapalogs,” which target mTORC1 alone, and mTOR kinase inhibitors, or “TORKinibs,” which target both mTORC1 and mTORC2. Two rapalogs, everolimus and temsirolimus, have recently been FDA-approved for the treatment of kidney cancer. Some TORKinibs also inhibit the structurally related lipid kinase PI3K. My studies in Chapter 3 validated farnesyl-containing small molecules such as salirasib (FTS) as a distinct class of mTOR inhibitors. Adapted from Petroulakis et al., Br J Canc 2006;94:195-9.
115 FTS may overcome some of these feedback problems. If FTS blocks both mTOR and
Ras activity, then FTS would be expected to prevent the upregulation of ERK
phosphorylation in response to mTORC1 inhibition (Figure 4-6). A dual Ras/mTORC1
inhibitor would be particularly attractive and may have broader efficacy than an inhibitor of either mTORC1 or Ras alone.
What are the targets of FTS required for its anti-tumor activity? As with FTI,
determining the key targets of FTS responsible for its anti-tumor activity will be crucial in
order to identify an appropriate patient population that may benefit from FTS therapy.
Identification of the key targets will also provide a reliable biomarker to monitor FTS activity
in order to validate that target inhibition is achieved in the patient. It is conceivable that
inhibition of Ras, mTOR, or perhaps both is responsible for FTS anti-tumor activity.
Alternatively, an as yet unidentified target of FTS may mediate its anti-tumor effects. To
determine whether Ras inhibition contributes to FTS anti-tumor activity, Ras inhibition may
be rescued by expression of myristylated Ras, which is predicted to be resistant to FTS
inhibition (62). If cells expressing myristylated Ras remain sensitive to FTS, this would
suggest that Ras is probably not the most important FTS target. To determine if mTOR
inhibition contributes to FTS activity, overexpression of both mTOR and raptor may prevent
FTS inhibition of mTOR-raptor binding (79), and the sensitivity of these cells to FTS may then be assessed. Another potential approach would be to knock down mTOR or raptor using shRNA and determine whether this synergizes with FTS treatment. If these approaches fail to identify the appropriate target(s) of FTS, then genome-wide shRNA screens may be used to identify genes that, when downregulated, result in synthetic lethality in combination
116 RTK
P P P P PI3K AKT P IRS1 Ras GTP
Raf TSC2P P MEK Rheb GTP ERK P FTS Raptor P Rapamycin mTOR mLST8
P S6K*
Figure 4-6. Feedback regulation of mTOR signaling. The mTOR substrate S6K phosphorylates and inhibits the adaptor protein IRS1, thereby blocking PI3K signaling to AKT and Ras. Repression of S6K activation by rapamycin relieves this negative regulation, leading to increased levels of AKT and ERK phosphorylation in rapamycin-treated tumors. FTS may prevent upregulation of ERK upon mTORC1 inhibition by also blocking Ras signaling. Adapted from Carracedo et al., J Clin Invest 2008;118:3065-74.
117 with FTS treatment (259). These experiments will assist in determining which patients will
benefit most from FTS therapy.
Summary. Although protein kinase inhibitors continue to be the favored avenue for
anti-cancer drug discovery, it is also acknowledged that this pipeline is finite and that we
may soon reach the limits of this approach. Therefore, there is considerable discussion and
debate in the pharmaceutical industry regarding what the next “big” classes of molecules will
be for targeted therapeutics. One such class of molecules is the Ras superfamily of small
GTPases. While my studies have added to the knowledge of the function of Ras family small
GTPases in breast cancer, much remains to be determined. These studies argue against a role for Rerg as a tumor suppressor in breast cancer, but future studies should determine whether
Rerg is involved in breast or other cancers in vivo. Although Rheb has also been implicated in breast cancer, the precise role Rheb plays in breast cancer progression remains to be defined. I investigated several strategies to block Rheb signaling in cancer and found that
FTS potently blocks mTOR activation. Since FTS is currently undergoing clinical
evaluation, this work has significant implications for the selection of patients that may
benefit from FTS. Much more regarding the functions of Ras family small GTPases in breast
cancer biology remains to be discovered, but such studies will likely lead to improved breast
cancer therapeutics and patient treatment.
118 REFERENCES
1. Peppercorn J, Perou CM, Carey LA. Molecular subtypes in breast cancer evaluation and management: divide and conquer. Cancer Invest 2008;26(1):1-10.
2. Sorlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H, Hastie T, Eisen MB, van de Rijn M, Jeffrey SS, Thorsen T, Quist H, Matese JC, Brown PO, Botstein D, Eystein Lonning P, Borresen-Dale AL. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci U S A 2001;98(19):10869-74.
3. Perou CM, Sorlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees CA, Pollack JR, Ross DT, Johnsen H, Akslen LA, Fluge O, Pergamenschikov A, Williams C, Zhu SX, Lonning PE, Borresen-Dale AL, Brown PO, Botstein D. Molecular portraits of human breast tumours. Nature 2000;406(6797):747-52.
4. Ali S, Coombes RC. Endocrine-responsive breast cancer and strategies for combating resistance. Nat Rev Cancer 2002;2(2):101-12.
5. Johnston SR, Dowsett M. Aromatase inhibitors for breast cancer: lessons from the laboratory. Nat Rev Cancer 2003;3(11):821-31.
6. Massarweh S, Schiff R. Unraveling the mechanisms of endocrine resistance in breast cancer: new therapeutic opportunities. Clin Cancer Res 2007;13(7):1950-4.
7. Rochefort H, Glondu M, Sahla ME, Platet N, Garcia M. How to target estrogen receptor-negative breast cancer? Endocr Relat Cancer 2003;10(2):261-6.
8. Nahta R, Esteva FJ. Trastuzumab: triumphs and tribulations. Oncogene 2007;26(25):3637-43.
9. Chen FL, Xia W, Spector NL. Acquired resistance to small molecule ErbB2 tyrosine kinase inhibitors. Clin Cancer Res 2008;14(21):6730-4.
10. Browne BC, O'Brien N, Duffy MJ, Crown J, O'Donovan N. HER-2 signaling and inhibition in breast cancer. Curr Cancer Drug Targets 2009;9(3):419-38.
11. Rexer BN, Engelman JA, Arteaga CL. Overcoming resistance to tyrosine kinase inhibitors: lessons learned from cancer cells treated with EGFR antagonists. Cell Cycle 2009;8(1):18-22.
12. Silva CM, Shupnik MA. Integration of steroid and growth factor pathways in breast cancer: focus on signal transducers and activators of transcription and their potential role in resistance. Mol Endocrinol 2007;21(7):1499-512.
13. Levin ER, Pietras RJ. Estrogen receptors outside the nucleus in breast cancer. Breast Cancer Res Treat 2008;108(3):351-61.
119 14. Campbell RA, Bhat-Nakshatri P, Patel NM, Constantinidou D, Ali S, Nakshatri H. Phosphatidylinositol 3-kinase/AKT-mediated activation of estrogen receptor alpha: a new model for anti-estrogen resistance. J Biol Chem 2001;276(13):9817-24.
15. Kato S, Endoh H, Masuhiro Y, Kitamoto T, Uchiyama S, Sasaki H, Masushige S, Gotoh Y, Nishida E, Kawashima H, Metzger D, Chambon P. Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase. Science 1995;270(5241):1491-4.
16. Martin MB, Franke TF, Stoica GE, Chambon P, Katzenellenbogen BS, Stoica BA, McLemore MS, Olivo SE, Stoica A. A role for Akt in mediating the estrogenic functions of epidermal growth factor and insulin-like growth factor I. Endocrinology 2000;141(12):4503-11.
17. Yamnik RL, Digilova A, Davis DC, Brodt ZN, Murphy CJ, Holz MK. S6 kinase 1 regulates estrogen receptor alpha in control of breast cancer cell proliferation. J Biol Chem 2009;284(10):6361-9.
18. Simoncini T, Hafezi-Moghadam A, Brazil DP, Ley K, Chin WW, Liao JK. Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol- 3-OH kinase. Nature 2000;407(6803):538-41.
19. Massarweh S, Schiff R. Resistance to endocrine therapy in breast cancer: exploiting estrogen receptor/growth factor signaling crosstalk. Endocr Relat Cancer 2006;13 Suppl 1:S15-24.
20. Normanno N, Di Maio M, De Maio E, De Luca A, de Matteis A, Giordano A, Perrone F. Mechanisms of endocrine resistance and novel therapeutic strategies in breast cancer. Endocr Relat Cancer 2005;12(4):721-47.
21. Wennerberg K, Rossman KL, Der CJ. The Ras superfamily at a glance. J Cell Sci 2005;118(Pt 5):843-6.
22. Vetter IR, Wittinghofer A. The guanine nucleotide-binding switch in three dimensions. Science 2001;294(5545):1299-304.
23. Karnoub AE, Weinberg RA. Ras oncogenes: split personalities. Nat Rev Mol Cell Biol 2008;9(7):517-31.
24. Cox AD, Der CJ. Ras family signaling: therapeutic targeting. Cancer Biol Ther 2002;1(6):599-606.
25. Konstantinopoulos PA, Karamouzis MV, Papavassiliou AG. Post-translational modifications and regulation of the RAS superfamily of GTPases as anticancer targets. Nat Rev Drug Discov 2007;6(7):541-55.
26. Winter-Vann AM, Casey PJ. Post-prenylation-processing enzymes as new targets in oncogenesis. Nat Rev Cancer 2005;5(5):405-12.
120 27. Sebti SM, Der CJ. Opinion: Searching for the elusive targets of farnesyltransferase inhibitors. Nat Rev Cancer 2003;3(12):945-51.
28. Jones S, Zhang X, Parsons DW, Lin JC, Leary RJ, Angenendt P, Mankoo P, Carter H, Kamiyama H, Jimeno A, Hong SM, Fu B, Lin MT, Calhoun ES, Kamiyama M, Walter K, Nikolskaya T, Nikolsky Y, Hartigan J, Smith DR, Hidalgo M, Leach SD, Klein AP, Jaffee EM, Goggins M, Maitra A, Iacobuzio-Donahue C, Eshleman JR, Kern SE, Hruban RH, Karchin R, Papadopoulos N, Parmigiani G, Vogelstein B, Velculescu VE, Kinzler KW. Core signaling pathways in human pancreatic cancers revealed by global genomic analyses. Science 2008;321(5897):1801-6.
29. Eckert LB, Repasky GA, Ulku AS, McFall A, Zhou H, Sartor CI, Der CJ. Involvement of Ras activation in human breast cancer cell signaling, invasion, and anoikis. Cancer Res 2004;64(13):4585-92.
30. von Lintig FC, Dreilinger AD, Varki NM, Wallace AM, Casteel DE, Boss GR. Ras activation in human breast cancer. Breast Cancer Res Treat 2000;62(1):51-62.
31. Dimri G, Band H, Band V. Mammary epithelial cell transformation: insights from cell culture and mouse models. Breast Cancer Res 2005;7(4):171-9.
32. Kasid A, Lippman ME, Papageorge AG, Lowy DR, Gelmann EP. Transfection of v- rasH DNA into MCF-7 human breast cancer cells bypasses dependence on estrogen for tumorigenicity. Science 1985;228(4700):725-8.
33. Kasid A, Lippman ME. Estrogen and oncogene mediated growth regulation of human breast cancer cells. J Steroid Biochem 1987;27(1-3):465-70.
34. Sommers CL, Papageorge A, Wilding G, Gelmann EP. Growth properties and tumorigenesis of MCF-7 cells transfected with isogenic mutants of rasH. Cancer Res 1990;50(1):67-71.
35. Sharif S, Moran A, Huson SM, Iddenden R, Shenton A, Howard E, Evans DG. Women with neurofibromatosis 1 are at a moderately increased risk of developing breast cancer and should be considered for early screening. J Med Genet 2007;44(8):481-4.
36. Guran S, Safali M. A case of neurofibromatosis and breast cancer: loss of heterozygosity of NF1 in breast cancer. Cancer Genet Cytogenet 2005;156(1):86-8.
37. Yu F, Yao H, Zhu P, Zhang X, Pan Q, Gong C, Huang Y, Hu X, Su F, Lieberman J, Song E. let-7 Regulates Self Renewal and Tumorigenicity of Breast Cancer Cells. Cell 2007;131(6):1109-23.
38. Repasky GA, Chenette EJ, Der CJ. Renewing the conspiracy theory debate: does Raf function alone to mediate Ras oncogenesis? Trends Cell Biol 2004;14(11):639-47.
121 39. Samuels Y, Ericson K. Oncogenic PI3K and its role in cancer. Curr Opin Oncol 2006;18(1):77-82.
40. Hollestelle A, Elstrodt F, Nagel JH, Kallemeijn WW, Schutte M. Phosphatidylinositol-3-OH Kinase or RAS Pathway Mutations in Human Breast Cancer Cell Lines. Mol Cancer Res 2007;5(2):195-201.
41. van der Weyden L, Adams DJ. The Ras-association domain family (RASSF) members and their role in human tumourigenesis. Biochim Biophys Acta 2007;1776(1):58-85.
42. Donninger H, Vos MD, Clark GJ. The RASSF1A tumor suppressor. J Cell Sci 2007;120(Pt 18):3163-72.
43. Shinozaki M, Hoon DS, Giuliano AE, Hansen NM, Wang HJ, Turner R, Taback B. Distinct hypermethylation profile of primary breast cancer is associated with sentinel lymph node metastasis. Clin Cancer Res 2005;11(6):2156-62.
44. Yeo W, Wong WL, Wong N, Law BK, Tse GM, Zhong S. High frequency of promoter hypermethylation of RASSF1A in tumorous and non-tumourous tissue of breast cancer. Pathology 2005;37(2):125-30.
45. Hu H, Bliss JM, Wang Y, Colicelli J. RIN1 is an ABL tyrosine kinase activator and a regulator of epithelial-cell adhesion and migration. Curr Biol 2005;15(9):815-23.
46. Milstein M, Mooser CK, Hu H, Fejzo M, Slamon D, Goodglick L, Dry S, Colicelli J. RIN1 is a breast tumor suppressor gene. Cancer Res 2007;67(24):11510-6.
47. Roberts PJ, Der CJ. Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer. Oncogene 2007;26(22):3291-310.
48. Yeh JJ, Der CJ. Targeting signal transduction in pancreatic cancer treatment. Expert Opin Ther Targets 2007;11(5):673-94.
49. Jaiswal BS, Janakiraman V, Kljavin NM, Eastham-Anderson J, Cupp JE, Liang Y, Davis DP, Hoeflich KP, Seshagiri S. Combined targeting of BRAF and CRAF or BRAF and PI3K effector pathways is required for efficacy in NRAS mutant tumors. PLoS ONE 2009;4(5):e5717.
50. Cox AD, Der CJ. Farnesyltransferase inhibitors: promises and realities. Curr Opin Pharmacol 2002;2(4):388-93.
51. Kelland LR, Smith V, Valenti M, Patterson L, Clarke PA, Detre S, End D, Howes AJ, Dowsett M, Workman P, Johnston SR. Preclinical antitumor activity and pharmacodynamic studies with the farnesyl protein transferase inhibitor R115777 in human breast cancer. Clin Cancer Res 2001;7(11):3544-50.
122 52. Reid TS, Terry KL, Casey PJ, Beese LS. Crystallographic analysis of CaaX prenyltransferases complexed with substrates defines rules of protein substrate selectivity. J Mol Biol 2004;343(2):417-33.
53. Johnston SR. Targeting downstream effectors of epidermal growth factor receptor/HER2 in breast cancer with either farnesyltransferase inhibitors or mTOR antagonists. Int J Gynecol Cancer 2006;16 Suppl 2:543-8.
54. O'Regan RM, Khuri FR. Farnesyl transferase inhibitors: the next targeted therapies for breast cancer? Endocr Relat Cancer 2004;11(2):191-205.
55. Kato K, Cox AD, Hisaka MM, Graham SM, Buss JE, Der CJ. Isoprenoid addition to Ras protein is the critical modification for its membrane association and transforming activity. Proc Natl Acad Sci U S A 1992;89(14):6403-7.
56. Wahlstrom AM, Cutts BA, Liu M, Lindskog A, Karlsson C, Sjogren AK, Andersson KM, Young SG, Bergo MO. Inactivating Icmt ameliorates K-RAS-induced myeloproliferative disease. Blood 2008;112(4):1357-65.
57. Bergo MO, Ambroziak P, Gregory C, George A, Otto JC, Kim E, Nagase H, Casey PJ, Balmain A, Young SG. Absence of the CAAX endoprotease Rce1: effects on cell growth and transformation. Mol Cell Biol 2002;22(1):171-81.
58. Wahlstrom AM, Cutts BA, Karlsson C, Andersson KM, Liu M, Sjogren AK, Swolin B, Young SG, Bergo MO. Rce1 deficiency accelerates the development of K-RAS- induced myeloproliferative disease. Blood 2007;109(2):763-8.
59. Winter-Vann AM, Baron RA, Wong W, dela Cruz J, York JD, Gooden DM, Bergo MO, Young SG, Toone EJ, Casey PJ. A small-molecule inhibitor of isoprenylcysteine carboxyl methyltransferase with antitumor activity in cancer cells. Proc Natl Acad Sci U S A 2005;102(12):4336-41.
60. Wang M, Tan W, Zhou J, Leow J, Go M, Lee HS, Casey PJ. A small molecule inhibitor of isoprenylcysteine carboxymethyltransferase induces autophagic cell death in PC3 prostate cancer cells. J Biol Chem 2008;283(27):18678-84.
61. Blum R, Cox AD, Kloog Y. Inhibitors of chronically active ras: potential for treatment of human malignancies. Recent Patents Anticancer Drug Discov 2008;3(1):31-7.
62. Haklai R, Weisz MG, Elad G, Paz A, Marciano D, Egozi Y, Ben-Baruch G, Kloog Y. Dislodgment and accelerated degradation of Ras. Biochemistry 1998;37(5):1306-14.
63. Paz A, Haklai R, Elad-Sfadia G, Ballan E, Kloog Y. Galectin-1 binds oncogenic H- Ras to mediate Ras membrane anchorage and cell transformation. Oncogene 2001;20(51):7486-93.
123 64. Gana-Weisz M, Haklai R, Marciano D, Egozi Y, Ben-Baruch G, Kloog Y. The Ras antagonist S-farnesylthiosalicylic acid induces inhibition of MAPK activation. Biochem Biophys Res Commun 1997;239(3):900-4.
65. Marom M, Haklai R, Ben-Baruch G, Marciano D, Egozi Y, Kloog Y. Selective inhibition of Ras-dependent cell growth by farnesylthiosalisylic acid. J Biol Chem 1995;270(38):22263-70.
66. Egozi Y, Weisz B, Gana-Weisz M, Ben-Baruch G, Kloog Y. Growth inhibition of ras-dependent tumors in nude mice by a potent ras-dislodging antagonist. Int J Cancer 1999;80(6):911-8.
67. Beiner ME, Niv H, Haklai R, Elad-Sfadia G, Kloog Y, Ben-Baruch G. Ras antagonist inhibits growth and chemosensitizes human epithelial ovarian cancer cells. Int J Gynecol Cancer 2006;16 Suppl 1:200-6.
68. Barkan B, Starinsky S, Friedman E, Stein R, Kloog Y. The Ras inhibitor farnesylthiosalicylic acid as a potential therapy for neurofibromatosis type 1. Clin Cancer Res 2006;12(18):5533-42.
69. Halaschek-Wiener J, Kloog Y, Wacheck V, Jansen B. Farnesyl thiosalicylic acid chemosensitizes human melanoma in vivo. J Invest Dermatol 2003;120(1):109-15.
70. Gana-Weisz M, Halaschek-Wiener J, Jansen B, Elad G, Haklai R, Kloog Y. The Ras inhibitor S-trans,trans-farnesylthiosalicylic acid chemosensitizes human tumor cells without causing resistance. Clin Cancer Res 2002;8(2):555-65.
71. Jansen B, Heere-Ress E, Schlagbauer-Wadl H, Halaschek-Wiener J, Waltering S, Moll I, Pehamberger H, Marciano D, Kloog Y, Wolff K. Farnesylthiosalicylic acid inhibits the growth of human Merkel cell carcinoma in SCID mice. J Mol Med 1999;77(11):792-7.
72. Jansen B, Schlagbauer-Wadl H, Kahr H, Heere-Ress E, Mayer BX, Eichler H, Pehamberger H, Gana-Weisz M, Ben-David E, Kloog Y, Wolff K. Novel Ras antagonist blocks human melanoma growth. Proc Natl Acad Sci U S A 1999;96(24):14019-24.
73. Weisz B, Giehl K, Gana-Weisz M, Egozi Y, Ben-Baruch G, Marciano D, Gierschik P, Kloog Y. A new functional Ras antagonist inhibits human pancreatic tumor growth in nude mice. Oncogene 1999;18(16):2579-88.
74. Zundelevich A, Elad-Sfadia G, Haklai R, Kloog Y. Suppression of lung cancer tumor growth in a nude mouse model by the Ras inhibitor salirasib (farnesylthiosalicylic acid). Mol Cancer Ther 2007;6(6):1765-73.
75. Tsimberidou AM, Rudek MA, Hong D, Ng CS, Blair J, Goldsweig H, Kurzrock R. Phase 1 first-in-human clinical study of S-trans, trans-farnesylthiosalicylic acid (salirasib) in patients with solid tumors. Cancer Chemother Pharmacol 2009.
124 76. Laheru D RM, Taylor G, Goldsweig H, Rajeshkumar NF, Linden S, Angenendt M, Le D, Donehower R, Jimeno A, Hidalgo M. Integrated development of s-trans, trans- farnesylthiosalicyclic acid (FTS, salirasib) in advanced pancreatic cancer. In: 2009 American Society of Clinical Oncology Annual Meeting; 2009 29 May - 2 Jun 2009; Orlando, FL: J Clin Oncol; 2009.
77. Yue W, Fan P, Wang J, Li Y, Santen RJ. Mechanisms of acquired resistance to endocrine therapy in hormone-dependent breast cancer cells. J Steroid Biochem Mol Biol 2007;106(1-5):102-10.
78. Yue W, Wang J, Li Y, Fan P, Santen RJ. Farnesylthiosalicylic acid blocks mammalian target of rapamycin signaling in breast cancer cells. Int J Cancer 2005;117(5):746-54.
79. McMahon LP, Yue W, Santen RJ, Lawrence JC, Jr. Farnesylthiosalicylic acid inhibits mammalian target of rapamycin (mTOR) activity both in cells and in vitro by promoting dissociation of the mTOR-raptor complex. Mol Endocrinol 2005;19(1):175-83.
80. Goldberg L, Haklai R, Bauer V, Heiss A, Kloog Y. New derivatives of farnesylthiosalicylic acid (salirasib) for cancer treatment: farnesylthiosalicylamide inhibits tumor growth in nude mice models. J Med Chem 2009;52(1):197-205.
81. Gourdeau H, McAlpine JB, Ranger M, Simard B, Berger F, Beaudry F, Farnet CM, Falardeau P. Identification, characterization and potent antitumor activity of ECO- 4601, a novel peripheral benzodiazepine receptor ligand. Cancer Chemother Pharmacol 2008;61(6):911-21.
82. Han Z, Slack RS, Li W, Papadopoulos V. Expression of peripheral benzodiazepine receptor (PBR) in human tumors: relationship to breast, colorectal, and prostate tumor progression. J Recept Signal Transduct Res 2003;23(2-3):225-38.
83. Aspuria PJ, Tamanoi F. The Rheb family of GTP-binding proteins. Cell Signal 2004;16(10):1105-12.
84. Arsham AM, Neufeld TP. Thinking globally and acting locally with TOR. Curr Opin Cell Biol 2006;18(6):589-97.
85. Inoki K, Li Y, Xu T, Guan KL. Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling. Genes Dev 2003;17(15):1829-34.
86. Castro AF, Rebhun JF, Clark GJ, Quilliam LA. Rheb binds tuberous sclerosis complex 2 (TSC2) and promotes S6 kinase activation in a rapamycin- and farnesylation-dependent manner. J Biol Chem 2003;278(35):32493-6.
87. Inoki K, Corradetti MN, Guan KL. Dysregulation of the TSC-mTOR pathway in human disease. Nat Genet 2005;37(1):19-24.
125 88. Astrinidis A, Henske EP. Tuberous sclerosis complex: linking growth and energy signaling pathways with human disease. Oncogene 2005;24(50):7475-81.
89. Huang J, Manning BD. A complex interplay between Akt, TSC2 and the two mTOR complexes. Biochem Soc Trans 2009;37(Pt 1):217-22.
90. Long X, Lin Y, Ortiz-Vega S, Yonezawa K, Avruch J. Rheb binds and regulates the mTOR kinase. Curr Biol 2005;15(8):702-13.
91. Sun Y, Fang Y, Yoon MS, Zhang C, Roccio M, Zwartkruis FJ, Armstrong M, Brown HA, Chen J. Phospholipase D1 is an effector of Rheb in the mTOR pathway. Proc Natl Acad Sci U S A 2008;105(24):8286-91.
92. Ma D, Bai X, Guo S, Jiang Y. The switch I region of Rheb is critical for its interaction with FKBP38. J Biol Chem 2008;283(38):25963-70.
93. Dunlop EA, Dodd KM, Seymour LA, Tee AR. Mammalian target of rapamycin complex 1-mediated phosphorylation of eukaryotic initiation factor 4E-binding protein 1 requires multiple protein-protein interactions for substrate recognition. Cell Signal 2009;21(7):1073-84.
94. Bai X, Ma D, Liu A, Shen X, Wang QJ, Liu Y, Jiang Y. Rheb activates mTOR by antagonizing its endogenous inhibitor, FKBP38. Science 2007;318(5852):977-80.
95. Wang X, Fonseca BD, Tang H, Liu R, Elia A, Clemens MJ, Bommer UA, Proud CG. Re-evaluating the roles of proposed modulators of mammalian target of rapamycin complex 1 (mTORC1) signaling. J Biol Chem 2008;283(45):30482-92.
96. Sato T, Nakashima A, Guo L, Tamanoi F. Specific Activation of mTORC1 by Rheb G-protein in Vitro Involves Enhanced Recruitment of Its Substrate Protein. J Biol Chem 2009;284(19):12783-91.
97. Uhlenbrock K, Weiwad M, Wetzker R, Fischer G, Wittinghofer A, Rubio I. Reassessment of the role of FKBP38 in the Rheb/mTORC1 pathway. FEBS Lett 2009;583(6):965-70.
98. Averous J, Proud CG. When translation meets transformation: the mTOR story. Oncogene 2006;25(48):6423-35.
99. Hsu YC, Chern JJ, Cai Y, Liu M, Choi KW. Drosophila TCTP is essential for growth and proliferation through regulation of dRheb GTPase. Nature 2007;445(7129):785-8.
100. Rehmann H, Bruning M, Berghaus C, Schwarten M, Kohler K, Stocker H, Stoll R, Zwartkruis FJ, Wittinghofer A. Biochemical characterisation of TCTP questions its function as a guanine nucleotide exchange factor for Rheb. FEBS Lett 2008;582(20):3005-10.
126 101. Lee MN, Ha SH, Kim J, Koh A, Lee CS, Kim JH, Jeon H, Kim DH, Suh PG, Ryu SH. Glycolytic flux signals to mTOR through glyceraldehyde-3-phosphate dehydrogenase-mediated regulation of Rheb. Mol Cell Biol 2009;29(14):3991-4001.
102. Li Y, Wang Y, Kim E, Beemiller P, Wang CY, Swanson J, You M, Guan KL. Bnip3 mediates the hypoxia-induced inhibition on mammalian target of rapamycin by interacting with Rheb. J Biol Chem 2007;282(49):35803-13.
103. Sancak Y, Peterson TR, Shaul YD, Lindquist RA, Thoreen CC, Bar-Peled L, Sabatini DM. The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science 2008;320(5882):1496-501.
104. Sancak Y, Sabatini DM. Rag proteins regulate amino-acid-induced mTORC1 signalling. Biochem Soc Trans 2009;37(Pt 1):289-90.
105. Kim E, Goraksha-Hicks P, Li L, Neufeld TP, Guan KL. Regulation of TORC1 by Rag GTPases in nutrient response. Nat Cell Biol 2008;10(8):935-45.
106. Maehama T, Tanaka M, Nishina H, Murakami M, Kanaho Y, Hanada K. RalA functions as an indispensable signal mediator for the nutrient-sensing system. J Biol Chem 2008;283(50):35053-9.
107. Clark GJ, Kinch MS, Rogers-Graham K, Sebti SM, Hamilton AD, Der CJ. The Ras- related protein Rheb is farnesylated and antagonizes Ras signaling and transformation. J Biol Chem 1997;272(16):10608-15.
108. Karbowniczek M, Robertson GP, Henske EP. Rheb inhibits C-raf activity and B- raf/C-raf heterodimerization. J Biol Chem 2006;281(35):25447-56.
109. Karbowniczek M, Cash T, Cheung M, Robertson GP, Astrinidis A, Henske EP. Regulation of B-Raf kinase activity by tuberin and Rheb is mammalian target of rapamycin (mTOR)-independent. J Biol Chem 2004;279(29):29930-7.
110. Im E, von Lintig FC, Chen J, Zhuang S, Qui W, Chowdhury S, Worley PF, Boss GR, Pilz RB. Rheb is in a high activation state and inhibits B-Raf kinase in mammalian cells. Oncogene 2002;21(41):6356-65.
111. Yee WM, Worley PF. Rheb interacts with Raf-1 kinase and may function to integrate growth factor- and protein kinase A-dependent signals. Mol Cell Biol 1997;17(2):921-33.
112. Zhou X, Ikenoue T, Chen X, Li L, Inoki K, Guan KL. Rheb controls misfolded protein metabolism by inhibiting aggresome formation and autophagy. Proc Natl Acad Sci U S A 2009;106(22):8923-8.
113. Mavrakis KJ, Zhu H, Silva RL, Mills JR, Teruya-Feldstein J, Lowe SW, Tam W, Pelletier J, Wendel HG. Tumorigenic activity and therapeutic inhibition of Rheb GTPase. Genes Dev 2008;22(16):2178-88.
127 114. Jiang H, Vogt PK. Constitutively active Rheb induces oncogenic transformation. Oncogene 2008;27(43):5729-40.
115. Basso AD, Mirza A, Liu G, Long BJ, Bishop WR, Kirschmeier P. The farnesyl transferase inhibitor (FTI) SCH66336 (lonafarnib) inhibits Rheb farnesylation and mTOR signaling. Role in FTI enhancement of taxane and tamoxifen anti-tumor activity. J Biol Chem 2005;280(35):31101-8.
116. Gromov PS, Madsen P, Tomerup N, Celis JE. A novel approach for expression cloning of small GTPases: identification, tissue distribution and chromosome mapping of the human homolog of rheb. FEBS Lett 1995;377(2):221-6.
117. Chakraborty S, Mohiyuddin SA, Gopinath KS, Kumar A. Involvement of TSC genes and differential expression of other members of the mTOR signaling pathway in oral squamous cell carcinoma. BMC Cancer 2008;8(1):163.
118. Nardella C, Chen Z, Salmena L, Carracedo A, Alimonti A, Egia A, Carver B, Gerald W, Cordon-Cardo C, Pandolfi PP. Aberrant Rheb-mediated mTORC1 activation and Pten haploinsufficiency are cooperative oncogenic events. Genes Dev 2008;22(16):2172-7.
119. Wood LD, Parsons DW, Jones S, Lin J, Sjoblom T, Leary RJ, Shen D, Boca SM, Barber T, Ptak J, Silliman N, Szabo S, Dezso Z, Ustyanksky V, Nikolskaya T, Nikolsky Y, Karchin R, Wilson PA, Kaminker JS, Zhang Z, Croshaw R, Willis J, Dawson D, Shipitsin M, Willson JK, Sukumar S, Polyak K, Park BH, Pethiyagoda CL, Pant PV, Ballinger DG, Sparks AB, Hartigan J, Smith DR, Suh E, Papadopoulos N, Buckhaults P, Markowitz SD, Parmigiani G, Kinzler KW, Velculescu VE, Vogelstein B. The genomic landscapes of human breast and colorectal cancers. Science 2007;318(5853):1108-13.
120. Schewe DM, Aguirre-Ghiso JA. ATF6alpha-Rheb-mTOR signaling promotes survival of dormant tumor cells in vivo. Proc Natl Acad Sci U S A 2008;105(30):10519-24.
121. Jiang WG, Sampson J, Martin TA, Lee-Jones L, Watkins G, Douglas-Jones A, Mokbel K, Mansel RE. Tuberin and hamartin are aberrantly expressed and linked to clinical outcome in human breast cancer: the role of promoter methylation of TSC genes. Eur J Cancer 2005;41(11):1628-36.
122. Cully M, You H, Levine AJ, Mak TW. Beyond PTEN mutations: the PI3K pathway as an integrator of multiple inputs during tumorigenesis. Nat Rev Cancer 2006;6(3):184-92.
123. Ma L, Chen Z, Erdjument-Bromage H, Tempst P, Pandolfi PP. Phosphorylation and functional inactivation of TSC2 by Erk implications for tuberous sclerosis and cancer pathogenesis. Cell 2005;121(2):179-93.
128 124. Roux PP, Ballif BA, Anjum R, Gygi SP, Blenis J. Tumor-promoting phorbol esters and activated Ras inactivate the tuberous sclerosis tumor suppressor complex via p90 ribosomal S6 kinase. Proc Natl Acad Sci U S A 2004;101(37):13489-94.
125. Stoica GE, Franke TF, Wellstein A, Czubayko F, List HJ, Reiter R, Morgan E, Martin MB, Stoica A. Estradiol rapidly activates Akt via the ErbB2 signaling pathway. Mol Endocrinol 2003;17(5):818-30.
126. Yu J, Henske EP. Estrogen-Induced Activation of Mammalian Target of Rapamycin Is Mediated via Tuberin and the Small GTPase Ras Homologue Enriched in Brain. Cancer Res 2006;66(19):9461-6.
127. Boulay A, Rudloff J, Ye J, Zumstein-Mecker S, O'Reilly T, Evans DB, Chen S, Lane HA. Dual inhibition of mTOR and estrogen receptor signaling in vitro induces cell death in models of breast cancer. Clin Cancer Res 2005;11(14):5319-28.
128. deGraffenried LA, Friedrichs WE, Russell DH, Donzis EJ, Middleton AK, Silva JM, Roth RA, Hidalgo M. Inhibition of mTOR activity restores tamoxifen response in breast cancer cells with aberrant Akt Activity. Clin Cancer Res 2004;10(23):8059-67.
129. Beeram M, Tan QT, Tekmal R, Russell D, Middleton A, Degraffenried L. Akt- induced endocrine therapy resistance is reversed by inhibition of mTOR signaling. Ann Oncol 2007;18(8):1323-1328.
130. Buerger C, DeVries B, Stambolic V. Localization of Rheb to the endomembrane is critical for its signaling function. Biochem Biophys Res Commun 2006;344(3):869- 80.
131. Gau CL, Kato-Stankiewicz J, Jiang C, Miyamoto S, Guo L, Tamanoi F. Farnesyltransferase inhibitors reverse altered growth and distribution of actin filaments in Tsc-deficient cells via inhibition of both rapamycin-sensitive and - insensitive pathways. Mol Cancer Ther 2005;4(6):918-26.
132. Urano J, Tabancay AP, Yang W, Tamanoi F. The Saccharomyces cerevisiae Rheb G- protein is involved in regulating canavanine resistance and arginine uptake. J Biol Chem 2000;275(15):11198-206.
133. Head J, Johnston SR. New targets for therapy in breast cancer: farnesyltransferase inhibitors. Breast Cancer Res 2004;6(6):262-8.
134. Johnston SR, Semiglazov VF, Manikhas GM, Spaeth D, Romieu G, Dodwell DJ, Wardley AM, Neven P, Bessems A, Park YC, De Porre PM, Perez Ruixo JJ, Howes AJ. A phase II, randomized, blinded study of the farnesyltransferase inhibitor tipifarnib combined with letrozole in the treatment of advanced breast cancer after antiestrogen therapy. Breast Cancer Res Treat 2008;110(2):327-35.
129 135. Petroulakis E, Mamane Y, Le Bacquer O, Shahbazian D, Sonenberg N. mTOR signaling: implications for cancer and anticancer therapy. Br J Cancer 2006;94(2):195-9.
136. Brachmann S, Fritsch C, Maira SM, Garcia-Echeverria C. PI3K and mTOR inhibitors: a new generation of targeted anticancer agents. Curr Opin Cell Biol 2009;21(2):194-8.
137. Johnston SR. Clinical efforts to combine endocrine agents with targeted therapies against epidermal growth factor receptor/human epidermal growth factor receptor 2 and mammalian target of rapamycin in breast cancer. Clin Cancer Res 2006;12(3 Pt 2):1061s-1068s.
138. Widakowich C, Dinh P, de Azambuja E, Awada A, Piccart-Gebhart M. HER-2 positive breast cancer: what else beyond trastuzumab-based therapy? Anticancer Agents Med Chem 2008;8(5):488-96.
139. Santen RJ, Song RX, Zhang Z, Kumar R, Jeng MH, Masamura A, Lawrence J, Jr., Berstein L, Yue W. Long-term estradiol deprivation in breast cancer cells up- regulates growth factor signaling and enhances estrogen sensitivity. Endocr Relat Cancer 2005;12 Suppl 1:S61-73.
140. Yu Y, Xu F, Peng H, Fang X, Zhao S, Li Y, Cuevas B, Kuo WL, Gray JW, Siciliano M, Mills GB, Bast RC, Jr. NOEY2 (ARHI), an imprinted putative tumor suppressor gene in ovarian and breast carcinomas. Proc Natl Acad Sci U S A 1999;96(1):214-9.
141. Lu Z, Luo RZ, Peng H, Huang M, Nishmoto A, Hunt KK, Helin K, Liao WS, Yu Y. E2F-HDAC complexes negatively regulate the tumor suppressor gene ARHI in breast cancer. Oncogene 2006;25(2):230-9.
142. Yuan J, Luo RZ, Fujii S, Wang L, Hu W, Andreeff M, Pan Y, Kadota M, Oshimura M, Sahin AA, Issa JP, Bast RC, Jr., Yu Y. Aberrant methylation and silencing of ARHI, an imprinted tumor suppressor gene in which the function is lost in breast cancers. Cancer Res 2003;63(14):4174-80.
143. Feng W, Lu Z, Luo RZ, Zhang X, Seto E, Liao WS, Yu Y. Multiple histone deacetylases repress tumor suppressor gene ARHI in breast cancer. Int J Cancer 2007;120(8):1664-8.
144. Wang L, Hoque A, Luo RZ, Yuan J, Lu Z, Nishimoto A, Liu J, Sahin AA, Lippman SM, Bast RC, Jr., Yu Y. Loss of the expression of the tumor suppressor gene ARHI is associated with progression of breast cancer. Clin Cancer Res 2003;9(10 Pt 1):3660- 6.
145. Shi Z, Zhou X, Xu L, Zhang T, Hou Y, Zhu W, Zhang T. [NOEY2 gene mRNA expression in breast cancer tissue and its relation to clinicopathological parameters]. Zhonghua Zhong Liu Za Zhi 2002;24(5):475-8.
130 146. Yang J, Hu A, Wang L, Li B, Chen Y, Zhao W, Xu W, Li T. NOEY2 mutations in primary breast cancers and breast hyperplasia. Breast 2009;18(3):197-203.
147. Yu Y, Luo R, Lu Z, Wei Feng W, Badgwell D, Issa JP, Rosen DG, Liu J, Bast RC, Jr. Biochemistry and Biology of ARHI (DIRAS3), an Imprinted Tumor Suppressor Gene Whose Expression Is Lost in Ovarian and Breast Cancers. Methods Enzymol 2005;407:455-68.
148. Bao JJ, Le XF, Wang RY, Yuan J, Wang L, Atkinson EN, LaPushin R, Andreeff M, Fang B, Yu Y, Bast RC, Jr. Reexpression of the tumor suppressor gene ARHI induces apoptosis in ovarian and breast cancer cells through a caspase-independent calpain- dependent pathway. Cancer Res 2002;62(24):7264-72.
149. Luo RZ, Fang X, Marquez R, Liu SY, Mills GB, Liao WS, Yu Y, Bast RC. ARHI is a Ras-related small G-protein with a novel N-terminal extension that inhibits growth of ovarian and breast cancers. Oncogene 2003;22(19):2897-909.
150. Nishimoto A, Yu Y, Lu Z, Mao X, Ren Z, Watowich SS, Mills GB, Liao WS, Chen X, Bast RC, Jr., Luo RZ. A Ras homologue member I directly inhibits signal transducers and activators of transcription 3 translocation and activity in human breast and ovarian cancer cells. Cancer Res 2005;65(15):6701-10.
151. Finlin BS, Gau CL, Murphy GA, Shao H, Kimel T, Seitz RS, Chiu YF, Botstein D, Brown PO, Der CJ, Tamanoi F, Andres DA, Perou CM. RERG is a novel ras-related, estrogen-regulated and growth-inhibitory gene in breast cancer. J Biol Chem 2001;276(45):42259-67.
152. Hanker AB, Morita S, Repasky GA, Ross DT, Seitz RS, Der CJ. Tools to study the function of the ras-related, estrogen-regulated growth inhibitor in breast cancer. Methods Enzymol 2008;439:53-72.
153. Key MD, Andres DA, Der CJ, Repasky GA. Characterization of RERG: An Estrogen-Regulated Tumor Suppressor Gene. Methods Enzymol 2005;407:513-27.
154. Itoh M, Nelson CM, Myers CA, Bissell MJ. Rap1 integrates tissue polarity, lumen formation, and tumorigenic potential in human breast epithelial cells. Cancer Res 2007;67(10):4759-66.
155. Reynet C, Kahn CR. Rad: a member of the Ras family overexpressed in muscle of type II diabetic humans. Science 1993;262(5138):1441-4.
156. Tseng YH, Vicent D, Zhu J, Niu Y, Adeyinka A, Moyers JS, Watson PH, Kahn CR. Regulation of growth and tumorigenicity of breast cancer cells by the low molecular weight GTPase Rad and nm23. Cancer Res 2001;61(5):2071-9.
157. Suzuki M, Shigematsu H, Shames DS, Sunaga N, Takahashi T, Shivapurkar N, Iizasa T, Minna JD, Fujisawa T, Gazdar AF. Methylation and gene silencing of the Ras-
131 related GTPase gene in lung and breast cancers. Ann Surg Oncol 2007;14(4):1397- 404.
158. Bodemann BO, White MA. Ral GTPases and cancer: linchpin support of the tumorigenic platform. Nat Rev Cancer 2008;8(2):133-40.
159. Lim KH, Baines AT, Fiordalisi JJ, Shipitsin M, Feig LA, Cox AD, Der CJ, Counter CM. Activation of RalA is critical for Ras-induced tumorigenesis of human cells. Cancer Cell 2005;7(6):533-45.
160. Yu Y, Feig LA. Involvement of R-Ras and Ral GTPases in estrogen-independent proliferation of breast cancer cells. Oncogene 2002;21(49):7557-68.
161. Sjoblom T, Jones S, Wood LD, Parsons DW, Lin J, Barber TD, Mandelker D, Leary RJ, Ptak J, Silliman N, Szabo S, Buckhaults P, Farrell C, Meeh P, Markowitz SD, Willis J, Dawson D, Willson JK, Gazdar AF, Hartigan J, Wu L, Liu C, Parmigiani G, Park BH, Bachman KE, Papadopoulos N, Vogelstein B, Kinzler KW, Velculescu VE. The consensus coding sequences of human breast and colorectal cancers. Science 2006;314(5797):268-74.
162. van Agthoven T, van Agthoven TL, Dekker A, van der Spek PJ, Vreede L, Dorssers LC. Identification of BCAR3 by a random search for genes involved in antiestrogen resistance of human breast cancer cells. Embo J 1998;17(10):2799-808.
163. Gotoh T, Cai D, Tian X, Feig LA, Lerner A. p130Cas regulates the activity of AND- 34, a novel Ral, Rap1, and R-Ras guanine nucleotide exchange factor. J Biol Chem 2000;275(39):30118-23.
164. Felekkis KN, Narsimhan RP, Near R, Castro AF, Zheng Y, Quilliam LA, Lerner A. AND-34 activates phosphatidylinositol 3-kinase and induces anti-estrogen resistance in a SH2 and GDP exchange factor-like domain-dependent manner. Mol Cancer Res 2005;3(1):32-41.
165. Cai D, Iyer A, Felekkis KN, Near RI, Luo Z, Chernoff J, Albanese C, Pestell RG, Lerner A. AND-34/BCAR3, a GDP exchange factor whose overexpression confers antiestrogen resistance, activates Rac, PAK1, and the cyclin D1 promoter. Cancer Res 2003;63(20):6802-8.
166. Tang Y, Olufemi L, Wang MT, Nie D. Role of Rho GTPases in breast cancer. Front Biosci 2008;13:759-76.
167. Burbelo P, Wellstein A, Pestell RG. Altered Rho GTPase signaling pathways in breast cancer cells. Breast Cancer Res Treat 2004;84(1):43-8.
168. Cheng KW, Lahad JP, Gray JW, Mills GB. Emerging role of RAB GTPases in cancer and human disease. Cancer Res 2005;65(7):2516-9.
132 169. Garcia M, Derocq D, Freiss G, Rochefort H. Activation of estrogen receptor transfected into a receptor-negative breast cancer cell line decreases the metastatic and invasive potential of the cells. Proc Natl Acad Sci U S A 1992;89(23):11538-42.
170. Dhanasekaran SM, Barrette TR, Ghosh D, Shah R, Varambally S, Kurachi K, Pienta KJ, Rubin MA, Chinnaiyan AM. Delineation of prognostic biomarkers in prostate cancer. Nature 2001;412(6849):822-6.
171. Lapointe J, Li C, Higgins JP, van de Rijn M, Bair E, Montgomery K, Ferrari M, Egevad L, Rayford W, Bergerheim U, Ekman P, DeMarzo AM, Tibshirani R, Botstein D, Brown PO, Brooks JD, Pollack JR. Gene expression profiling identifies clinically relevant subtypes of prostate cancer. Proc Natl Acad Sci U S A 2004;101(3):811-6.
172. Ramaswamy S, Ross KN, Lander ES, Golub TR. A molecular signature of metastasis in primary solid tumors. Nat Genet 2003;33(1):49-54.
173. Morgenstern JP, Land H. Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res 1990;18(12):3587-96.
174. Fiordalisi JJ, Johnson RL, 2nd, Ulku AS, Der CJ, Cox AD. Mammalian expression vectors for Ras family proteins: generation and use of expression constructs to analyze Ras family function. Methods Enzymol 2001;332:3-36.
175. Baines AT, Lim KH, Shields JM, Lambert JM, Counter CM, Der CJ, Cox AD. Use of retrovirus expression of interfering RNA to determine the contribution of activated k- ras and ras effector expression to human tumor cell growth. Methods Enzymol 2005;407:556-74.
176. Novina CD, Murray MF, Dykxhoorn DM, Beresford PJ, Riess J, Lee SK, Collman RG, Lieberman J, Shankar P, Sharp PA. siRNA-directed inhibition of HIV-1 infection. Nat Med 2002;8(7):681-6.
177. Clark GJ, Cox AD, Graham SM, Der CJ. Biological assays for Ras transformation. Methods Enzymol 1995;255:395-412.
178. Ring BZ, Seitz RS, Beck R, Shasteen WJ, Tarr SM, Cheang MC, Yoder BJ, Budd GT, Nielsen TO, Hicks DG, Estopinal NC, Ross DT. Novel prognostic immunohistochemical biomarker panel for estrogen receptor-positive breast cancer. J Clin Oncol 2006;24(19):3039-47.
179. Feig LA. Tools of the trade: use of dominant-inhibitory mutants of Ras-family GTPases. Nat Cell Biol 1999;1(2):E25-7.
180. Reuter JA, Khavari PA. Use of conditionally active ras fusion proteins to study epidermal growth, differentiation, and neoplasia. Methods Enzymol 2005;407:691- 702.
133 181. Tarutani M, Cai T, Dajee M, Khavari PA. Inducible activation of Ras and Raf in adult epidermis. Cancer Res 2003;63(2):319-23.
182. Eilers M, Picard D, Yamamoto KR, Bishop JM. Chimaeras of myc oncoprotein and steroid receptors cause hormone-dependent transformation of cells. Nature 1989;340(6228):66-8.
183. Pritchard CA, Samuels ML, Bosch E, McMahon M. Conditionally oncogenic forms of the A-Raf and B-Raf protein kinases display different biological and biochemical properties in NIH 3T3 cells. Mol Cell Biol 1995;15(11):6430-42.
184. Lim KH, Counter CM. Reduction in the requirement of oncogenic Ras signaling to activation of PI3K/AKT pathway during tumor maintenance. Cancer Cell 2005;8(5):381-92.
185. Huang J, Manning BD. The TSC1-TSC2 complex: a molecular switchboard controlling cell growth. Biochem J 2008;412(2):179-90.
186. Takahashi K, Nakagawa M, Young SG, Yamanaka S. Differential membrane localization of ERas and Rheb, two Ras-related proteins involved in the phosphatidylinositol 3-kinase/mTOR pathway. J Biol Chem 2005;280(38):32768-74.
187. Chenette EJ, Mitin NY, Der CJ. Multiple sequence elements facilitate Chp Rho GTPase subcellular location, membrane association, and transforming activity. Mol Biol Cell 2006;17(7):3108-21.
188. Urano J, Comiso MJ, Guo L, Aspuria PJ, Deniskin R, Tabancay AP, Jr., Kato- Stankiewicz J, Tamanoi F. Identification of novel single amino acid changes that result in hyperactivation of the unique GTPase, Rheb, in fission yeast. Mol Microbiol 2005;58(4):1074-86.
189. Urano J, Sato T, Matsuo T, Otsubo Y, Yamamoto M, Tamanoi F. Point mutations in TOR confer Rheb-independent growth in fission yeast and nutrient-independent mammalian TOR signaling in mammalian cells. Proc Natl Acad Sci U S A 2007;104(9):3514-9.
190. Bivona TG, Perez De Castro I, Ahearn IM, Grana TM, Chiu VK, Lockyer PJ, Cullen PJ, Pellicer A, Cox AD, Philips MR. Phospholipase Cgamma activates Ras on the Golgi apparatus by means of RasGRP1. Nature 2003;424(6949):694-8.
191. Schalm SS, Blenis J. Identification of a conserved motif required for mTOR signaling. Curr Biol 2002;12(8):632-9.
192. Roberts PJ, Mitin N, Keller PJ, Chenette EJ, Madigan JP, Currin RO, Cox AD, Wilson O, Kirschmeier P, Der CJ. Rho Family GTPase modification and dependence on CAAX motif-signaled posttranslational modification. J Biol Chem 2008;283(37):25150-63.
134 193. Sun J, Blaskovich MA, Knowles D, Qian Y, Ohkanda J, Bailey RD, Hamilton AD, Sebti SM. Antitumor efficacy of a novel class of non-thiol-containing peptidomimetic inhibitors of farnesyltransferase and geranylgeranyltransferase I: combination therapy with the cytotoxic agents cisplatin, Taxol, and gemcitabine. Cancer Res 1999;59(19):4919-26.
194. Falsetti SC, Wang DA, Peng H, Carrico D, Cox AD, Der CJ, Hamilton AD, Sebti SM. Geranylgeranyltransferase I inhibitors target RalB to inhibit anchorage- dependent growth and induce apoptosis and RalA to inhibit anchorage-independent growth. Mol Cell Biol 2007;27(22):8003-14.
195. Rotblat B, Ehrlich M, Haklai R, Kloog Y. The ras inhibitor farnesylthiosalicylic Acid (salirasib) disrupts the spatiotemporal localization of active ras: a potential treatment for cancer. Methods Enzymol 2008;439:467-89.
196. Saito K, Araki Y, Kontani K, Nishina H, Katada T. Novel role of the small GTPase Rheb: its implication in endocytic pathway independent of the activation of mammalian target of rapamycin. J Biochem (Tokyo) 2005;137(3):423-30.
197. Choy E, Chiu VK, Silletti J, Feoktistov M, Morimoto T, Michaelson D, Ivanov IE, Philips MR. Endomembrane trafficking of ras: the CAAX motif targets proteins to the ER and Golgi. Cell 1999;98(1):69-80.
198. Michaelson D, Silletti J, Murphy G, D'Eustachio P, Rush M, Philips MR. Differential localization of Rho GTPases in live cells: regulation by hypervariable regions and RhoGDI binding. J Cell Biol 2001;152(1):111-26.
199. Heo WD, Meyer T. Switch-of-function mutants based on morphology classification of Ras superfamily small GTPases. Cell 2003;113(3):315-28.
200. Michaelson D, Ali W, Chiu VK, Bergo M, Silletti J, Wright L, Young SG, Philips M. Postprenylation CAAX processing is required for proper localization of Ras but not Rho GTPases. Mol Biol Cell 2005;16(4):1606-16.
201. Nakase Y, Fukuda K, Chikashige Y, Tsutsumi C, Morita D, Kawamoto S, Ohnuki M, Hiraoka Y, Matsumoto T. A defect in protein farnesylation suppresses a loss of Schizosaccharomyces pombe tsc2+, a homolog of the human gene predisposing to tuberous sclerosis complex. Genetics 2006;173(2):569-78.
202. Sato T, Umetsu A, Tamanoi F. Characterization of the Rheb-mTOR Signaling Pathway in Mammalian Cells: Constitutive Active Mutants of Rheb and mTOR. Methods Enzymol 2008;438:307-20.
203. Kloog Y, Cox AD. Prenyl-binding domains: potential targets for Ras inhibitors and anti-cancer drugs. Semin Cancer Biol 2004;14(4):253-61.
204. Chou MM, Blenis J. The 70 kDa S6 kinase complexes with and is activated by the Rho family G proteins Cdc42 and Rac1. Cell 1996;85(4):573-83.
135 205. Whyte DB, Kirschmeier P, Hockenberry TN, Nunez-Oliva I, James L, Catino JJ, Bishop WR, Pai JK. K- and N-Ras are geranylgeranylated in cells treated with farnesyl protein transferase inhibitors. J Biol Chem 1997;272(22):14459-64.
206. Rowell CA, Kowalczyk JJ, Lewis MD, Garcia AM. Direct demonstration of geranylgeranylation and farnesylation of Ki-Ras in vivo. J Biol Chem 1997;272(22):14093-7.
207. Wienecke R, Maize JC, Jr., Shoarinejad F, Vass WC, Reed J, Bonifacino JS, Resau JH, de Gunzburg J, Yeung RS, DeClue JE. Co-localization of the TSC2 product tuberin with its target Rap1 in the Golgi apparatus. Oncogene 1996;13(5):913-23.
208. Drenan RM, Liu X, Bertram PG, Zheng XF. FKBP12-rapamycin-associated protein or mammalian target of rapamycin (FRAP/mTOR) localization in the endoplasmic reticulum and the Golgi apparatus. J Biol Chem 2004;279(1):772-8.
209. Du G, Altshuller YM, Vitale N, Huang P, Chasserot-Golaz S, Morris AJ, Bader MF, Frohman MA. Regulation of phospholipase D1 subcellular cycling through coordination of multiple membrane association motifs. J Cell Biol 2003;162(2):305- 15.
210. Law BK, Norgaard P, Moses HL. Farnesyltransferase inhibitor induces rapid growth arrest and blocks p70s6k activation by multiple stimuli. J Biol Chem 2000;275(15):10796-801.
211. Collins MO, Choudhary JS. Mapping multiprotein complexes by affinity purification and mass spectrometry. Curr Opin Biotechnol 2008;19(4):324-30.
212. Bauer A, Kuster B. Affinity purification-mass spectrometry. Powerful tools for the characterization of protein complexes. Eur J Biochem 2003;270(4):570-8.
213. Wang AG, Fang W, Han YH, Cho SM, Choi JY, Lee KH, Kim WH, Kim JM, Park MG, Yu DY, Kim NS, Lee DS. Expression of the RERG gene is gender-dependent in hepatocellular carcinoma and regulated by histone deacetyltransferases. J Korean Med Sci 2006;21(5):891-6.
214. Fujii S, Luo RZ, Yuan J, Kadota M, Oshimura M, Dent SR, Kondo Y, Issa JP, Bast RC, Jr., Yu Y. Reactivation of the silenced and imprinted alleles of ARHI is associated with increased histone H3 acetylation and decreased histone H3 lysine 9 methylation. Hum Mol Genet 2003;12(15):1791-800.
215. Glaser KB. HDAC inhibitors: Clinical update and mechanism-based potential. Biochem Pharmacol 2007;74(5):659-71.
216. Yoo CB, Jones PA. Epigenetic therapy of cancer: past, present and future. Nat Rev Drug Discov 2006;5(1):37-50.
136 217. Sharma D, Saxena NK, Davidson NE, Vertino PM. Restoration of Tamoxifen Sensitivity in Estrogen Receptor-Negative Breast Cancer Cells: Tamoxifen-Bound Reactivated ER Recruits Distinctive Corepressor Complexes. Cancer Res 2006;66(12):6370-8.
218. Bayliss J, Hilger A, Vishnu P, Diehl K, El-Ashry D. Reversal of the Estrogen Receptor Negative Phenotype in Breast Cancer and Restoration of Antiestrogen Response. Clin Cancer Res 2007;13(23):7029-7036.
219. Chu I, Arnaout A, Loiseau S, Sun J, Seth A, McMahon C, Chun K, Hennessy B, Mills GB, Nawaz Z, Slingerland JM. Src promotes estrogen-dependent estrogen receptor alpha proteolysis in human breast cancer. J Clin Invest 2007;117(8):2205-15.
220. Singh A, Greninger P, Rhodes D, Koopman L, Violette S, Bardeesy N, Settleman J. A Gene Expression Signature Associated with "K-Ras Addiction" Reveals Regulators of EMT and Tumor Cell Survival. Cancer Cell 2009;15(6):489-500.
221. Wang L, Lawrence JC, Jr., Sturgill TW, Harris TE. Mammalian Target of Rapamycin Complex 1 (mTORC1) Activity Is Associated with Phosphorylation of Raptor by mTOR. J Biol Chem 2009;284(22):14693-7.
222. Acosta-Jaquez HA, Keller JA, Foster KG, Ekim B, Soliman GA, Ballif BA, Fingar DC. Site-specific mTOR phosphorylation promotes mTORC1-mediated signaling and cell growth. Mol Cell Biol 2009.
223. Klein S, Levitzki A. Targeted cancer therapy: promise and reality. Adv Cancer Res 2007;97:295-319.
224. Jordan NJ, Gee JM, Barrow D, Wakeling AE, Nicholson RI. Increased constitutive activity of PKB/Akt in tamoxifen resistant breast cancer MCF-7 cells. Breast Cancer Res Treat 2004;87(2):167-80.
225. Shin I, Arteaga CL. Expression of active Akt protects against tamoxifen-induced apoptosis in MCF-7 Cells. IUBMB Life 2006;58(11):664-9.
226. Knowlden JM, Hutcheson IR, Jones HE, Madden T, Gee JM, Harper ME, Barrow D, Wakeling AE, Nicholson RI. Elevated levels of epidermal growth factor receptor/c- erbB2 heterodimers mediate an autocrine growth regulatory pathway in tamoxifen- resistant MCF-7 cells. Endocrinology 2003;144(3):1032-44.
227. Bishop WR, Kirschmeier P, Baum C. Farnesyl transferase inhibitors: mechanism of action, translational studies and clinical evaluation. Cancer Biol Ther 2003;2(4 Suppl 1):S96-104.
228. Baum C, Kirschmeier P. Preclinical and clinical evaluation of farnesyltransferase inhibitors. Curr Oncol Rep 2003;5(2):99-107.
137 229. Sparano JA MS, Kazi A, Coppola D, Negassa A, Vahdat L, Li T, Christine P, Fineberg S, Munster P, Malafa M, David L, Hoschander S, Hopkins U, Hershman D, Wright JJ, Kleer C, Merjver S, Sebti SM. Phase II trial of the farnesyl transferase inhibitor tipifarnib plus neoadjuvant doxorubicin-cyclophosphamide in patients with clinical stage IIB-IIIC breast cancer. In: 100th Annual Meeting of the American Association for Cancer Research; 2009 18-22 Apr 2009; Denver, Colorado; 2009.
230. Zhu K, Hamilton AD, Sebti SM. Farnesyltransferase inhibitors as anticancer agents: current status. Curr Opin Investig Drugs 2003;4(12):1428-35.
231. Pan J, Yeung SC. Recent advances in understanding the antineoplastic mechanisms of farnesyltransferase inhibitors. Cancer Res 2005;65(20):9109-12.
232. Law BK, Norgaard P, Gnudi L, Kahn BB, Poulson HS, Moses HL. Inhibition of DNA synthesis by a farnesyltransferase inhibitor involves inhibition of the p70(s6k) pathway. J Biol Chem 1999;274(8):4743-8.
233. Meier FE NH, Flaherty K, Schadendorf D, Sinnberg T, Schittek B, Garbe C. Effect of the farnesyl transferase inhibitor lonafarnib on sensitivity of melanoma cells to the multikinase inhibitor sorafenib and on Rheb farnesylation and mTOR signaling. In: 2009 American Society of Clinical Oncology Annual Meeting; 2009 29 May - 2 Jun 2009; Orlando, FL: J Clin Oncol; 2009.
234. Sepp-Lorenzino L, Ma Z, Rands E, Kohl NE, Gibbs JB, Oliff A, Rosen N. A peptidomimetic inhibitor of farnesyl:protein transferase blocks the anchorage- dependent and -independent growth of human tumor cell lines. Cancer Res 1995;55(22):5302-9.
235. Willumsen BM, Cox AD, Solski PA, Der CJ, Buss JE. Novel determinants of H-Ras plasma membrane localization and transformation. Oncogene 1996;13(9):1901-9.
236. Hancock JF, Magee AI, Childs JE, Marshall CJ. All ras proteins are polyisoprenylated but only some are palmitoylated. Cell 1989;57(7):1167-77.
237. Kato-Stankiewicz J, Hakimi I, Zhi G, Zhang J, Serebriiskii I, Guo L, Edamatsu H, Koide H, Menon S, Eckl R, Sakamuri S, Lu Y, Chen QZ, Agarwal S, Baumbach WR, Golemis EA, Tamanoi F, Khazak V. Inhibitors of Ras/Raf-1 interaction identified by two-hybrid screening revert Ras-dependent transformation phenotypes in human cancer cells. Proc Natl Acad Sci U S A 2002;99(22):14398-403.
238. Zeeh JC, Antonny B, Cherfils J, Zeghouf M. In vitro assays to characterize inhibitors of the activation of small G proteins by their guanine nucleotide exchange factors. Methods Enzymol 2008;438:41-56.
239. Bos JL, Rehmann H, Wittinghofer A. GEFs and GAPs: Critical Elements in the Control of Small G Proteins. Cell 2007;129(5):865-77.
138 240. Gao Y, Dickerson JB, Guo F, Zheng J, Zheng Y. Rational design and characterization of a Rac GTPase-specific small molecule inhibitor. Proc Natl Acad Sci U S A 2004;101(20):7618-23.
241. Shutes A, Onesto C, Picard V, Leblond B, Schweighoffer F, Der CJ. Specificity and mechanism of action of EHT 1864, a novel small molecule inhibitor of Rac family small GTPases. J Biol Chem 2007;282(49):35666-78.
242. Sampson JR. Therapeutic targeting of mTOR in tuberous sclerosis. Biochem Soc Trans 2009;37(Pt 1):259-64.
243. Yu J, Astrinidis A, Howard S, Henske EP. Estradiol and tamoxifen stimulate LAM- associated angiomyolipoma cell growth and activate both genomic and nongenomic signaling pathways. Am J Physiol Lung Cell Mol Physiol 2004;286(4):L694-700.
244. Rubinsztein DC. The roles of intracellular protein-degradation pathways in neurodegeneration. Nature 2006;443(7113):780-6.
245. Kondo Y, Kondo S. Autophagy and cancer therapy. Autophagy 2006;2(2):85-90.
246. Barlund M, Monni O, Kononen J, Cornelison R, Torhorst J, Sauter G, Kallioniemi O- P, Kallioniemi A. Multiple genes at 17q23 undergo amplification and overexpression in breast cancer. Cancer Res 2000;60(19):5340-4.
247. Barlund M, Forozan F, Kononen J, Bubendorf L, Chen Y, Bittner ML, Torhorst J, Haas P, Bucher C, Sauter G, Kallioniemi OP, Kallioniemi A. Detecting activation of ribosomal protein S6 kinase by complementary DNA and tissue microarray analysis. J Natl Cancer Inst 2000;92(15):1252-9.
248. Santen RJ, Lynch AR, Neal LR, McPherson RA, Yue W. Farnesylthiosalicylic acid: inhibition of proliferation and enhancement of apoptosis of hormone-dependent breast cancer cells. Anticancer Drugs 2006;17(1):33-40.
249. Berstein LM, Zheng H, Yue W, Wang JP, Lykkesfeldt AE, Naftolin F, Harada H, Shanabrough M, Santen RJ. New approaches to the understanding of tamoxifen action and resistance. Endocr Relat Cancer 2003;10(2):267-77.
250. Guertin DA, Stevens DM, Saitoh M, Kinkel S, Crosby K, Sheen JH, Mullholland DJ, Magnuson MA, Wu H, Sabatini DM. mTOR complex 2 is required for the development of prostate cancer induced by Pten loss in mice. Cancer Cell 2009;15(2):148-59.
251. Nardella C, Carracedo A, Alimonti A, Hobbs RM, Clohessy JG, Chen Z, Egia A, Fornari A, Fiorentino M, Loda M, Kozma SC, Thomas G, Cordon-Cardo C, Pandolfi PP. Differential Requirement of mTOR in Postmitotic Tissues and Tumorigenesis. Sci Signal 2009;2(55):ra2.
139 252. Feldman ME, Apsel B, Uotila A, Loewith R, Knight ZA, Ruggero D, Shokat KM. Active-site inhibitors of mTOR target rapamycin-resistant outputs of mTORC1 and mTORC2. PLoS Biol 2009;7(2):e38.
253. Mahalingam D, Sankhala K, Mita A, Giles FJ, Mita MM. Targeting the mTOR pathway using deforolimus in cancer therapy. Future Oncol 2009;5(3):291-303.
254. Shah OJ, Hunter T. Turnover of the active fraction of IRS1 involves raptor-mTOR- and S6K1-dependent serine phosphorylation in cell culture models of tuberous sclerosis. Mol Cell Biol 2006;26(17):6425-34.
255. O'Reilly KE, Rojo F, She QB, Solit D, Mills GB, Smith D, Lane H, Hofmann F, Hicklin DJ, Ludwig DL, Baselga J, Rosen N. mTOR inhibition induces upstream receptor tyrosine kinase signaling and activates Akt. Cancer Res 2006;66(3):1500-8.
256. Shah OJ, Wang Z, Hunter T. Inappropriate activation of the TSC/Rheb/mTOR/S6K cassette induces IRS1/2 depletion, insulin resistance, and cell survival deficiencies. Curr Biol 2004;14(18):1650-6.
257. Kinkade CW, Castillo-Martin M, Puzio-Kuter A, Yan J, Foster TH, Gao H, Sun Y, Ouyang X, Gerald WL, Cordon-Cardo C, Abate-Shen C. Targeting AKT/mTOR and ERK MAPK signaling inhibits hormone-refractory prostate cancer in a preclinical mouse model. J Clin Invest 2008;118(9):3051-64.
258. Carracedo A, Ma L, Teruya-Feldstein J, Rojo F, Salmena L, Alimonti A, Egia A, Sasaki AT, Thomas G, Kozma SC, Papa A, Nardella C, Cantley LC, Baselga J, Pandolfi PP. Inhibition of mTORC1 leads to MAPK pathway activation through a PI3K-dependent feedback loop in human cancer. J Clin Invest 2008;118(9):3065-74.
259. Whitehurst AW, Bodemann BO, Cardenas J, Ferguson D, Girard L, Peyton M, Minna JD, Michnoff C, Hao W, Roth MG, Xie XJ, White MA. Synthetic lethal screen identification of chemosensitizer loci in cancer cells. Nature 2007;446(7137):815-9.
140