SOSTDC1: A BMP/WNT DUAL PATHWAY ANTAGONIST IN BREAST AND RENAL CANCERS
BY
KIMBERLY ROSE BLISH
A Dissertation Submitted to the Graduate Faculty of WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES
in Partial Fulfillment of the Requirements
for the Degree of
DOCTOR OF PHILOSOPHY
Molecular Medicine and Translational Science
May 2009
Winston-Salem, North Carolina
Approved By:
Suzy V. Torti, Ph.D. Advisor ______
Examining Committee:
Mark C. Willingham, M.D., Chairman ______
Gregory A. Hawkins, Ph.D. ______
Raymond Penn, Ph.D. ______
William Jeffrey Petty, M.D. ______ACKNOWLEDGEMENTS
At the end of a long research and writing process, I come at last to the part
where I can reflect upon the journey with gratitude. However, as I am typing
these words last, I am hampered by a diminished vocabulary and I know I shall
fall short on the most important words of the document! To all whom I salute
below: I sincerely could not have accomplished anything worthwhile without you.
These words will not do you justice for your support has meant the world to me.
This project would be nonexistent without the opportunities, guidance, and experience offered from my advisor, Suzy Torti and co-advisor, Frank Torti. You both challenged me to become a better scientist. I am indebted to you for giving me and my ideas a “home” for the past 5 years.
I am grateful to my committee members (Mark Willingham, Greg Hawkins,
Anthony Atala, Ray Penn, Jeff Petty) and many other faculty collaborators who took my ideas seriously, encouraged new directions, and gave of their time and laboratory resources. I have acknowledged specific contributions where appropriate throughout the text. Mark Willingham, Tim Kute, Greg Hawkins, and members of their laboratories were colleagues with indispensible aid and inexhaustible advice.
The entire Torti Lab contributed through advice, technical assistance, and at times, “colleague commiseration”. Particular mention goes to Samer Kalakish,
ii Matt Triplette, Wei Wang, Seon-Hi Jang, Lan Coffman, Zandra Pinnix, Alice
Mims, and Jonathan Storey for their direct contributions to this project. All students rely heavily on laboratory technicians and I was no exception. My thanks especially go to Rong Ma and Julie Brown for keeping the lab running smoothly, yet finding time to teach and answer endless questions. I am particularly indebted to Julie Brown for her hours of support, technical, moral, and spiritual on this project.
I would not have arrived at Wake Forest in the first place without a lot of help. I had not considered a PhD degree until the mentoring I received in college from
Lowell Hall and Cynthia Davis, both of whom invested in me – not only as one of their students, but as a person. Mark Payne, David Bass, Cash McCall, Jamal
Ibdah, Paul Laurienti, Linda McPhail, Bridget Brosnihan, and Kevin High in the
Molecular Medicine and MD/PhD programs provided career and administrative support during my transition into and throughout the dual-degree program. I have had the honor of working with an incredibly high caliber of colleagues in the
Molecular Medicine, MD/PhD, and Cancer Biology programs and I am proud to be part of these departments.
Personally, there was a formidable army of friends and family who would not let me back down and were constantly rooting for me including:
• My prayer partners and cheerleaders- may you be abundantly blessed!
iii • My extended family at Harvest Point Church, especially the youth group
who tried to keep me smiling through it all.
• The cancer survivors who shared their stories and their enthusiasm for
life: Susan Blish, Kate Hacker, and Bob Smith
• The Rose, Pomante, Smith, Blish, LaBarr, and Stotler families for
understanding my preoccupation, missed holidays, and late birthday cards
and forgiving me when finishing this thesis work became overwhelming.
At the end, I couldn’t have made it without the support network that helped
sustain me day to day (and provided excellent child care)!
• Mom and Dad- There are no better examples of self-sacrificing love and
support than you. I am the person I am, having achieved these things-
because of you. When the battle was long, you held up my arms.
• My husband, Drew. You have put up with everything you promised and
more! “Time after Time.” I can’t imagine anyone else I’d rather share this
with.
I dedicate this work to:
Evelyn Smith & Elaine Rose, because I am in awe of all that has been done,
Drew, because every day is worth it,
and Ethan, because there’s hope for the future.
Glory to the One who was, and is, and is to come (Revelations 1:8)
iv TABLE OF CONTENTS
Page LIST OF ABBREVIATIONS ………………………………………………….. vi
LIST OF ILLUSTRATIONS …………………………………………………... xi
ABSTRACT…………………………………………………………………….. xiv
Chapter
I. INTRODUCTION: BMP/WNT PATHWAYS SIGNALING IN CANCER AND SOSTDC1 AS A POTENTIAL DUAL-PATHWAY INHIBITOR……………………………………………………………….. 1
II. THE HUMAN BONE MORPHOGENETIC PROTEIN ANTAGONIST SOSTDC1 IS DOWNREGULATED IN RENAL CANCERS Published in Molecular Biology of the Cell, February 2008…………. 39
III. LOSS OF HETEROZYGOSITY AFFECTING SOSTDC1 IN RENAL TUMORS For Submission to Cancer Epidemiology Biomarkers and Prevention ……………………………………………………………….. 77
IV. SOSTDC1, AN ANTI-PROLIFERATIVE BMP ANTAGONIST, IS POORLY EXPRESSED IN PATIENTS WITH ADVANCED BREAST CANCER In Preparation…………………………………………………………….. 106
V. GENERAL DISCUSSION……………………………………………….. 142
SCHOLASTIC VITA……………………………………………………………. 167
v LIST OF ABBREVIATIONS
AHR Aryl Hydrocarbon Receptor
AKT Akt/Protein Kinase B Oncogene Family
APC Adenomatous Polyposis Coli
ALK Activin-Like Kinase
AUS Antigen Unmasking Solution
BHD Birt-Hogg-Dubé
BMP Bone Morphogenetic Protein
BMPA BMP Antagonist
BMPR BMP Receptor
CCN Family of proteins including: CTGF, cyr61, and nov
CEU Population of Utah residents with northern or western
European ancestry (HapMap designation)
CK-1,-2 Casein kinase-1, -2
CTCK C-Terminal Cystine Knot
CTGF Connective Tissue Growth Factor
Cyr61 Cysteine rich 61
DAN Differential screening-selected gene Aberrative
in Neuroblastoma
DCIS Ductal Carcinomas In Situ (Breast)
DSH/DVL Disheveled
DKK Dickkopf
vi EGF/R Epidermal Growth Factor/Receptor
FBS Fetal Bovine Serum
FGF Fibroblast Growth Factor
FH Fumerate Hydratase
FWT Familial Wilms Tumor locus
FRZ Frizzled
FSH Follicle Stimulating Hormone
GAPDH GlycerAldehyde-3-Phosphate DeHydrogenase
GSK-3β Glycogen synthase kinase-3β
HapMap International HapMap Project- partnership to
database of frequencies of human SNPs hCG human Chorionic Gonadotropin
HMEC Human Mammary Epithelial Cell
HMER HMECs overexpressing ras oncogene
IGF/R Insulin-like Growth Factor/Receptor
IHC ImmunoHistoChemistry
INT MMTV Integration gene (M. musculus)
LCIS Lobular Carcinoma In Situ (Breast)
LDLR Low-Density Lipoprotein Receptor
LH Luteinizing Hormone
LOH Loss-Of-Heterozygosity
LRP LDLR Related Protein
MAD Mothers against decapentaplegic (D. melanogaster)
vii MAPK Mitogen-Activated Protein Kinase
MEOX2 MEsenchymal homeobOX-2
MMTV Mouse Mammary Tumor Virus
NDP Norrie Disease Protein
NGF Nerve Growth Factor
Nov Nephroblastoma overexpressed
PPAR Peroxisome Proliferator-Activated Receptor
PC Pearson’s Coefficient
PCP Planar Cell Polarity (Wnt signaling pathway)
PI3K PhosphoInositide-3 Kinase
P/S Penicillin/Streptomycin
PTCH Patched
PTEN Phosphatase and TENsin homolog
RPTEC Renal Proximal Tubule Epithelial Cells
RCC Renal Cell Carcinoma
RCC-Clear RCC-Clear Cell Type rh recombinant, human
R-Smad Regulatory-Smad
RT-CES Real Time-Cell Expansion System® (ACEA
Biosciences) sFRP secreted Frizzled-Related Protein
SBE Smad Binding Element
SDS Sodium Dodecyl Sulfate
viii SDS-PAGE SDS-PolyAcrylamide Gel Electrophoresis
Smad Mothers against decapentaplegic and SMA family
Member (Common name for MAD and SMA
homologs)
SNP Single Nucleotide Polymorphism
SOST SclerOSTin
SOSTDC1 SclerOSTin Domain-Containing 1 (H. sapiens)
TCF/LEF T-Cell Factor/Lymphoid Enhancing Factor
TGF-β Transforming Growth Factor-β
TMA Tissue MicroArray
Tsg twisted gastrulation
UEP UnExtended Primer
USAG-1 Uterine Sensitization-Association Gene-1 (R.
Norvegius)
VHL Von Hippl-Lindau vWF von Willebrand Factor
WAGR Wilms tumor, Aniridia, Genitourinary malformation,
and mental Retardation syndrome
Wg Wingless (D. melanogaster)
WIF Wnt Inhibitory Factor
WISP Wnt Inducible Signaling Pathway
Wnt Wingless-type MMTV integration site family member
(Common name for Wg and INT homologs)
ix WT Wilms Tumor locus
YRU Population of Nigeria residents with Yoruban
ancestors (HapMap designation)
x LIST OF ILLUSTRATIONS
CHAPTER I
Figure Page
1. Amino acid composition of SOSTDC1 22
2. CTCK motif in various secreted signaling molecules 25
3. SOSTDC1 alignment with sclerostin 26
CHAPTER II
Figure Page
1. Downregulation of Human SOSTDC1 mRNA in 52 Kidney Tumors
2. Immunohistochemical analysis of SOSTDC1 expression In normal kidney and renal cancer subtypes 55
3. Quantification of SOSTDC1 staining 58
4. Normal human SOSTDC1 is secreted and binds to matrix or cell surface proteins around neighboring cells 60
5. rhSOSTDC1 antagonizes the production of phosphorylated Smad in response to BMP-7 signaling 62
6. SOSTDC1 antagonizes Wnt-3a signaling in renal carcinoma cells 64
7. Overexpression of SOSTDC1 suppresses proliferation of 769-P RCC-clear cell cultures 66
Table Page
I. Quantitation of SOSTDC1 Immunohistochemistry 56
xi CHAPTER III
Figure Page
1. SOSTDC1 Locus 85
2. Genes in 2 Mbp Region of Interest on 7p 86
3. LOH Results in Wilms and Renal Cell Carcinoma 88
4. OncoMine Database Shows Downregulation of SOSTDC1 In Wilms Tumor 92
Table Page
I. SNPs in Direct Sequencing 89
SI. Primers for Direct Sequencing 96
SII. Primers for LOH Sequenom Analysis 97
CHAPTER IV
Figure Page
1. Immunohistochemistry of SOSTDC1 in Normal and Breast Tumor Tissues 118
2. Secreted SOSTDC1 Decreases in Mammary Cells with Increasing Transformation 121
3. Quantification of SOSTDC1 Message in Breast Cancer Patients 123
4. SOSTDC1 Protein Levels by Immunohistochemistry for 81 Breast tumors 126
5. SOSTDC1 Inhibits BMP-7-initiated Phosphorylation of Intracellular R-Smads-1,-5,-8 131
6. Cells Overexpressing hSOSTDC1-FLAG Show Proliferation Suppression 133
xii Table Page
I. SOSTDC1 Staining of TMAs- Significant Correlations 127
II. Comparisons Between SOSTDC1 Staining Intensity and Other Tumor Markers in the TMA Tissues 129
xiii ABSTRACT Blish, Kimberly Rose
SOSTDC1: A BMP/WNT DUAL ANTAGONIST IN BREAST AND RENAL CANCERS
Dissertation under the direction of Suzy V. Torti, Ph.D., Associate Professor of Biochemistry
The bone morphogenetic protein (BMP) pathway and the Wnt pathway are signaling networks associated with carcinogenesis. BMPs and Wnts are powerful morphogens and downstream signaling affects critical cell functions such as proliferation, differentiation, and viability. Proper regulation of these pathways is essential to prevent of transformation in normal cells.
SOSTDC1 (SclerOSTin Domain-Containing-1) is an inhibitor of BMP/Wnt signaling in mouse disease models; however, little is known about the expression or actions of SOSTDC1 in human tissue. This work expands upon the observation that SOSTDC1 downregulation occurs in 85-90% of breast and kidney tumors. It was hypothesized that extracellular BMP regulation by SOSTDC1 is protective against cancer; therefore, loss of SOSTDC1 may lead to tumorigenesis initiation or progression. This was investigated through studies of the SOSTDC1 gene locus on chromosome 7, examination of the expression and actions of SOSTDC1 in cell culture models, and evaluation of SOSTDC1 within clinical samples.
Human SOSTDC1 protein is expressed in normal kidney and breast tissue. Mature SOSTDC1 is secreted from normal kidney and breast cells but is not detectable from breast cancer cell lines. The distinction is relevant as SOSTDC1 in the extracellular space effectively antagonizes BMP signaling in breast and kidney cancer cells. Additionally, SOSTDC1 antagonizes Wnt signaling in cell models of kidney cancer. When expression of SOSTDC1 is restored to kidney or breast cancer cell models, striking inhibition of culture proliferation is achieved and cannot be rescued with BMP treatment alone. This suggests that therapeutic potential of exogenous SOSTDC1 may come through concurrent dual BMP and Wnt pathway inhibition.
Genetic studies revealed loss-of-heterozygosity (LOH) at the SOSTDC1 locus in 10% of renal carcinomas and pediatric Wilms Tumors. Searches for a putative Wilms Tumor suppressor at this locus are ongoing and SOSTDC1 may be a viable candidate. Evaluation of SOSTDC1 protein in clinical renal cell carcinomas and breast tumors reveals a significant loss of protein in renal clear cell carcinomas and breast tumors of advanced stage. Loss of SOSTDC1 may be a biomarker for more advanced breast disease.
xiv CHAPTER I
GENERAL INTRODUCTION
The BMP and Wnt Pathways and SOSTDC1 as a Potential Dual-Pathway
Inhibitor
K. BLISH
1 Cancer is a Complex Disease
Cancer incidence is rising, despite the exponential expansion of research into the
etiology, development, and treatment of various neoplasms. This is in part due
to a fundamental problem at the root of cancer research and treatment: the many
diseases collectively known as cancer are incredibly diverse and complex.
The first level of complexity arises from the location of the cancer. Separate
tissues are developed and maintained by many different transcriptomes; thus the
proteins and pathways susceptible to oncogenic stimuli differ from one organ to
another. The next layer of complexity to consider is the variety of causes that
initiates transformation. From heritable genetic mutations in protooncogenes or
tumor suppressor genes, to epigenetic changes, to environmental insults
chemical or viral, the list of causative agents is always expanding. Additionally,
the timing and potential additive effects of multiple exposures play a role. Next within tissues, there are numerous interactions between cells and within their surrounding matrix. Tumorigenesis cannot occur without disruptions in cell-cell communication and many changes to cell-matrix interactions. Finally, there are incredibly complex interactions within the cells themselves. Carcinogenesis even within the cell is a non-linear, multi-step process. Despite cellular “programming” for homeostasis, entire networks of intracellular signaling pathways are susceptible to many classes of perturbations. In the framework of this vast and multi-tiered complexity there is great need for models of carcinogenesis that identify the key processes and players of cancer; models that simplify, provide
2 understanding, and ultimately lead to practical, efficacious, and safe preventions
and treatments.
Cancer Models that Simplify the Disease, Identify Key Pathways, and Lead
to More Effective Treatments
Models of carcinogenesis tend to represent two schools of thought (1): the first
stipulates that fully differentiated cells all contain some genetic predisposition to
cancer (heritable germ line mutations result in familial cancer syndromes; random somatic mutations to sporadic disease) and then over time, stresses to the cell cause a number of carcinogenic changes, transforming it into a self- renewing and invasive entity. It has been proposed that the necessary and
sufficient carcinogenic changes can be categorized into six groups: evasion of apoptosis, self-propelled growth, insensitivity to anti-growth signals, ability to cause angiogenesis, unlimited replication potential, and the ability to invade into
normal tissues. This model suggests that between four and seven mutations
affecting these groups are necessary to cause transformation (2). The second
paradigm suggests that within all tissues reside less-differentiated stem cells,
capable of expansion as needed within the demands of tissue homeostasis.
Mutations within these populations of stem cells that prevent them from
differentiating into their proper lineage and instead cause them to proliferate
inappropriately could be responsible for tumor formation (3).
3 Common ground between these two models identifies the importance of certain core signaling pathways of development: Wnt (wingless/int), hedgehog, notch secreted growth factors (particularly epidermal growth factor/EGF, platelet- derived growth factor/PDGF and fibroblast growth factor/FGF) and subsequent
PI3K/PTEN, and TGF-β/BMP. All of these pathways include the actions of secreted morphogens functioning in both cell autonomous and paracrine capacities to control cell cycle progression, proliferation, protein synthesis, viability, and interactions with the surrounding matrix. Research has shown that these pathways are not only pivotal in the maintenance or differentiation of stem- cell populations, but also changes in these pathways in adult tissues can induce imbalances leading to all six previously discussed properties of transformed cells
(4).
Given the importance of these pathways, it is essential that the regulatory mechanisms governing them remain intact to prevent those transformative imbalances. Indeed, many of the characterized tumor suppressors to date are genes that have regulatory functions in one of these pathways (e.g. PTEN in
PI3K signaling which can influence p53 and AKT (5), APC in Wnt pathway (6),
PTCH in hedgehog signaling (7), Smad4 in BMP pathway (8). Naturally, the goal of studying these regulatory mechanisms is to develop synthetic strategies to restore proper regulation to cancer cells.
4 Studies of secreted protein antagonists, mutated receptors or monoclonal antibodies against receptors, and small-molecule inhibitors of these pathways have demonstrated the possibility of extracellular regulation. Regulation occurring extracellularly holds therapeutic potential as it bypasses the need to safely deliver the compounds to the intracellular spaces. Noteworthy examples of therapies “targeted” directly to extracellular modulation of these pathways include: receptor tyrosine-kinase inhibitors (EGFR inhibitors gefitinib/Iressa®,
AstraZeneca, Waltham, MA and erlotinib/Tarceva®, Genentech, South San
Francisco, CA) and monoclonal antibodies against receptors (HER2/neu receptor and trastuzumab/Herceptin®, Genentech).
Building on the success of these targeted anti-neoplastic drugs, there has been interest in identification of other such extracellular targets. The work presented
here grew out of collaboration between Wake Forest University and Human
Genome Sciences, Inc. (Rockville, MD) to run screens identifying secreted or
extracellular proteins that are differentially expressed in tumors. Identified
proteins associated with any of these known pathways might represent novel
extracellular regulatory mechanisms that could serve as templates for future
therapeutics. One protein identified by this screen is SOSTDC1 (SclerOSTin
Domain-Containing-1), which has associations with the Wnt and BMP (bone
morphogenetic protein) signaling pathways. Accordingly, the rest of this general
review will focus on these particular pathways and their roles in cancer.
5 The Wnt Pathway
Studies in Drosophila melanogaster identified the wingless (Wg) gene,
responsible for body patterning during fruit fly embryogenesis. The murine
ortholog, Int-1 lies near the integration site for mouse mammary tumor virus
(MMTV) and may be overactivated by the viral oncogene. After the proteins
were found to be homologous via protein comparison, the combined name of
Wnt was used to describe the members of this family of secreted growth,
differentiating, and morphogenetic proteins. There are currently 19 known
members of the vertebrate Wnt family and these proteins are able to exert their
effects through several mechanisms; including both classical and non-classical
signaling pathways (9). Traditionally, Wnt ligands are grouped into “canonical”
ligands that tend to active the classical β-catenin pathway (10) (most ubiquitous
are Wnts -1, -3, and -8) and “non-canonical” or “β-catenin-independent Wnts”
(Wnt-4, Wnt-5a, 5b, and Wnt 11) that tend to activate one of the non-classical
signaling pathways. Less is known about the non-canonical pathways as details are still to be elucidated, but they involve one of the following mechanisms:
intracellular Ca2+ modulation , changes in cell polarity (sometimes referred to as
the planar cell polarity, PCP pathway , and co-activity of protein kinase A)
(11;12).
All Wnt family members contain an N-terminal secretion signal sequence, at least
one N-liked glycosylation site, upwards of 20 conserved cysteine residues, which
are spaced similarly in all family members and may be sites of palmitoylation.
6 Along with a presumed role in protein folding, the cysteine residues often play
crucial roles in the amount of post-translational modification, and subsequent
activity of the proteins. For example, two cysteine residues in Wnt-1 are necessary for its ability to transform C57MG mammary epithelial cells (13). Most of what is known about structure-function relationship in the Wnt family comes
from studies of non-functional mutants or partial-protein chimeras, as Wnt
proteins are notoriously insoluble and full-molecule crystal structures have not
been obtained. It is thought that the C-terminal region of Wnts is responsible for
signaling activity, while the N-terminal end provides the specificity for cognate
receptors (14).
Wnt proteins play a role in a variety of normal and disease processes (15);
indeed, many normal developmental roles of Wnts are mimicked in cancer
pathologies (16). In organogenesis, Wnts often cause differentiation of
mesenchymal stem cells (17); Wnt 4 expressed from the uteric bud is sufficient to
induce metanephric blastema into early kidney tubules (18). Cancer stem cell
self-renewal is controlled by aberrant Wnt signaling (19), and it has been shown
that a gene commonly mutated in renal cancers, VHL, is under the control of Wnt
signaling (20).
As research into Wnt ligands expands the knowledge base, the diversity and
complexity of Wnt signaling can be appreciated. Not only are there a rather large
number of Wnt ligands, there are also multiple receptors, co-receptors, and
7 intracellular signaling machinery that transmit the signals. A brief review of the main signaling pathway follows here, but many variations on this basic scheme
have been reported (21).
Classical Wnt Signaling
In the absence of active Wnt ligand in the extracellular space, intracellular β- catenin expression and localization is tightly controlled. In this state, β-catenin is found either bound to cell surface cadherins or in a complex of proteins responsible for the ubiquitination and subsequent degradation of β-catenin. The
degradation complex includes: axin, APC(Adenomatous polyposis coli), GSK-3β
(glycogen synthase kinase-3β), and sometimes CK-1 (casein kinase-1). The exact role of each protein in the complex is unknown and provides material for
ongoing research. The key steps seems to be that in the presence of axin as a
scaffolding protein and APC, GSK-3β and sometimes CK-1 play the pivotal role
of “marking” β-catenin for ubiquitination by phosphorylation of critical N-terminal
serine/threonine residues (22;23).
Wnt ligands function by signaling through receptors of the frizzled (Fzd) family.
Fzd proteins are a subfamily of 7 transmembrane, G coupled-protein receptors.
There are currently 8 known members of the mammalian Fzd family and studies
thus far show differing ligand specificity amongst the Fzds. Importantly, the
Wnt/Fzd interaction is facilitated and stabilized by LRP (low-density lipoprotein
8 related-peptides) -4, -5,or -6 co-receptors, presumably forming a ternary complex
(physical association of the protein has not been proven, yet cells expressing
LRP mutants cannot transmit Wnt signals) (24). Current models suggest that
downstream Wnt signaling activity (such as whether β-catenin independent
pathways are activated) is specified by the Wnt/Fzd/LRP combination and not the
Wnt ligand alone (25).
Upon Wnt binding to a complex of frizzled with LRP co-receptors, the second- messenger disheveled (DSH- xenopus or DVL- vertebrate) is phosphorylated.
Activated DVL causes stabilization on the intracellular face of the cell membrane of the multi-protein complex involving DVL, Frizzled, and co-receptors. Most importantly, activation of DSH/DVL is known to destabilize the interactions of the
APC/Axin and GSK-3β in the degradation system (26). The subsequent dissolution of the degradation system leads to a rise in intracellular stable (non-
phosphorylated) β-catenin levels. Stable β-catenin is a viable signaling
molecule, capable of translocation to the nucleus with other signaling proteins,
and transcription of a multitude of downstream target genes through the
TCF/LEF promoter. Well-characterized target genes include: c-myc, cyclin D1, id2, PPARd, among others (27). The downstream effects of active intracellular β- catenin are profound: including changes in the cell cycle and the viability/apoptosis axis. The tight regulation of β-catenin through this pathway is a very basic cell function and is highly conserved throughout evolution. Many
9 additional molecules contribute to this regulation and are still being identified
(28).
Wnt Regulatory Molecules
There are four known types of extracellular Wnt antagonists: WIF1 (Wnt inhibitory factor 1) , secreted Frizzled-related protein (SFRP) family (29), DKK
(dickkopf) family (30), and multi-pathway Wnt antagonists such as cerberus and sclerostin. Each family is hypothesized to inhibit Wnt signal transduction by some manner of disruption of the Wnt/Fzl receptor signaling complex; however, each set of proteins has its own binding sites and mechanism of inhibition.
Current data demonstrates that WIF1, cerberus, and SFRPs bind primarily to the
Wnt ligands themselves resulting in either sequestration of the ligand away from the receptors or disruption of the ligand-receptor interaction. DKK proteins and sclerostin do not have high affinities for Wnt ligands, but instead bind to the co- receptors LRP-5 and LRP-6 (12). DKKs cross link LRPs away from the Fzds and to another type of cell-membrane proteins called Kremins (31), while sclerostin is thought to bind to the LRP is such a way as to prevent its association with Fzd
(32). Other β-catenin pathway regulatory molecules exist including: Intracellular antagonists such as WTX- a new tumor suppressor in Wilms Tumor (pediatric kidney lesion), which has been cloned on the X-chromosome and has been show to also increase the degradation of β-catenin (33) or extracellular non-Wnt
10 ligands that activate β-catenin: such as Norrie disease protein (NDP, (34), and R-
spondins .
Wnt Pathway Proteins and Antagonists in Cancer
β-catenin
The earliest connection of the Drosophila wingless genes with their vertebrate
orthologs (Wnts) came through the study of mouse mammary tumor virus
(MMTV). From this time, it has been well-established that the viral activation of
Wnts can lead to oncogenic transformation. Much effort has gone into
understanding the mechanisms the Wnt pathways and downstream effects in
tumorigenesis- branching out of the mouse model of breast cancer to almost every type of neoplasm in all organ systems. Overexpression of active, nuclear
β-catenin has been documented in between 20-33% of various tumors (35).
However, the first connection between Wnt signaling and human cancer occurred
with the discovery of the interactions between β-catenin and APC and the
development of colorectal cancers. In intestinal epithelium, β-catenin is a proto-
oncogene and APC as a member of the β-catenin degradation complex is a
tumor suppressor.
Many types of mutations occur to activate the β-catenin proto-oncogene, including changes in the regulatory site serine/threonine residues that render the mutant β-catenin less susceptible to phosphorylation and subsequent ubiquitination (36). Additionally, β-catenin has Wnt-independent roles that are
11 oncogenic when perturbed. In normal cells, β-catenin plays a role with surface cadherins in the establishment of the adherens junctions between epithelial cells lining organs (37). Surprisingly, mutant β-catenin is not only likely to stimulate
Wnt-like signaling responses, but it has also been shown to bind less well to cadherins in the adherens junctions (38). The relationship between the pool of β- catenin associated with cadherins and the pool available for nuclear signaling is under investigation.
APC/Axin:
Along with β-catenin, many color cancers experience changes in the expression or function of APC. Familial adenomatous polyposis (FAP) is a hereditary condition resulting in numerous polyp formations in the large intestine and early- onset colo-rectal cancer. Positional cloning assigned the responsible gene, APC, on the long arm of chromosome 5 . Now, APC is a known tumor suppressor gene (39) and the common inactivating mutations have been characterized as ones that prematurely terminate the protein, abrogating its negative regulatory effects on β-catenin (40). In the assembly of the β-catenin degradation complex, the amount of axin together with APC appears to dictate the amount of degradation complexes available. When axin is lost, spurious β-catenin signaling can result. Thus, axin has tumor suppressive functions. Furthermore, axin has additional roles regulating p53 and myc complexes (41;42).
12 Others:
In addition to these widely studied mechanisms, over-activity of β-catenin can be initiated by a change in the extracellular signaling environment with the overexpression of Wnts and/or receptors (43;44) or loss of natural antagonists
(45). In contrast, Wnt5a shows tumor suppressive capabilities (46;47). The many mutations and dysregulation of Wnts, Fzd receptors, and antagonists identified vary greatly from one tumor to another. After the past 20 years of research concerning the Wnt pathway and carcinogenesis, two things are clear: this is a key pathway with the potential to cause cancer if misappropriated and ongoing study is needed to identify which proteins have the most critical roles and which might be good targets for future therapies.
The Bone Morphogenetic Protein Pathway
Like the Wnt pathway, the BMP pathway is an essential pathway of development and studies concerning it have been ongoing for the past couple of decades.
The BMPs were initially described for their ability to induce ectopic bone formation in cartilage (48). Since then, BMPs have been found to be powerful whole-body morphogens. The BMP family is part of the TGF-β superfamily and contains over 20 cysteine-rich molecules extracellular signaling molecules. An important structural fold in the cysteine rich regions is the C-terminal cystine-knot domain (CTCK) and is found in all BMPs. BMP ligands are expressed in diverse tissues and exert pleiotropic effects including cellular differentiation, adhesion, viability, extracellular matrix formation, angiogenesis, and proliferation (49-51).
13
BMP Signaling
The BMP signaling pathway bears many similarities to TGF-β signaling and is initiated when a BMP homodimer ligand binds a hetero-tetrameric receptor complex that consists of separate type I and type II moieties (52). Specific receptors in the BMP pathway include for type I: (activin-like kinase) ALK-3
(BMPR-IA), ALK-6 (BMPR-IB), and ALK-2 and for type II: BMP receptor-II
(BMPR-II) and ActII-R. Various proteoglycan co-receptors will stabilize this receptor upon ligand binding. If uninhibited binding of the BMP to its receptor tetramer occurs, the constitutively active type II receptors phosphorylate the C- terminal serine/threonine kinase on the type I receptors. Next, the type I
receptors recruit an appropriate receptor-regulated Smad (R-Smad) and this R-
Smad will be phosphorylated and activated. R-Smads pertinent to this pathway
include Smad-1, Smad-5, and Smad-8. Activated R-Smads have a greater
affinity for other Smads.
Depending upon the availability of other Smads in the cells, either 1) the main
common partner Smad (Co-Smad), Smad-4 or -4a will associate with the R-
Smad causing further multimerization with appropriate transcription factors, co- activators or co-repressors molecules, and DNA binding molecules. This new multimer forms a transcription apparatus which translocates into the nucleus through nuclear pore complexes. In the nucleus, these transcription complexes
14 can bind to a large variety of genes that regulate cell cycle and growth; or
dephosphorylation of the activated R-Smad can occur, and an inhibitory Smad (I-
Smad-6, -7) can bind to and inhibit the R-Smad from further interactions (53). If
transcription complexes are formed and enter the nucleus unimpeded,
expression of genes containing Smad Binding Elements (SBEs) in their
promoters can be altered (54).
It is important to note that signaling overlap between BMP and TGF-β signaling
does not just occur by the shared use of Smad-4. Connections between these
analogous pathways are constantly being identified, with many occurring at the
level of receptor cross-talk. Thus far, many of the activin-like (ALK) receptors
have been classified as BMP-binding or TGF-β binding, but there are an
increasing number of examples where BMP-ligands activate TGF-β receptors or
co-receptors and vice versa. This is an important source of pathway regulation and can be confounding when it comes to understanding the contributions of
each pathway in carcinogenesis (55).
BMP Antagonists
It has been proposed that BMPs are released as free signals with paracrine and
autocrine effects on cells, depending upon the microenvironment into which they are released. BMPs and their receptors tend to be rather ubiquitously expressed
in many tissues, so fine-tuning of the pathway must come from BMPs
15 extracellular interactions with other molecules. BMPs are fairly “sticky” molecules and can bind heparin sulfates and other proteoglycans.
Concentrations gradients are established based on the availability of such proteoglycans in the extracellular matrix, free BMP receptor presence on nearby cells, and whether antagonists have been released in the area. The presence of antagonists (BMPAs) in the local environment can affect the ability of BMPs reach the receptor or have an available binding site, thus the antagonists act as local “on/off” switches (56).
There are a variety of molecules with regulatory or inhibitory effects on BMP pathway signaling including both intra- and extracellular varieties. A brief review follows, with particular focus on the extracellular inhibitors as this group presents the most feasible group of therapeutic targets or models for mimetics.
Intracellular Regulatory Molecules
Smads-6 and -7 function as regulatory Smads and their expression is regulated by other Smad signaling complexes as a feedback mechanism (57). Various reports exist on the mechanisms of Smad-6 and -7, whether its binding of phosphorylated Smad complexes and targeting them for degradation with the help of E3 ubiquitin ligases SMURF1 and SMURF2 or inactivation of the ALK receptors by dephosphorylation (58;59). Other intracellular antagonists exist at
16 the transcriptional levels (such as cSki, SnoN) or kinase-dead receptors (BAMBI
(60;61).
Extracellular Antagonists
All known extracellular BMPAs contain structural similarities to the BMPs themselves, mostly within the CTCK domain. Among the secreted antagonists, differences in function appear to be due in part to differences in the arrangements within the CTCK. All extracellular BMPAs are thought to bind to
BMPs directly.
The noggin and chordin family
Members of the noggin family contain a variation of the cystine knot signature with a 9- , 10-, or 12-membered ring (see below for discussion on the CTCK).
Noggin has binding preferences for BMPs-2, and -4 and is the only BMPA to have a solved crystal structure. It has been show that homodimers of noggin
“clip” onto BMP dimers and block receptor-binding epitopes on both parts of the
BMP dimer (62).
Chordin is a larger protein than many of the BMPs and BMPAs (120 kDa as opposed to an average size of 35 kDa) and is a functional antagonist of BMPs when bound to BMPs alone (63). However, chordin can partner with a unique
BMPA, twisted gastrulation (tsg) that can interestingly serve as both a BMP
17 agonist and BMP antagonist. Tsg’s antagonist actions occur in the stabilization
of the BMP-chordin interaction, guaranteeing a more successful sequestration of
BMP ligands from the receptor. Ironically, if BMP-1/Tolloid is present, the BMP-
chordin/tsg ternary complex is more susceptible than chordin/BMP alone to
cleavage by tolloid, releasing free BMP. Therefore, in the presence of tolloid,
chordin, and BMP, tsg can behave as a BMP agonist (64).
DAN Family
The DAN family, contains the BMPAs known as DAN (Differential screening-
selected gene aberrative in neuroblastoma (65)), PRDC (Protein Related to Dan
and Cerberus (66)), gremlin (67), and the dual-Wnt-pathway antagonists:
Cerberus/coco, sclerostin, and SOSTDC1. All contain 8-membered rings in the
CTCK as the BMPs themselves do; however, outside of the CTCK the members
do not display great amounts of homology. Functionally, all inhibit BMP
signaling, particularly BMP-2 and BMP-4. The dual-pathway antagonists play
critical roles in development and are remarkable for the variety of manners in
which the dual-pathway specificity is accomplished.
Roles of BMPs and Antagonists in Cancer
Smad4
Smad-4 was originally identified as DPC4 (Deleted in Pancreatic Cancer) and mutations in Smad-4 are indeed associated with pancreatic and colon cancers
(68). Heritable germ line mutations of Smad4 cause familial juvenile polyposis
18 syndrome (69), showing that Smad4 has tumor suppressive properties in several
disease types. Based on these data, one might hypothesize that TGF-β/BMP
signaling resulting in Smad4 activation is protective/growth-inhibitory. However,
the roles of BMPs are quite diverse in cancers.
BMP downstream targets include multiple cell cycle check point proteins and
proto-oncogenes, including c-myc (53) and p53/p21. Alterations in these
pathways have been observed when expression of BMP antagonists is increased
(70-72). In some tumors, BMP ligand expression is decreased, in others there is
overexpression (73). In some systems, overexpression of BMPAs is beneficial
(74), in others it seems to accompany a more transformed phenotype (75). At this point the picture is quite complex, but there is a growing body of evidence that the BMPs and their antagonists may be pivotal in our understanding of cancer.
As mentioned above, array experiments in collaboration with Human Genome revealed a novel gene product that is downregulated in breast and kidney cancer tissues compared to adjacent normal tissue, SOSTDC1. This protein is a putative dual-regulator of both BMP and Wnt signaling. A brief discussion of the other known dual-pathway antagonists will precede more information about
SOSTDC1.
19 Dual BMP/Wnt Pathway Antagonists
Cerberus/coco
Cerberus acts in Xenopus embryos as a tri-pathway inhibitor with separate
binding sites for Nodal, Wnts, and BMPs (63). Identification of the rodent or
mammalian orthologs for cerberus has been difficult. One candidate is coco,
whose expression is induced by Smad-7 and shows BMP/Wnt pathway
modulation (76). Much remains to be determined regarding these proteins.
Sclerostin
Sclerostin was first identified as the genetic link to the rare bone overgrowth
disorder known as sclerosteosis, which is a continued deposition of new bone
due to increased activity of osteoblasts (77;78). It was determined that families
suffering from sclerosteosis showed mutations in the SOST gene resulting in loss of function mutations. Additionally, deletion of SOST regulatory elements causes a related bone dysplasia known as van Buchem disease (79). By either mechanism, the resulting lack of active sclerostin being secreted from osteocytes causes a loss of inhibitory paracrine signal to the osteoblasts (80;81).
Originally, sclerostin was thought to be a non-classical antagonist of the BMP pathway as it has sequence similar to members of the DAN subfamily of BMP antagonists (82). Some groups reported that Sclerostin binds to BMPs, particularly BMP-6 and -7, and inhibits Smad phosphorylation (83-85). However, further reports showed sclerostin was unable to inhibit ALP production (alkaline
20 phosphatase, a marker of osteocyte activity induced by BMP signaling) as most
BMP antagonists do (80). Thus, sclerostin was hypothesized to be a non-
classical BMP antagonist. The basis for the diverse findings has been under scrutiny. Additionally, sclerostin was shown to antagonize the workings of noggin when the two are co-expressed in a novel method of pathway regulation (86).
More recent work has shown that sclerostin interferes with both Wnt and BMP signaling, in a tissue-context dependent manner (32;87;88). As mentioned
above, sclerostin binds LRP 5 and 6, known co-receptors in the Wnt/Fzd receptor
complex. Sclerostin is also unique from other members of the DAN family of
BMPAs in that it is missing an additional Cys residue that is thought to play a role
in multimerization of the BMPAs (Figure 2). Current data show that sclerostin is
secreted and active as a monomer.
SOSTDC1- A “novel” dual-pathway inhibitor
The human SOSTDC1 gene is located on the short arm of chromosome 7. The
known protein product of this locus is encoded over 2 exons and is 206 amino
acids in length. Analysis of the protein’s primary structure revealed a high usage
of cysteine residues (in the top 5% of all known proteins). SOSTDC1 protein is
positively charged (Arg and Lys residues outnumber Glu and Asp by a differential
of 18-Figure 1) and the computed pI of the entire chain is 9.9 (Proteomics and
Sequencing Tools, Primary Structure Analysis, ExPASy, www.expasy.ch). This
protein was predicted to be secreted from the cell with a high probability that the
first 23 residues comprised a secretory signal peptide (SignalP prediction server-
21 ExPASy, www.expasy.ch). Furthermore, the many cysteine residues within the
protein are arranged in a c-terminal cystine knot pattern (CTCK domain).
Other 30% Cystine 5% Proline Arginine 6% 8%
Lysine Hydrophobic 7% 17% Acidic
6% Hydrophilic
21%
Figure 1. Amino acid profile of the 206 amino acid SOSTDC1 propeptide.
Secondary structure is likely influenced by the high incidence of cysteine
(10 residues) and proline (12 residues). The protein will have an overall
positive charge due to the incidence of basic residues (17 arginine and 14 lysine) compared to acidic (5 aspartate and 8 glutamate).
22 The CTCK domain motif is comprised of at least 6 cysteine residues that form 3 disulfide bonds in such a manner where a two dimensional “ring” formed by
cysteines #2,3,5, and 6 in the primary motif signature is crossed in a third plane
by the disulfide bond between cysteines 1 and 4. The result is a “knot” at the
center of the domain. This arrangement forces amino acids interspersed between the cysteines into three loops or “fingers” from the knot. In crystallized
proteins containing the CTCK (noggin, PDGF, and BMP7), these loops often
assume short β-sheets. In many cases, the cystine knot is a rigid core which
initiates folding of the more flexible loops around it.
In addition to the role in protein folding, the CTCK domain is thought to play a
large role in protein-protein interactions for two reasons: first, hydrophobic
residues that might otherwise be buried in the protein core can be exposed by
the knot structure and secondly, the CTCK domain usually occurs in a very
cysteine-rich area, so other cysteine residues are available for binding between
monomers; many cysteine-knot proteins are active as homo- or heterodimers.
The superfamily of cystine-knot proteins contains a wide array of secreted factors
with essential roles in development, growth, and differentiation. The CTCK
domain is not seen in yeast, but is contained in proteins in C. elegans and
Drosophila, suggesting the roles of CTCK-containing proteins are geared towards intercellular communication. There are three types of cystine-knot motifs: the growth factor knot, the inhibitor knot seen in many types of naturally
23 occurring toxins, and the cyclic knot found in cyclotides (89). All motifs are based
around six core cysteine residues, however only the growth factor knot has the
bond between cysteine #1 and cysteine #4 as the transverse bond (90). The
family containing the growth-factor motif is the largest group of proteins: including
glycoprotein hormones (such as luteinizing hormone, human chorionic
gonadotropin, and follicle stimulating hormone), the TGF-β superfamily (including
TGF-β, activins, and BMPs), the PDGF family, von Willebrand factor (vWF),
nerve growth factor (NGF), norrie disease factor (NDF), the CCN family, mucins,
and slit-like homologues.
Among the cysteine knot superfamily, homology searches and alignments
(Figure 2) identified SOSTDC1 as bearing the most similarity to the CCN family
(28%) and the BMP antagonist (BMPA) family, particularly sclerostin (54%-Figure
3). The highly similar region between sclerostin and SOSTDC1 (SclerOSTin
Domain-Containing 1) is now being referred to as a “sclerostin domain”.
Recently, a solved NMR crystal structure of sclerostin defined probable LRP binding sites, showed a previously un-indentified heparin-binding domain, and
identified greater flexibility than originally anticipated in the cystine knot structure
(91).
24
Figure 2. Alignment of 8-membered ring cystine knot proteins. * = protein with NMR crystal structure known. Shaded boxes show the 8 residues in the ring of the cystine knot. Di-sulfide bonds exist between Cys1 and Cys4,
Cys2 and Cys 5, and Cys3 and Cys6. Arrow points to extra cysteine residue present in many family members, thought to potentiate dimerization. This residue is missing in sclerostin and SOSTDC1.
25
Figure 3. Alignment of SOST (sclerostin) and SOSTDC1 protein sequences.
Orange box shows largest area of 100% identity. Red lines show end of
signal peptide. Yellow circles highlight the 6 conserved cysteines and green box shows the entirety of the cystine knot (CTCK) domain. Overall homology between the two is 54% with homology in the CTCK at 64%
26 From these structural similarities, it can be inferred that SOSTDC1 may have common functional activities to sclerostin. Additionally, extended homology searches to the proteomes of other organisms identifies the following orthologs: ectodin (Mus musculus, 95% homology); USAG-1 (Uterine Sensitization
Associated Gene, Rattus norvegicus, 94%), wise (Xenopus laevis 78%), and zgc:110293 (Danio rerio, 72%).
Known Functions of SOSTDC1 homologs in Animal Models
Wise (Xenopus)
Wise was first identified as both an inhibitor and an activator of Wnt signaling that interacted with Wnt ligand and was able to bind to LRP6, causing some Wnt- independent signaling (92). Later, a mechanism for both BMP and Wnt binding was observed (93). It has lately been shown that another mechanism for the dual agonist/antagonist pathway is the cellular localization of Wise as it is able to bind LRP6 in the endoplasmic reticulum, sequestering it away from Wnt pathway signaling (94). This represents a novel Wnt regulatory paradigm.
Ectodin/USAG-1/SOSTDC1 (Murinae)
In rodents, USAG-1 was first identified as a circulating factor involved in the pre- implantation changes of the uterine epithelium. Shortly thereafter, USAG-1 was shown to be highly expressed in the adult kidney (95) while ectodin was noticed in the developing tooth buds of embryos (96) and was able to affect multiple pathways, including BMP and Wnt signaling simultaneously. Knock-out models
27 have confirmed the importance of ectodin in these tissues as these mice exhibit supernumerary teeth (97;98) and greater resistance to healing after kidney injury
(through antagonism of the pro-healing molecule BMP-7) (99). Little is known about the effects of SOSTDC1 in normal human homeostasis and disease.
There exist no published data concerning SOSTDC1 in cancer. Collaborative data with Human Genome Sciences for this project showed that there was less
SOSTDC1 in tumors compared to normal tissues (particularly in breast and
kidney), so it is logical to hypothesize that it might have tumor suppressor roles
supporting normal homeostasis in adult tissues. While diverse in their etiologies and properties, breast and kidney cancers remain difficult to detect at an early stage. This is crucial as both have the propensity for devastating metastasis as the disease progresses. Thus, the identification of secreted protein products that may be useful in earlier diagnosis or treatment, such as SOSTDC1, is a crucial area for further exploration.
Thesis Goal and Hypothesis
This work examines facets of the BMP and Wnt pathways within the framework of two diseases: renal and breast cancer. The hypothesis is that one molecule,
SOSTDC1, has the ability to modulate both of these pathways in breast and kidney tissue. Furthermore, this ability of SOSTDC1 prevents improper activation of either (or both) pathways. Thus, the end result of SOSTDC1’s actions is to suppress the transformation of normal cells through the spectrum of
28 carcinogenic changes (i.e. hyperproliferation, evasion of death, invasiveness,
and metastatic potential).
The hypothesis is addressed with a three-pronged approach. 1) The expression
of SOSTDC1 in kidney and breast normal and tumor tissues is evaluated as an early step to explore the potential of SOSTDC1’s use as an extracellular marker or therapeutic target of cancer. 2) Characterization of the SOSTDC1 locus and possible dysregulation is addressed through studies of the SOSTDC1 gene and subsequent mRNA expression in kidney and breast cancers. 3) Cell culture models of SOSTDC1 are developed and the phenotypes of SOSTDC1 manipulation in vitro will be assessed to start elucidating SOSTDC1’s functional
capabilities.
Through these studies, the goal of this work is to address the consequences of
SOSTDC1 downregulation in cancer progression, assess the possible affects of
the downregulation of Wnt/BMP signaling, and determine whether SOSTDC1
might be a useful marker or therapeutic target in breast and/or renal lesions.
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38 CHAPTER II:
A HUMAN BONE MORPHOGENETIC PROTEIN ANTAGONIST IS DOWNREGULATED IN RENAL CANCER
Kimberly Rose Blish, Wei Wang, Mark C. Willingham, Wei Du, Charles E. Birse, Surekha R. Krishnan, Julie C. Brown, Gregory A. Hawkins, A. Julian Garvin, Ralph B. D’Agostino, Jr. Frank M. Torti, Suzy V. Torti
The following manuscript was published in Molecular Biology of the Cell
(19(2):457-464, February 2008). Variations in style are due to the requirements of the journal. Figure and manuscript preparation by K.R. Blish. Experiments performed and data gathered by K.R. Blish and Wei Wang (Wnt luciferase assay) with assistance and expertise from W. Du (immunostaining), M.C. Willingham
(immunostaining quantitation and analysis) and J.C. Brown (cell culture). Dot blot and preparation of rhSOSTDC1 by C.E. Birse and S.R. Krishnan at Human
Genome Sciences, Inc. A.J. Garvin and G.A. Hawkins served advisory roles in experimental design. Statistical analyses for immunostaining by R.B. D’Agostino,
Jr. F.M. Torti and S.V. Torti served advisory and editorial roles related to experimental design, data analysis, and manuscript preparation.
39 ABSTRACT
We analyzed expression of candidate genes encoding cell surface or secreted proteins in normal kidney and kidney cancer. This screen identified a BMP
antagonist, SOSTDC1 (SclerOSTin Domain-Containing-1) as downregulated in
kidney tumors. To confirm screening results, we probed cDNA dot blots with
SOSTDC1. SOSTDC1 message was decreased in 20/20 kidney tumors
compared to normal kidney tissue. Immunohistochemistry confirmed significant
decrease of SOSTDC1 protein in clear cell renal carcinomas relative to normal
proximal renal tubule cells (p<0.001). Expression of SOSTDC1 was not
decreased in papillary and chromophobe kidney tumors. SOSTDC1 was
abundantly expressed in podocytes, distal tubules, and transitional epithelia of
the normal kidney. Transfection experiments demonstrated that SOSTDC1 is
secreted and binds to neighboring cells and/or the extracellular matrix.
SOSTDC1 suppresses both BMP-7-induced phosphorylation of R-Smads-1, -5
and -8 and Wnt-3a signaling. Restoration of SOSTDC1 in renal clear carcinoma
cells profoundly suppresses proliferation. Collectively, these results
demonstrate that SOSTDC1 is expressed in the human kidney and decreased in
renal clear cell carcinoma. Since SOSTDC1 suppresses proliferation of renal
carcinoma cells, restoration of SOSTDC1 signaling may represent a novel target
in treatment of renal clear cell carcinoma.
40 INTRODUCTION
Bone morphogenetic proteins (BMPs) are members of the transforming
growth factor-β (TGF-β) superfamily that function as extracellular signaling
proteins (Urist, 1965; Wozney, 1992; Reddi, 2001). BMPs are pleiotropic whole-
body morphogens involved in proliferation, differentiation, and maintenance of
diverse tissue types (ten Dijke et al., 1994; Yamashita et al., 1995). BMP
signaling is primarily mediated though binding to a heterotetrameric complex of
cognate type I and type II receptors (BMPRs). Upon ligand binding, the
serine/threonine kinase on the Type II receptors phosphorylates the Type I
receptors, triggering intracellular Smad signal transduction cascades.
Phosphorylation of the BMP-specific intracellular Smads (Smad-1, -5, and -8)
allows them to bind the partnering co-Smad, Smad4 (Yamashita et al., 1995;
Kawabata et al., 1998; Miyazono et al., 2000; Miyazono et al., 2001; Moustakas
et al., 2001; Chen et al., 2004). This complex of Smads and other DNA binding proteins undergoes nuclear translocation and triggers transcriptional activation of
target genes, including cell cycle regulators or apoptosis mediators such as
p21/Cip1/Waf1, bax, and p53 (Chen et al., 2002; Pouliot and Labrie, 2002;
Fukuda et al., 2006).
Regulation of BMP signaling is accomplished in a tissue-specific manner
via BMP antagonists (Yanagita, 2005; 2006). Classical BMP antagonists bind
BMPs in the extracellular space, preventing the association of BMPs with their
receptors. BMP antagonists may also prevent the activity of BMPs by fostering
41 their retention in the ER (Guidato and Itasaki, 2007). BMP antagonists have been postulated to act as the key ‘on/off’ switches that spatially and temporally
regulate local BMP activity. Thus, BMP antagonists may represent local targets
to control specific tumors.
In addition to their effects on BMP pathways, BMP antagonists have also
been reported to impinge on Wnt signaling, For example, sclerostin exerts its
inhibitory effects on bone formation in mice primarily through inhibition of Wnt
signaling (van Bezooijen et al., 2007); in Xenopus, the BMP antagonist Wise
inhibits or stimulates Wnt signaling in a manner dependent on context (Itasaki et
al., 2003) and cellular localization (Guidato and Itasaki, 2007).
Several genes have been implicated in the development of renal clear cell
carcinoma. These include VHL, an ubiquitin ligase and tumor suppressor, as
well as c-Met and BHD (Linehan et al., 2005; Brugarolas, 2007). To identify novel genes whose expression is altered in renal cancer, we probed arrays of secreted and cell surface cDNAs with cDNA libraries prepared from tumor tissue and matched normal tissue from 20 kidney cancer patients. This screen identified SOSTDC1, a BMP antagonist, as a candidate gene with reduced expression in renal cancer.
SOSTDC1 is orthologous to a recently characterized murine antagonist of
BMPs-2, -4, and -7 termed ectodin/USAG-1 (Mus musculus) (Yanagita et al.,
42 2004). In other species, this protein is known as USAG-1 (Rattus norvegius)
(Yanagita et al., 2004), ectodin (Mus musculus) (Laurikkala et al., 2003; Kassai
et al., 2005), and wise (Xenopus laevis) (Itasaki et al., 2003). Recent mouse
knock-outs of ectodin/USAG-1 have shown that this molecule is important in
tooth formation and modulation of the renoprotective effects of BMP-7 (Kassai et
al., 2005; Yanagita et al., 2006). The human protein is 98% identical to the
rodent protein, and like most members of the BMP and BMP antagonist families,
is predicted to contain a C-terminal cystine-knot motif (Avsian-Kretchmer and
Hsueh, 2004). However, the role of SOSTDC1 in human tissues or human
cancers has not been reported.
In this report, we demonstrate that SOSTDC1 is highly expressed in cells of the normal human kidney, but is significantly decreased in clear cell renal carcinoma.
MATERIALS & METHODS
Recombinant proteins and reagents
Recombinant human BMP-7 (rhBMP-7), noggin-Fc chimera, recombinant mouse Wnt-3a (rmWnt-3a), and DKK1 were purchased from R & D Systems
(Minneapolis, MN). Mammalian expression vectors encoding the open reading frame (ORF) for BMP-7 (pReceiver-M2-BMP7), dickkopf-1 (DKK1; pReceiver-
M2-DKK1), noggin (pReceiver-M02-noggin), and Wnt-3a (pReceiver-M02-Wnt3a) were purchased from GeneCopoeia, Inc. (Germantown, MD). The TCF/LEF
43 luciferase reporter construct Super 8X TOPFlash and the corresponding mutant control Super 8X FOPFlash vectors were obtained from the Addgene Repository
(Cambridge, MA). Plasmids deposited by Dr. Randall Moon (Veeman et al.,
2003).
The following commercial antibodies were obtained: anti-phospho-Smad-1,-5,-8
(Cell Signaling Technologies, Danvers, MA), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Research Diagnostics International, Flanders, NJ), and mouse monoclonal anti-FLAG (Sigma Aldrich, St. Louis, MO).
Production of anti-hSOSTDC1 antisera
Production and affinity purification of an anti-SOSTDC1 peptide polyclonal rabbit anti-sera directed against the 18 C-terminal amino acids of the SOSTDC1 sequence was performed by Quality Control Biochemicals Custom Services
(Hopkinton, MA).
Cells and culture conditions
Human embryonic kidney cells, HEK293, were maintained in Dulbecco’s
Modified Eagle Medium (DMEM; Gibco, Invitrogen,) supplemented with 10% fetal bovine serum (FBS; Gibco, Invitrogen) and 1% penicillin/streptomycin
(Invitrogen). The following kidney cancer cell lines were obtained from the
American Type Culture Collection (ATCC, Rockville, MD): 786-O (CRL-1932, renal clear cell adenocarcinoma), ACHN (CRL-1611, renal cell adenocarcinoma,
44 pleural effusion), 769-P (CRL-1933, derived from primary renal clear cell
adenocarcinoma), A-704 (HTB-45, adenocarcinoma), and Caki-1 (HTB-46, clear
cell carcinoma skin metastasis). All kidney cancer cell lines were grown in
McCoy’s 5A basal media (Gibco) supplemented with 10% FBS, 1%
penicillin/streptomycin, and 200mM L-glutamine. Renal epithelial proximal tubule
cells (RPTECs) were purchased from Cambrex (Hopkinton, MA) and were grown
in Renal Epithelial Basal Medium (REBM) with supplements (Cambrex).
Mammalian Vector Construction and preparation of recombinant, human
SOSTDC1 (rhSOSTDC1)
The open reading frame encoding the SOSTDC1 protein (NCBI:
NM_015464) without the final stop codon was amplified from cDNA by polymerase chain reaction (PCR) and cloned into the BamH1/Asp718 restriction
sites of the pFLAG-CMV5a vector (Invitrogen). The resulting recombinant
human SOSTDC1 (rhSOSTDC1) has the FLAG (DYKDDDDK) epitope added to
its C-terminus.
HEK293 cells were transiently transfected with the pFLAG-CMV5A
SOSTDC1 vector using LipofectAMINE™ reagent (Invitrogen). Conditioned
media was harvested after 2 days and purified using an anti-FLAG M2 affinity
column (Sigma, St. Louis, MO).
45 Tissue Samples and Immunohistochemistry
All tissue samples were used with approval from the Institutional Review
Board at Wake Forest University. Paraffin blocks from the pathology archives
were sectioned at 5 micron intervals. After heat-induced antigen retrieval with
Antigen Unmasking Solution (AUS, Vector Labs, Burlingame, CA), slides were stained with anti-SOSTDC1 polyclonal sera followed by affinity-purified goat anti- rabbit-IgG-HRP (Jackson ImmunoResearch) and diaminobenzidine (DAB) HRP substrate. The slides were counterstained with hematoxylin, viewed under brightfield conditions, and photographed at the same magnification.
Intracellular SOSTDC1 Relative Quantification
SOSTDC1 staining was quantified in Image J (NIH freeware (Rasband,
1997-2007) as mean pixel intensity. Brightfield digital images of the peroxidase-
stained tissues were collected at equal exposure conditions. To avoid bias of cell
selection, an identical grid consisting of 9 equal squares was superimposed over
every image digitally. Only tumor cells crossing predetermined lines of the grid
were included in analysis. Analysis in Image J was completed via the “measure
line” function as follows: a 5 pixel straight line selection was drawn in the
perinuclear region of each candidate cell. Then, the “Measure” command was
chosen from the Analyze menu. The mean pixel intensity value was averaged for 30 separate cells and reported as a percentage of maximal density. Mix pixel intensity was also quantified using Adobe Photoshop (Lehr et al., 1997), which
gave virtually identical results.
46 Statistical Analysis
Statistical analysis was performed by the Biostatistics core of the
Comprehensive Cancer Center of Wake Forest University. A one-way ANOVA was fit to compare the mean percent maximal density of the five groups. Pair wise comparisons were then performed between groups using a conservative p- value of 0.005 for significance (using a Bonferroni adjustment to allow for all 10 pair wise comparisons between groups to be made). Standard deviations for the outcome were compared across the five groups to assure that they were comparable.
cDNA microarray and RNA dot blot
Microarray analyses were performed on tissues from matched normal and tumor tissues from 20 kidney cancer patients and cDNAs from other normal and tumor tissue types. Labeled SOSTDC1 probes were hybridized to the array and resulting signals were scanned and quantified. To independently verify these results, the BD Clontech™ Cancer Profiling Array I, also having 20 matched normal and tumor kidney tissues, was probed with radiolabeled SOSTDC1. The majority of these tissues (~70%) are from patients with renal cell carcinoma, clear type.
rhSOSTDC1 transient transfection and immunofluorescence
pFLAG-CMV5a-SOSTDC1 was transiently transfected into HEK293 cells via LipofectAMINE™ reagent. Twenty-four hours after transfection, cells were
47 washed once in room temperature PBS, and then fixed in 100% methanol at 4°C
for 15 minutes. Cells were labeled with anti-FLAG mouse monoclonal antibody
(Sigma Aldrich) conjugated to rhodamine. Nuclei were stained with 4',6-
Diamidino-2-phenylindole (DAPI, CalBiochem, EMD Biosciences, Inc., San
Diego, CA) and cells were visualized through fluorescent microscopy.
Stimulation of phosphorylated Smad (p-Smad) production with BMP-7 and p-Smad immunoblot
Renal cancer cells were plated in phenol red-free McCoy’s 5A with 5%
FBS and 1% penicillin/streptomycin and allowed to attach overnight. Media was replaced with phenol-red free McCoy’s 5A (Promocell GmBH, Heidelberg,
Germany) containing 0.5% FBS and 1% penicillin/streptomycin with additions of
rhBMP-7 (40 ng/mL), rhnoggin-Fc chimera (150 ng/mL) and/or rhSOSTDC1 (150
ng/mL). Cells were placed at 37°C for either 1 or 3 hours. At harvest, cells were
washed with PBS, and 150 uL of sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) loading buffer added (0.5M Tris pH 6.8, 20%
glycerol, 10% SDS, and 5% beta-mercaptoethanol). Lysates were
electrophoresed on 10% SDS-PAGE gels, transferred to nitrocellulose, and p-
Smad detected by Western blot analysis according to the instructions provided
by the antibody supplier. p-Smad-1,-5,-8 was visualized with PicoWest
chemiluminescent substrate (Pierce Biotechnology, Rockford, IL). GAPDH was
used as a loading control.
48 Wnt signaling luciferase assay
Transfections were performed using Fugene6 (Roche Applied Science,
Indianapolis, IN) according to the manufacturer’s instructions. For each condition,
300,000 HEK293 cells were transfected with 1.76 μg of total plasmid DNA. Each
group was some combination of: 0.25 μg TOPFlash reporter plasmid, 0.01 μg of
internal control pRL-Tk, 0.5 μg pReceiver-M02-Wnt3a expression vector, 1 μg pFLAG-CMV5a-SOSTDC1, or 1 μg pReceiver-M02-DKK1. Empty pFLAG-
CMV5a vector was used to maintain a consistent level of plasmid DNA in each
transfection. Luciferase activity was measured twenty-four hours after
transfection using a Dual Luciferase Assay Kit according to the manufacturer’s
instructions (Promega, Madison, WI).
Transient transfection of SOSTDC1 in kidney cancer cell lines
pFLAG-CMV5a (empty) and pFLAG-CMV5a-SOSTDC1 were transiently
transfected into 769-P renal cancer cell lines using LipofectAMINE™ 2000
(Invitrogen) via manufacturer’s instructions. Twenty-four hours after transfection,
FBS was added to the transfected cell cultures to a final concentration of 5%. To
evaluate the success of transient SOSTDC1 overexpression, cells were
harvested for real-time RT PCR 36 hours after transfection.
Real-time RT PCR evaluation of SOSTDC1 transfected cells
Approximately 2.5 x 106 transfected cells were trypsinized, washed twice
with PBS, and then suspended in Trizol solution (Invitrogen) for preparation of
49 total RNA. RNA was treated with RQ1 RNase-free DNase (Promega) and then purified with the Absolutely RNA® MiniPrep kit (Stratagene, La Jolla, CA).
Reverse transcriptase reactions were performed with the Taqman® Reverse transcription kit (Applied Biosystems, Foster City, CA) per kit instructions.
Resulting cDNA was used in real-time PCR reactions on the ABI Prism 7000
Sequence Detection System machine with SYBR Green PCR Master Mix
(Applied Biosystems) according to manufacturer’s instructions. SOSTDC1 primers used in the real-time analysis were: Forward; 5’-
TGGATTGGAGGAGGCTATGGAACA-3’; Reverse, 5’-
ACTTGCAGGCAGTGACTACTGTGA-3’. Primers to the housekeeping gene
GAPDH were used to standardize for total RNA levels: Forward, 5’-
GAAGGTGAAGGTCGGAGTC- 3’; Reverse, 5’- GAAGATGGTGATGGGATTTC-
3’.
Effect of SOSTDC1, noggin, and DKK1 on proliferation
50,000 cells of the 769-P cell line were plated per well of a 24 well plate.
Cells were transfected with 0.4 μg either pFLAG-CMV5a empty vector control, pFLAG-CMV5a-SOSTDC1, pReceiver-M02-Noggin, or pReceiver-M02-DKK1 with LipofectAMINE™ 2000 as described above. Twenty-four hours after transfection, FBS was added to the transfected cell cultures to a final concentration of 5%. Cells were allowed to recover from transfection for an additional 2 hours before trypsinization and reseeding at 5,000 cells per well in an E-plate for use on the ACEA RT-CES® system (ACEA Biosciences, Inc., San
50 Diego, CA). This system allows real-time monitoring of cultures grown in a 96-
well plate format. Each type of transfected cell was replated in its own
conditioned media.
RESULTS
Downregulation of SOSTDC1 in renal tumors.
To identify novel genes whose expression is altered in cancer, we probed
cDNA arrays of secreted and cell surface genes with cDNA libraries prepared
from tumor tissue and matched normal tissue from 20 renal cell carcinoma
patients. High SOSTDC1 expression was observed in normal kidney tissue, but
mean SOSTDC1 expression decreased by more than one standard deviation in
renal tumors (data not shown). To confirm this finding, a Cancer Profiling Array
(Clontech, Mountain View, CA) containing 500 cDNAs from a variety of tissues,
tumors, and cancer cell lines was probed with radiolabeled SOSTDC1
oligonucleotide. As seen in Figure 1, SOSTDC1 was downregulated in all 20 of
matched normal and tumor kidney sample pairs on this array. Of note, these
matched samples were from a different set of kidney cancer patients than the 20 patients included in the microarray analysis. These results suggest that
SOSTDC1 mRNA is downregulated in a majority of kidney cancers.
51 Figure 1.
Figure 1. Downregulation of human SOSTDC1 mRNA in kidney tumors. cDNA of matched normal and tumor kidney tissue from the same patient was hybridized with radiolabeled DNA oligonucleotides to human
SOSTDC1. N, normal (lane 1); T, tumor (lane 2). SOSTDC1 cDNA decreases in normal versus tumor tissue in 20 out of 20 patients; 17 out of
20 (85%) of tumors show virtually no detectable SOSTDC1 mRNA.
52 Expression of SOSTDC1 within the normal kidney and in kidney cancer.
To test whether expression of the SOSTDC1 protein was similarly
affected, we prepared an anti-SOSTDC1 antibody and performed
immunohistochemical analyses. We first evaluated the distribution of SOSTDC1
protein within the normal human kidney. IHC revealed the presence of
intracellular SOSTDC1 within most epithelial cells of the kidney, including
proximal and distal tubules and transitional urothelium (Figure 2, top panels).
Strong SOSTDC1 signal was observed within the distal tubules, collecting ducts
and podocytes of the glomerulus, with more moderate staining in the cytoplasm of proximal tubule epithelial cells. Quantification of cytoplasmic SOSTDC1 staining intensity in each of these cell types demonstrated that an increase in expression of SOSTDC1 was generally correlated with progression through the nephron and into the collecting ducts and urothelium (Table 1). These results
are consistent with the strong distal tubule expression observed for USAG-1, the
murine homolog of SOSTDC1 (Yanagita et al., 2004).
53 Figure 2. Immunohistochemical analysis of SOSTDC1 expression in normal kidney and renal cancer subtypes. Representative images of IHC
staining with anti-SOSTDC1 antiserum are shown. All images are obtained at
equal magnification and exposure. Left panel represents procedural background
control (without anti-SOSTDC1 primary antibodies) and the right panel depicts
the same tissue stained with anti-SOSTDC1 antiserum. Top row: normal kidney.
G = glomerulus, arrowhead = distal tubule, arrow = proximal tubule. Bottom
rows: renal epithelial or urothelial cancers. RCC, renal cell carcinoma; TCC,
transitional cell carcinoma.
54 Figure 2.
55 Table 1. Quantification of intracellular anti-SOSTDC1 by
immunohistochemical staining.
Normal Cell Type Staining Intensity
(% of Maximum)
Podocyte 45 ± 6
Proximal Tubule 25 ± 4
Distal Tubule 49 ± 4
Collecting Duct 53 ± 4
Renal Urothelium 48 ± 3
Bladder Urothelium 61 ± 5
Table 1. Quantification of intracellular anti-SOSTDC1 by
immunohistochemical staining. Staining intensity was analyzed as
described in Materials and Methods. Means and standard deviations are reported.
We then assessed the expression of SOSTDC1 in a variety of kidney tumor types. Over 75% of kidney tumors are of the clear cell type. Papillary and chromophobe types are also seen. These represent approximately 15 and 5% of all kidney cancers, respectively, and have a more favorable prognosis than clear cell (Iliopoulos, 2006). We investigated SOSTDC1 protein expression in these kidney tumor types and compared them to normal controls. In addition, we
56 examined expression of SOSTDC1 in transitional cell carcinoma of the bladder.
Representative images are shown in Figure 2, and results are quantified in
Figure 3.
As seen in Figure 2, SOSTDC1 expression is markedly diminished in clear
cell renal carcinoma (RCC-clear cell) when compared to normal kidney. In
papillary (RCC-papillary), transitional cell carcinoma (TCC) and chromophobe
types (data not shown), SOSTDC1 staining was intracellular and of higher signal
intensity than the staining in clear cell renal carcinoma. Results of SOSTDC1
protein quantification in various renal cell carcinomas are shown in Figure 3.
Because multiple comparisons were performed, we required a conservative p
value <0.005 for significance (see Materials and Methods). ANOVA analysis
demonstrated a large group difference, p<0.001. RCC-clear cell had significantly lower SOSTDC1 immunostaining than any other group, including normal renal
urothelium (p < 0.001 for all comparisons). Additionally, no RCC-clear cell
sample had higher staining than its likely cell of origin, the proximal tubule cell (p
value also <0.001 see also Table 1). Normal renal urothelium and bladder TCC
groups were not significantly different from one another (p=.137), but were
significantly higher than the proximal tubule and RCC-clear cell groups. The
RCC-Papillary group was significantly higher than RCC-clear cell (p<0.001) and
lower than normal renal urothelium (p=0.0008), but not significantly different from proximal tubules (p=0.03) or bladder TCC (p=.008) based on the Bonferroni correction. Comparison of standard deviations across the five groups indicated
57 that variability was comparable between groups. Thus, immunohistochemical analysis is concordant with SOSTDC1 mRNA analysis, and indicates that
SOSTDC1 is profoundly decreased in RCC-clear cell tumors.
Figure 3.
Figure 3. Quantification of SOSTDC1 staining. Relative SOSTDC1 staining of normal and tumor tissues was quantified as described in Materials and
Methods in 3-9 individuals per group; means and standard deviations are shown. RCC-clear cell has significantly less SOSTDC1 than the other tissues. * = p < 0.001.
58 SOSTDC1 is secreted and antagonizes signaling of BMP-7 in kidney cancer cells
We next assessed functional consequences of SOSTDC1 expression. As classic BMP antagonists function extracellularly, we first tested whether the localization of SOSTDC1 was compatible with an extracellular mode of action. cDNA for C-terminally FLAG-tagged SOSTDC1 was cloned into an expression vector and transiently transfected into HEK293 cells. rhSOSTDC1-FLAG was detected via immunofluorescence after binding of anti-FLAG-rhodamine antibodies to fixed cells (Figure 4). This experiment revealed the presence of
SOSTDC1 predominantly in the secretory apparatus of transfected, highly expressing cells. We transfected cells at low efficiency for this experiment so that each highly expressing transfectant was relatively isolated, enabling visualization of extracellular SOSTDC1-specific signal on the outside of low- expressing cells (Figure 4, right panels). Greatest staining intensity was seen closest to highly expressing cells, with staining intensity decreasing with distance
(Figure 4, center panel). Western blotting also demonstrated the presence of
SOSTDC1 in the media of cultured cells (data not shown). These results demonstrate that SOSTDC1 is secreted and binds to the extracellular matrix and/or cell surface, consistent with an ability to function in an autocrine or paracrine manner.
59 Figure 4.
Figure 4. Normal human SOSTDC1 is secreted and binds to matrix or cell surface proteins around neighboring cells. HEK293 cells were transfected with SOSTDC1-FLAG and expression analyzed using rhodamine- conjugated anti-FLAG antibody (mouse anti-FLAG-rh). Nuclei were stained with DAPI. Top row: color fluorescent image. Bottom row: Corresponding black and white channel of same images. All images obtained at the same magnification and exposure. Left panels: a highly expressing cell (arrow) shows strong signal in the cytoplasm, especially the endoplasmic reticulum and Golgi apparatus. Center panels: secreted SOSTDC1 (small arrowheads) accumulates around both expressing and non-expressing cells. Right panels: Empty vector control transfected cells show no background rhodamine fluorescence.
60 We next tested whether SOSTDC1 acts as a functional antagonist of BMP-7
signaling. BMP-7 binds to BMPRs leading to receptor activation and
phosphorylation of intracellular R-Smads (Smad-1,-5,-8). We predicted that if
SOSTDC1 functions as an extracellular BMP antagonist, it should bind to BMP-7, prevent the BMP-receptor interaction, and stop BMP-7 induced Smad phosphorylation. To test this, cells were treated with BMP-7 in presence and absence of SOSTDC1, and phosphorylation of the BMP-specific R-Smads measured.
As shown in Figure 5, treatment with rhBMP-7 alone caused a rapid and robust increase in phosphorylation of R-Smads-1,-5, and -8 (p-Smad) in cultured kidney cancer (786-O) cells. A similar rapid increase in p-Smad levels was also observed in other kidney cell lines, including ACHN, 769-P, A-704, Caki-1, as well as normal RPTEC cells (data not shown). To test the ability of rhSOSTDC1 to antagonize this effect, rhSOSTDC1 was added to the cells simultaneously with rhBMP-7. Noggin, a classical antagonist of BMP-7, was used as a positive control (Avsian-Kretchmer and Hsueh, 2004; Kawabata et al, 1998). At both 1 hour and 3 hours, rhSOSTDC1 antagonized the BMP-7 induced production of p-
Smad at least as efficiently as noggin (Figure 5). Thus, SOSTDC1 effectively
antagonizes BMP-7 signaling.
61 Figure 5.
Figure 5. rhSOSTDC1 antagonizes the production of phosphorylated Smad
in response to BMP-7 signaling. 786-O cells were treated BMP-7,
SOSTDC1, or noggin as indicated and Smad phosphorylation analyzed by western blotting. SOSTDC1 inhibited the production of p-Smad-1, -5, and -
8 by 3.5 fold. GAPDH was used as a loading control. Note: for clarity, lanes from the same blot have been rearranged. Similar results were observed in the 769-P and A-704 renal adenocarcinoma cell lines (data not shown).
62 SOSTDC1 suppresses Wnt signaling in kidney cells
Since Wise, an ortholog of SOSTDC1, can both stimulate and inhibit the
Wnt pathway (Itasaki et al., 2003), we asked whether SOSTDC1 would affect
Wnt signaling in human kidney cells. Cells were transfected with TOPFlash, a construct containing 8 TCF/LEF transcription factor binding sites upstream of the luciferase gene. Cells were co-transfected with Wnt-3a (to activate the classical
Wnt/β-catenin/TCF signaling pathway) with and without co-transfection with
SOSTDC1 expression vector. Co-transfection with DKK1, a known Wnt inhibitor,
was used as a control. As seen in Figure 6, Wnt-3a increased expression of the
TOPFlash luciferase, and this response was blocked by DKK1. SOSTDC1
inhibited Wnt signaling to an extent comparable to DKK1. Thus, SOSTDC1
suppresses Wnt-3a signaling as well as BMP signaling in human kidney cells.
63 Figure 6.
Figure 6. SOSTDC1 antagonizes Wnt-3a signaling in renal carcinoma cells.
HEK293 cells were transiently transfected with Super 8X TOPFlash luciferase reporter construct plus Wnt-3a and antagonist expression constructs as outlined in Materials & Methods. After 24 hours, resulting luciferase activity was measured for each transfection group and reported as relative luciferase units. Means and standard errors of triplicate experiments are shown.
64 SOSTDC1 suppresses proliferation of RCC-clear cell cultures
Clear cell tumors are the most prevalent histopathologic type of kidney
cancer. Because these tumors showed the greatest change in SOSTDC1 protein expression (Figure 2,3), we asked what functional effects SOSTDC1
might have on these cells. To address this question, the renal cancer clear cell
line 769-P was transiently transfected with the pFLAG-CMV5a-SOSTDC1
expression plasmid, and the effect of SOSTDC1 on proliferation measured. As
shown in Figure 7, SOSTDC1 strikingly inhibited proliferation of 769-P cells:
SOSTDC1 was cytostatic to these cells, while cells transfected with empty vector
proliferated normally. Since SOSTDC1 inhibits BMP and Wnt signaling (Figure
5, 6), we asked whether other inhibitors of these pathways would exert similar effects. Cells were transfected with an expression vector for noggin, an inhibitor
of the BMP pathway, or a vector encoding DKK1, an inhibitor of the Wnt
pathway, and effects on proliferation measured. As seen in Figure 7, inhibition of
either of these pathways also inhibited proliferation, although the effect was more
modest than that seen with SOSTDC1.
65 Figure 7.
Figure 7. Overexpression of SOSTDC1 suppresses proliferation of 769-P
RCC-clear cell cultures. 769-P cells were transiently transfected with pFLAG-CMV5a-SOSTDC1 (open circles), pFLAG-CMV5a empty vector control (darkened circles), pReceiver-M02-noggin (open triangles), or pReceiver-M02-DKK1 (darkened triangles). After transfection recovery, cells were reseeded onto the 96 well ACEA E-plate and loaded into the
ACEA RT-CES system for continuous cell growth monitoring. All treatment groups were seeded in triplicate, means and standard deviations for each group at selected times for a representative experiment are shown here.
66 DISCUSSION
Differential expression of genes in normal and cancer tissue has been
used to study processes involved in malignant change, to identify new tumor
markers, and to identify new targets in tumor therapy. Motivated by these goals,
we sought to identify secretory or cell surface proteins altered in kidney cancer.
Several genes have been implicated in the development of renal clear cell
carcinoma, including VHL, a ubiquitin ligase and tumor suppressor, as well as c-
Met, BHD and sFRP1 (Linehan et al., 2005; Brugarolas, 2007); (Gumz et al.,
2007); however current molecular understanding of the disease is incomplete. To
identify novel genes whose expression is altered in renal cancer, we probed
arrays of secreted and cell surface cDNAs with cDNA libraries prepared from
tumor tissue and matched normal tissue from 20 kidney cancer patients. This
screen identified SOSTDC1, a BMP antagonist, as a candidate gene with
reduced expression in renal cancer.
Studies have previously linked BMPs, the target of BMP antagonists, as
well as BMP receptors, to cancer. For example, aberrant BMP or BMPR
expression has been noted in cancers, including osteosarcomas, breast, kidney,
colon, prostate (Miyazaki et al., 2004; Hsu et al., 2005; Alarmo et al., 2006).
Smad4, the main intracellular target of both BMP and TGF-β signaling, is a known tumor suppressor in pancreatic and intestinal cancer (Alberici et al.,
2006).
67 SOSTDC1 is a BMP antagonist, as evidenced both by its sequence (Vitt et
al., 2001; Avsian-Kretchmer and Hsueh, 2004)and function (Figure 5). Although
BMP antagonists have not been previously implicated in kidney cancer, they
have been implicated in malignant processes in other tissue types. For example,
noggin (a BMP antagonist) suppresses growth of implanted prostate cancer cells
(Feeley et al., 2006). DAN, another BMP antagonist discovered in v-mos
transformed cells, inhibits neoplastic transformation (Chen et al., 2002).
However, the activities of BMP antagonists are complex and may vary. For
example the BMP antagonist gremlin 1 is expressed by stromal cells associated
with esophageal, pancreatic and other cancers (not including the kidney), and
may promote tumor cell proliferation (Sneddon et al., 2006). Our data demonstrate that in renal clear cell cancer, decreases in SOSTDC1 mRNA and
protein are associated with malignant change. These data are the first link
between the BMP antagonist SOSTDC1 and the process of carcinogenesis.
A number of intersections between BMP signaling and regulatory
pathways important in carcinogenesis have been recently identified. These include such key cancer pathways as Wnt/β-catenin, PI3-K/PTEN, and
MAPK/ERK (Aubin et al., 2004; He et al., 2004; Moustakas and Heldin, 2005;
Pardali et al., 2005). Further, BMP signaling directly impinges on the cell cycle
regulatory proteins p21 and Rb: BMP signaling regulates p21/Cip1/Waf1
expression in prostate cancer (Haudenschild et al., 2004), thyroid carcinomas
68 (Franzen and Heldin, 2001), and inhibits phosphorylation of Rb protein in breast
cancer (Ghosh-Choudhury et al., 2000).
We found that in human kidney cells, SOSTDC1 inhibits both BMP-7
(Figure 5,6) and Wnt-3a (Figure 6) signaling. Similarly, previous reports have
indicated that orthologs of SOSTDC1 can affect both BMP and Wnt pathways in
complex ways that are dependent on context and cellular localization. USAG-1,
the murine ortholog of SOSTDC1, binds to BMPs-2, -4 and -7 and antagonizes
BMP activity in Xenopus embryos (Yanagita et al., 2004). Wise, another ortholog
of SOSTDC1 identified in chick and Xenopus, can both activate and antagonize
Wnt signaling during Xenopus development (Itasaki et al., 2003; Guidato and
Itasaki, 2007).
Recent work has directly implicated the Wnt pathway in kidney cancer.
WTX, a negative regulator of Wnt signaling, was identified as a new tumor
suppressor in Wilms tumor, a pediatric form of kidney cancer (Major et al., 2007).
In a separate study using genomic profiling of adult renal cell carcinoma tumors,
loss of sFRP1, a secreted form of the frizzled receptor that acts as an inhibitor of the Wnt pathway, was identified in 15/15 renal cell carcinoma patients (Gumz et al., 2007). Our results indicate that SOSTDC1, a BMP antagonist that can also negatively regulate the Wnt pathway (Figure 6), is similarly downregulated in kidney cancer. Collectively, these results suggest that upregulation of Wnt signaling via reduction of sFRP1, SOSTDC1 or perhaps other as yet unidentified
69 mechanisms, may make a critical contribution to the development of kidney
cancer.
We observed that SOSTDC1 exerts an antiproliferative effect on clear cell
renal carcinoma cells (Figure 7). Similarly, sFRP1 was also reported to
negatively affect cell proliferation (Gumz et al., 2007). Considering that
SOSTDC1 is downregulated in virtually all of the 20 cancer specimens we
analyzed (Figure 1), we speculate that the pervasive decrease in SOSTDC1 in
kidney cancers may arise due to the requirement of the malignant cell to overcome this antiproliferative activity of SOSTDC1. Our evidence further
suggests that inhibition of both BMP and Wnt pathways may underlie the
antiproliferative effect of SOSTDC1. Thus, the BMP antagonist noggin as well as
the Wnt antagonist DKK1 also inhibited proliferation in RCC cells (Figure 7).
The ability of SOSTDC1 to simultaneously antagonize both Wnt and BMP
signaling may explain its enhanced antiproliferative activity when compared to
noggin and DKK alone (Figure 7).
In this manuscript, we identify SOSTDC1 as a BMP antagonist that is
down regulated in renal cancer, particularly clear cell carcinoma, the most
common kidney cancer. In contrast, expression of SOSTDC1 was not decreased
in papillary and chromophobe kidney tumors, which have a more favorable prognosis than clear cell carcinoma. We show a pattern of extracellular secretion in human tissue sections. We observe that reestablishment of SOSTDC1
70 expression in clear cell renal cancer cells profoundly inhibits proliferation of these cells, suggesting that SOSTDC1 is involved in regulating the proliferative capacity of these cancer cells. Finally, we show that SOSTDC1 expression leads to inhibition of both BMP and Wnt signaling. The observations that SOSTDC1 is significantly downregulated in clear cell renal carcinoma and that overexpression of SOSTDC1 inhibits proliferation of clear cell carcinoma cells suggest that modulation of SOSTDC1 may represent a novel therapeutic approach for renal cancers.
71 ACKNOWLEDGEMENTS
1. KRB is supported by the Department of Defense Breast Cancer Research
Program under award number W81XWH-05-1-0287. Views, opinions, and
endorsements by the author(s) do not reflect those of the US Army or the
Department of Defense.
2. Supported by NIH R21 CA119181 (SVT). The authors also gratefully
acknowledge support from the Ben Mynatt family and the Brown
Foundation.
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76 CHAPTER III
LOSS OF HETEROZYGOSITY AFFECTING SOSTDC1 IN RENAL TUMORS
Kimberly R. Blish, Abdoulaye Diallo, Shelley Smith, Seon-Hi Jang, Julie C. Brown, A. J. Garvin, M.C. Willingham, Gregory A. Hawkins, Frank M. Torti, Suzy V. Torti
The following manuscript is for submission to Cancer Epidemiology, Biomarker, and Prevention. Stylistic variations are due to the requirements of the journal.
Figure and manuscript preparation by K.R. Blish. Sample preparation, experiments performed and data gathered by K.R. Blish with DNA sequencing assistance from S. Jang, J.C. Brown, A. Diallo and LOH analysis from S. Smith.
A.J. Garvin and M.C. Willingham served as expert consultants on Wilms Tumor and experiment design. G.A. Hawkins, F.M. Torti, and S.V. Torti served advisory and editorial roles related to experimental design, data analysis, and manuscript preparation.
77 Abstract Loss of heterozygosity (LOH) at 7p21-22 affects 10% of pediatric Wilms renal tumors. SOSTDC1, a bone morphogenetic protein antagonist dysregulated in adult renal tumors, lies within this area of observed LOH. We hypothesized that
LOH or other genetic abnormalities in this area may affect SOSTDC1 in Wilms
and adult renal tumors. Normal and tumor tissue from 25 Wilms patients, 34
adult renal clear cell carcinoma (RCC) patients, and 2 adult oncocytoma patients
were used to obtain genomic DNA. Fifty-one single nucleotide polymorphisms
(SNPs) were genotyped across approximately 2 million bases (MB) surrounding
SOSTDC1 to assess LOH. Direct sequencing of SOSTDC1 exons was also
performed.
We observed LOH within all or part of the 2 MB region in 4/25 (16%) of Wilms
tumors. Additionally, LOH was observed in 5/36 (13.8 %) of adult RCC samples.
Of these samples, LOH within SOSTDC1 was observed in 3 Wilms tumors and 2
adult samples- 1 RCC and 1 oncocytoma. Interestingly, LOH was discontinuous
throughout this region. Direct sequencing confirmed LOH observations. No
additional genetic variations were found from direct sequencing. We conclude
from these studies that LOH in the short arm of chromosome 7 occurs in adult as
well as pediatric renal tumors, and includes SOSTDC1.
78 Introduction
Renal tumors affecting both adults and children are often idiopathic in origin. The clinical presentation, disease history, and treatments of renal tumors differ between children and adults. In children, the majority of renal masses are pediatric Wilms tumors (referred to less often as nephroblastomas). Wilms tumor is the 6th most common malignancy of childhood; affecting approximately 500
children in the U.S. per year (1). Children most often present with palpable
abdominal mass. While lesions respond quite well to treatment, the causes contributing to the tumor are not fully known (2). 1-2% of tumors are associated with familial predisposition and are associated with loci at 17q, FWT1 (3), and
19q FWT2 (4). Over 90% of Wilms tumor cases are sporadic in origin, with the remaining 5-10 % of tumors arising from developmental syndromes which include Beckwith-Wiedemann syndrome, hemihypertrophy, Denys-Drash syndrome, and WAGR (Wilms tumor, Aniridia, Genitourinary malformation, and mental Retardation) syndrome. These syndromes have been instrumental in identifying Wilms tumor suppressor loci (5): WT1 gene, a zinc-finger transcription factor at 11p13 (6, 7) and the putative WT2 locus containing multiple genes at
11p15 (8). Additional studies have highlighted an increased incidence of loss of heterozygosity (LOH) at 16q (9), 1p (10), and 7p (11-13) as other potential loci for Wilms tumor suppressor genes.
Unlike pediatric tumors, adult renal cancers can be difficult to detect and treat.
The most prevalent type of renal tumor is renal clear cell carcinoma (RCC, 80-85
79 % of cases); followed by papillary (5-10%), chromophobe, medullary, and
oncocytic (<5%) types. Incidence and mortality from renal carcinoma has
increased in the past two decades, in part due to the asymptomatic nature of the
tumor and lack of reliable early detection strategies (14). Similar to Wilms tumor, familial tumor syndromes have aided in the discovery of certain genes
involved in the pathogenesis of RCC. These include von Hippel-Lindau on 3p14
(VHL, (15); hereditary papillary renal carcinoma, c-met, on 7q (16); Birt-Hogg-
Dube on 17p (BHD; (17); and fumarate hydratase (FH, (18)). Other genetic
abnormalities noted in kidney cancer include trisomy of 5q, 12, and 20; and
partial or total loss of chromosomes 7, 8, 9, 13q, 14q (19).
Regions of LOH associated with both pediatric and adult renal cancers represent
candidate regions for the location of tumor suppressors whose inactivation may
be critical for the initiation or progression of renal cancer. In both adult and
pediatric tumors, cytogenetic changes have been noted on the short arm of
chromosome 7. These include a 10% incidence of LOH on 7p in Wilms Tumor
(12), loss of 7p and duplication of 7q (20) and consistent gains of chromosome 7
in adult late stage RCC (21) and RCC-papillary subtypes (22, 23). In Wilms
Tumor, the smallest consensus region of LOH has been narrowed to a 2 million base pair (MB) segment within 7p21 (12) containing 10 known genes, including the aryl hydrocarbon receptor (AHR), the mesenchyme homeobox 2 gene
(MEOX2), and SOSTDC1.
80 In this study, we focused on SOSTDC1. SOSTDC1 encodes an extracellular human bone morphogenetic protein (BMP) antagonist (24, 25) that is predominantly expressed in the renal epithelia of the distal tubules, collecting ducts, and urothelium (26). We have previously shown the SOSTDC1 product is
downregulated in approximately 90% of clear cell RCC tumors (14). SOSTDC1
affects two key signaling pathways in the kidney: BMP and Wnt. As changes in
BMP signaling have been noted in a variety of tumors (27-29), including renal
tumors (30), an extracellular modulator of BMP signaling could have potential
tumor suppressor roles within normal kidney epithelia. Similarly, dysregulation of the Wnt pathway often plays a role in tumorigenesis (31). In particular, in Wilms tumor, mutations have been observed in β-catenin, the main intracellular effector of classical Wnt signaling (5); alterations in Wnt signaling have also been implicated in adult renal carcinoma (32).
As SOSTDC1 is a secreted molecule with the potential to modify two cell signaling pathways that are critical to renal development and function, we asked whether SOSTDC1 is contained within a region of LOH at 7p noted in kidney tumors.
81 Materials and Methods
Collection of Tissues
Approval was obtained from the Institutional Review Board at Wake Forest
University for the retrieval of matched normal and tumor tissues from the Tumor
Bank of the Wake Forest University Comprehensive Cancer Center. Matched
tissues were collected for 36 adult kidney cancer patients and 7 pediatric Wilms tumor patients. Information concerning the patients’ primary diagnosis was collected; however no patient identifiers were obtained. An additional 18 matched normal and Wilms tumor tissues were obtained from the Cooperative
Human Tissue Network (CHTN), which is funded by the National Cancer
Institute. Other investigators may have received samples from these same
tissues. Diagnosis and treatment information were made available for each
tissue. All tissues were collected as snap frozen specimens stored at -80°C.
Sample preparation and Genomic DNA isolation
Each snap frozen tissue was sectioned on a bed of dry ice to ensure minimal
thawing during sample preparation. An approximately 30-50 mg piece of tissue
was cut and an adjacent piece of tissue was cut and embedded in OCT
cryomounting material (Tissue-Tek, Elkhart, IN) to make frozen blocks for future
cryosectioning and histology if desired. Genomic DNA was isolated from tissue samples via homogenization in ice cold lysis buffer (10mM Tris pH 8.0, 0.1M
EDTA, 0.5% SDS, 100 µg/ml Proteinase K, 25 µg/ml RNAase); followed by subsequent phenol chloroform extraction as has been previously described (33).
82 Integrity and concentration of each resulting DNA sample was assessed by
agarose gel electrophoresis.
Sequencing Primer Design
The known coding region of SOSTDC1 is contained in two exons. However,
other potentially transcribed areas have been identified in the University of
California Santa Clara Genome database (UCSC; http://genome.ucsc.edu, (34,
35)). Two of these areas occur upstream of the coding region and one small area occurs between the known coding exons for a total of 5 putative exons or regulatory regions at this locus (Figure 1). Primers were designed for direct sequencing of each putative exon or regulatory region for a total of 14 pairs direct sequencing primers (Table S1). All primers were synthesized by IDT (Coralville,
Iowa).
PCR Amplification and Direct Sequencing
Each direct sequencing primer pair was used to amplify all 5 putative regions of interest in each normal and tumor sample using PCR. PCR and sequencing
reactions were performed with 60 ng of genomic DNA template at 60°C for 40
cycles. Resulting data from the ABI 3730XL was analyzed using Sequencher
software (GeneCodes, Ann Arbor, MI v. 4.7).
83 Loss of Heterozygosity Analysis
To examine the area surrounding SOSTDC1 for loss of heterozygosity, we genotyped single nucleotide polymorphisms (SNPs). All SNPs within the 2.4 MB region as found at HapMap (www.hapmap.org) were examined and 51 SNPs with high percentage of heterozygosity (>0.45) were chosen. These SNPs are fairly well distributed over the region (Figure 2). Primers for each SNP were designed for analysis on the MassARRAY system (Sequenom, San Diego, CA
(Table S2). All primers were synthesized by IDT. 5 ng genomic DNA from each sample was used in the genotyping reactions.
84 Figure 1.
Figure 1. SOSTDC1 locus. Summarized from the Genome Browser hosted at UCSC. Diagram shows relative positions of designed primer pairs to potential regions of interest within the gene (arrows). Gene translation start codon is in exon 3 and stop codon is in exon 5. All known coding sequence in contained in exons 3 and 5.
(Next page)
Figure 2. Map of known gene loci over 2 million base pair region of interest in LOH study. Approximate locations of genes and the 51 SNP markers used in this study shown on chromosome 7p21 map from physical location
15400000 to 18000000. Terminal location is at the top, while centrosomal end of map is at the bottom.
85 Figure 2.
86 Results and Discussion
Loss of Heterozygosity in 7p21 Wilms tumors
In Wilms tumors, SNP genotyping over the 2 MB region at 7p21.1 to 7p21.2
revealed LOH in 3 of the 25 tumors we genotyped (Figure 3). These samples
included a patient with hemihypertrophy being evaluated for Beckwidth-
Wiedemann syndrome with a Stage II tumor that showed complete LOH at every
informative SNP in the region (W733); a patient with multifocal Wilms also
showing complete LOH at every informative SNP (W8188); and a patient with
anaplastic Wilms (W8194), showing one instance of LOH at SNP rs15711849, near MEOX2.
Direct sequencing of the SOSTDC1 allele revealed one additional patient,
W8197, with one instance of LOH affecting the 3’ UTR in Exon 5 of SOSTDC1; all other sequences in this patient showed no informative SNPs. Direct sequencing also confirmed that LOH directly affects SOSTDC1 in patients W733 and W8188, as every heterozygous SNP in the normal was lost in the tumor
(Table 1). Patient W8194 had no informative SNPs seen in the direct sequence of SOSTDC1, so it was not possible to ascertain whether this patient exhibited
LOH at SOSTDCI. Sequence analysis revealed no mutations within known exons (3 and 5) or candidate exons (1, 2, and 4) of the remaining SOSTDC1 allele.
87 Figure 3.
Figure 3. LOH analysis in 2MB region. Results from positive samples aligned with the 7p21.1 to 7p21.2 SNP map from Figure 2. For each patient’s row, black boxes indicate regions where all genotyped SNPS show LOH in the tumor samples. Gray blocks indicate regions of uninformative SNPs in between observed LOH; therefore, these areas may be regions of LOH as well. Unmarked areas of each sample indicate informative SNPs where no LOH was observed. The dotted lines highlight the region covered by SOSTDC1. We note 3 samples (2 Wilms and 1 RCC show a large region of LOH that includes either the entire genotyped region, or a ~1 MB region including SOSTDC1 (RCC-614). LOH does not appear to center around a particular gene. RCC = adult renal carcinoma samples; W = pediatric Wilms tumor samples.
88 Table 1. Results of direct sequencing of SOSTDC1 locus. All SNPs found in the direct sequences are summarized here. All other samples sequences showed no LOH or other mutations. SNP location relative to sequenced exons and chromosome 7 base pair location provided where known. The existence of heterozygous SNPs (informative, but no LOH present) in the sample is shown via yes/no designation. RCC = adult renal carcinoma samples, W = pediatric Wilms tumors.
Table 1. Direct Sequencing of SOSTDC1 Results Sample Location Informative SNPs Normal Tumor without LOH RCC-129 End of Exon 1- rs35324397 Yes A/G G RCC-614 Beginning of Exon 1: Yes G/T, A/G T, A 16,536,670; 16,536,667 between rs10240242 and rs35324397 RCC-614 Beginning of Exon 1: Yes C/G C 16,536,641 between rs10240242 and rs35324397 RCC-614 End of Exon 1- rs35324397 Yes C/G C RCC-614 End of Exon 1- 5 bp Yes A/G G downstream of rs35324397 RCC-635 Beginning of Exon 1: Yes C/G C 16,536,641 between rs10240242 and rs35324397 RCC-737 Exon 5: 16468252 closest to rs Yes G/T T 6959246 W-733 Before Exon 1: rs7781903 No C/T C W-733 Beginning of Exon 1 between No C/G G rs10240242 and rs35324397 W-733 Beginning of Exon 2: No C/T C rs7801569 W-8188 Beginning of Exon 2: No C/T C rs7801569 W-8197 Exon 5: 16468252 closest to rs No G/T T 6959246
89 To assess whether expression levels of SOSTC1 are affected in Wilms tumor patients, we queried the Oncomine database (36). As seen in Figure 4, there is a highly significant (p< 0.00001) loss of SOSTDC1 in Wilms Tumors compared to normal fetal kidney and adult clear cell sarcomas (median of normal tissue =
1.116 compared to Wilms media value of -0.948, Figure 4).
Loss of Heterozygosity in adult renal tumors
Three of the 36 adult patients we analyzed showed LOH in the 2MB region of interest (Figure 3). Two of these patients had clear cell renal carcinoma (RCC-1,
RCC-614); while one had a far less common oncocytoma (RCC-635). Patient
RCC-614 showed LOH over much of the area, while RCC-1 and RCC-635 showed LOH at approximately 15-20% of informative SNPs.
Direct sequencing of SOSTDC1 exons in adult tumors also showed LOH in patients RCC-614 and RCC-635 in several locations of exon 1. Additionally, patients RCC-129 and RCC-737 also showed LOH in one SNP each. Similar to
what was observed on the large scale LOH study, all of the adult tumors showed
LOH at some heterozygous loci, but not others- even within the SOSTDC1 gene
itself- while all of the Wilms tumors showed complete LOH at every heterozygous
allele. Among all samples (adult and pediatric), LOH within SOSTDC1 was
observed mostly in exon 1; with no observed heterozygosity in the known
transcribed parts of the gene. Whether adult or Wilms, for each SNP that
90 Figure 4. Oncomine database shows significant SOSTDC1 downregulation
in adult renal clear cell tumors and pediatric Wilms tumors. The
ONCOMINE database was queried for all studies involving markers in
SOSTDC1. Results of studies by Cutcliffe et al (Wilms tumors vs. normal or adult tumors, (43)) and Bittner et al (Adult clear cell carcinoma vs. other
RCC subtypes, http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE2109 ) were
compared using the software available on the site. Dots above and below
the boxes show sample maximum and minimum values respectively. The
horizontal lines show the spread of the values from starting at the 10%
value through the 90%, with the box highlighting the range of 25% to 75%.
Dark boxes show the normal or control tissues for each study, white boxes
show adult clear cell RCC and Wilms tumor respectively. The horizontal
black bar through each box shows the median value for the sample. ** = p<
0.00001, normal or control tissue compared to adult RCC or Wilms tumors.
91 Figure 4.
92 showed LOH in more than one sample, the same allele was lost. For example, at the beginning of exon (position 16,536,641) the G is lost out of the C/G.
Comparison between LOH in Wilms and adult renal tumors
Overall, we observed LOH at the SOSTDC1 gene in 4/25 (16%) of Wilms tumor patients. This frequency is comparable to WT1, the prototype of Wilms tumor suppressor genes, which occurs in sporadically in Wilms tumor, often associated with WAGR Syndrome. We also observed LOH including the SOSTDC1 in adult
RCC at a similar frequency (5/36 or 14%). It should be borne in mind that current treatment of Wilms tumors sometimes involves pre-surgical chemotherapy or radiation. Differences in treatment status within the patient population may have effects on the resulting tissues used to obtain genomic DNA and thus the results of the LOH studies.
LOH in Wilms tumors appears to occur in large sections on the short arm of chromosome 7, as seen in W733 and W8188. This is concordant with previous studies. Interestingly, two patients (W8194 and W8197) showed examples of just one instance of LOH each. Due to distances between LOH markers for patient W8194 (approximately 100 KB), and a lack of informative SNPs in
SOSTDC1, it is hard to estimate whether this region of LOH extends beyond the
SOSTDC1 locus. Patient W8197 showed 1 incidence of LOH in the direct sequence; since no other informative SNPs were found within the direct sequence, this may represent either LOH affecting SOSTDC1 or a point
93 mutation. It is noteworthy that tumor size, stage, histology, and treatment status
varied among these patients.
In contrast to what we observed in Wilms tumors, in adult tumors, regions of LOH
were noncontiguous or “patchy” as SNPs showing LOH were broken up by
heterozygous alleles. Due to the high incidence of aneuploidy in these tumors, this phenomenon may be partially explained by chromosomal copy number variation. Indeed, multiple studies referenced in the Database of Genomic
Variants show variations in copy number that affect parts of the 2 MB region; including the area around SOSTDC1 (http://projects.tcag.ca/cgi-
bin/variation/tbrowse?source=hg18&table=Locus&rnum=50&rstart=101; 37).
We have previously reported downregulation of both the message (90% of
patients) and protein of SOSTDC1 in RCC-clear cell tumors. Results presented
here demonstrate for the first time that LOH in SOSTDC1 affects pediatric as well
as adult (Fig. 3) renal tumors, and that SOSTDC1 expression is downregulated in
pediatric tumors (Fig. 4). However, while LOH may play a role in the regulation
of this locus in some patients, other mechanisms, including epigenetic regulation, must also be considered. For example, promoter methylation has been shown to have an important role in regulation of the IGF2 gene (38-40) and loci at 11p13 and 11p15 (41) in Wilms tumor. Improper splicing, a mechanism that contributes
to dysregulation of the Wilms tumor suppressor gene WT1, must also be
considered (42). Future studies on the role and regulation of SOSTDC1 in
94 pediatric and adult kidney cancer may be particularly important given the potential utility of SOSTDC1 (or SOSTDC1 mimetics) as a therapeutic agent that targets both BMP and Wnt pathway signaling.
95 Supplemental Data
Table S1. Direct Sequencing Primers for SOSTDC1. All primers designed to potential exons or regulatory regions of SOSTDC1 and were optimized for 60°C reaction temperatures. Target exon, forward and reverse primer sequence and amplicon size shown.
Table S1. Primers for Direct Sequencing of SOSTDC1 Title Sequence Amplicon (Exon, #) 1-1 F CCAGCCATTCTACCTCCAGG 1-1 R TGAAGTGTGTGCATTTTGTATTCA 785 1-2 F TGCATAGTGTTTGGGGTGG 1-2 R TGAAGTGTGTGCATTTTGTATTCA 877 2-1 F GGCAGTTCCCCTGCACAT 2-1 R GGCCTGAAGGGAGGTGAAG 604 2-2 F TGGTTTGACCAGTCCCCACT 2-2 R ATAGTTCTCCACACAATCTCCTCA 656 3-1 F TAGATTCAGGAAAGGAAATGGC 3-1 R ACTTACTGTTCCGATCCAGTCC 572 3-2 F TCCCACCCCTTCTCTGTGTT 3-2 R ATGGTCATTTTGCATGATTTTG 680 3-3 F ACACCTGAATGAACGCCAAACCTC 3-3 R TAGGGAAGAATGCCAACCTGCACA 495 4 F CTTACACAAATCTTTTGCCTCTCC 4 R ATCAGGAGTTTCACTTCATCTCTG 520 5-1 F CATGAAAGTGTCCCTATACTATCCA 5-1 R CTAACTCATGCTGTGCTTGCT 597 5-2 F GTACTGGAGCAGGAGGAGCT 5-2 R AGGAAGATCACTCATGGCTGC 750 5-3 F TCAGGACCTTCTTTGGGAATAG 5-3 R GGTCAAGACACCTTCTGATTGC 810 5-4 F CGCTTGGAATGGAATGCC 5-4 R AATGAGCAGCAGACTTGGCA 668 5-5 F CCTGCCAGTGCTCCCTAACT 5-5 R CATTCCAAGCGAGGGTCAG 902
96 Table S2. Primers for LOH SNP Genotyping. All primers designed for use on the Sequenom MassARRAY platform. Percentage of heterozygosity
(informative SNPs) shown in two populations from the International
HapMap Project: CEU = Utah residents with Northern and Western
European Ancestry; YRI = Samples from Yoruban descent Ibadan, Nigeria.
UEP = Unextended Primer.
Table S2. Primers for Loss of Heterozygosity Analysis Marker % Informative Sequence CEU YRI rs4598143 1-ACGTTGGATGCAGTGTTTTTCTTTACAGAG 2- ACGTTGGATGGAGAACTCAATTTGCTTCCTC 53.4 64.5 UEP- TTGCTTCCTCTTGTTTATCCTTC rs6944475 1- ACGTTGGATGGAGACTATATTTTCTAGCCCC 2- ACGTTGGATGGTTATTATCACTCACAGTGC 55 69.5 UEP- gggAACAGTTCCGTAAACCTATCAAG rs11982985 1- ACGTTGGATGGCATTACTTATCTGTAGTTTC 2- ACGTTGGATGACCAGAGCTGGCATTCTTTC 45.6 90.8 UEP- ctGTGTTTGCCCTCTCTTT rs7783003 1- ACGTTGGATGGTCCGGATTCCATACTTTGC 2- ACGTTGGATGTCCCTGGTAACCTTACCTAC 47.4 81.4 UEP- gcACCTTACCTACAATTGTTTACATA rs10950562 1- ACGTTGGATGATGGTACCAGCTCGTCTTTG 2- ACGTTGGATGCCTACCAACCAAAAAACGCC 47.4 76.3 UEP- AAACGCCTAGGATCAAAC rs6946099 1- ACGTTGGATGGACAAACTCAGGTATTATG 2- ACGTTGGATGCGACCCCAATTACTTCCTAC 45.8 87.5 UEP- TGTCAGACTATATTTTCCTCTTCA rs6942413 1- ACGTTGGATGCTCCCGAGAAAACTTGTTGG 2- ACGTTGGATGCCTCCCTCCCTCAATTTATG 48.3 27.6 UEP- cccCCCTCAATTTATGAATTACAGAA rs37410 1- ACGTTGGATGGAGTTCATAGTGAATGATTCC 2- ACGTTGGATGCCCCAAACCCAATAAACCAC 50.8 17.5 UEP- AACCACTAATCTGCTTTCAGTC rs38176 1- ACGTTGGATGCTCAACGTTAAGCATGGTTC 2- ACGTTGGATGGAGCTCTGCAGAATGGAAAC 53.3 95.8 UEP- GAAACCCACTTCAAGTTGT rs13231687 1- ACGTTGGATGAGTTGAACTGTAGCATCCGC 2- ACGTTGGATGCTGCGAATTCACAGCAGTAG 47.5 60 UEP- CAGCAGTAGGTTGGGAT
97 rs9648211 1- ACGTTGGATGTGTTGCCCTATCTCATAAGC 2- ACGTTGGATGCCTTTGCTAACCTTACGGAC 55 88.3 UEP- GGACCATTTCCCAATACTG rs10277151 1- ACGTTGGATGCATTGCAGAATGTTCTCTCC 2- ACGTTGGATGCCATAATAAAGCTGGAAGAG 50 75.4 UEP- ttgGCTGGAAGAGTATTGACTCAG rs7777150 1- ACGTTGGATGATAAAAGGGTGGTACAAAGG 2-ACGTTGGATGTCTTTACATCCCAGAGTAGG 53.4 70.8 UEP- TCCCAGAGTAGGAATCACCTAT rs6963052 1- ACGTTGGATGCATGTCTAAACCACTGGAAA 2- ACGTTGGATGTAGCAGGTACACTTGCTCAC 54.6 76.8 UEP- cTGCTCACTGATATCCCTCTT rs12699778 1- ACGTTGGATGGAAGAAAGGAATGGAAGCAG 2- ACGTTGGATGCTTCCTGGATCTTTGGTTGG 47.5 96.7 UEP- TTGGTTGGTTTCTAACATTGATT rs2178598 1- ACGTTGGATGGGGTATCTCTGAGTTCTGTC 2- ACGTTGGATGGCCACAATAATCTCAACAGG 46.6 47.1 UEP- CTCAACAGGAAAGATTTCATATAA rs2704674 1- ACGTTGGATGTGGAAGTTGTCCATGTGCTC 2- ACGTTGGATGTGGTAGACACAGAGATGCAC 52.5 46.7 UEP- cCAAGGAAGGGCTTGTTG rs10270965 1- ACGTTGGATGCTATTTATAATGCAGAAACC 2- ACGTTGGATGTACAGGCATGAGCCATCGT 56.6 48 UEP- atAGTTTTCCTGGTTTTTTTATATTG rs6959566 1- ACGTTGGATGGGGACAGTGTAAAGCACAAT 2- ACGTTGGATGCTGGAGATAGTCTGACTAGC 45 23.3 UEP- TGTTTGACCTCAAGAAAAATT rs1524362 1- ACGTTGGATGTCACTTTTGTCATGTCTTG 2- ACGTTGGATGTGTTAATAGGCCACATGACC 54.4 55.9 UEP- TCTGTCTGTAATTTTCCATGA rs6968649 1- ACGTTGGATGGTATGGAGCAGAAGTAGAAG 2- ACGTTGGATGGCTTGCTAGGCTTTCCAAAC 55 40 UEP- cACCTGGCCTAGCCCTTTTGC rs1524358 1- ACGTTGGATGTTCGTCTTCTCTTTCCCCTC 2- ACGTTGGATGGTTACTTAACCAAGGATTAGC 45.8 40.8 UEP- AACCAAGGATTAGCAGAAAAA rs578621 1- ACGTTGGATGGGCGGAGTGATTCAAAATAG 2- ACGTTGGATGCAAAACAGTTTTGACAGGATG 49.2 62.5 UEP- cACAGTTTTGACAGGATGTTCTCAT rs479202 1- ACGTTGGATGTGTCTTTGGCTTGGAAGTGG 2- ACGTTGGATGATGAATGTGAGAGTGGCAGG 53.9 / UEP- CCAAATGCCTAGAGAGAA rs818488 1- ACGTTGGATGTATGTCCCCTTGGGAATGTG 2- ACGTTGGATGCCTGGTGATTTGGATGCAAG 45.2 8.5 UEP- ATGCAAGCTTGGGCTTA rs706059 46.6 5.3 1- ACGTTGGATGCCTGGCCTGTAGGCATTTTT
98 2- ACGTTGGATGGTACAGTGAAAAATACGAGAC UEP- acAAATACGAGACAAATGTGAGAGAC rs12112188 1- ACGTTGGATGCCTTACACAGTAACAGGGAC 2- ACGTTGGATGAGCCCACCTGGGTAATAATC 47.3 86.7 UEP- GAGACCCTTAACCACAC rs2389710 1- ACGTTGGATGGGCTGCAATAGATACACCAC 2- ACGTTGGATGGACATTTCCTCATTCCTCCC 50.8 / UEP- cTCCTCATTCCTCCCATTATACT rs860196 1- ACGTTGGATGGGCACTAATCCAGGAGTTTG 2- ACGTTGGATGTGCTATGACTTACTCTTTGG 45.5 0 UEP- TGACTTACTCTTTGGAACTAT rs17137106 1- ACGTTGGATGGTTCCTATGTTATCTCTCTG 2- ACGTTGGATGTACCTATGTTTTTCTTCAC 52.8 90 UEP- ggTAGGTGGTACCAGGCTCA rs7799920 1- ACGTTGGATGTTCCTGATGTTGGCAATTAG 2- ACGTTGGATGACGAAAAGCTGTTTCTTTG 50.8 / UEP- gaTGTTTCTTTGAATGATCAATAAGA rs4719491 1- ACGTTGGATGCCTACATTCTTTTTACCCAG 2- ACGTTGGATGTTGGGATGGAGTGTTGAGTG 47.5 77.5 UEP- tgTCTGAAAGTTTTCTCTTTACTCTC rs6957270 1- ACGTTGGATGAGACTTGGGAAAGCTAAGTG 2- ACGTTGGATGAGGAGAGGACTCTATGAGAC 52.5 74.2 UEP- CCAAACCTGAACTCTACCA rs11971406 1- ACGTTGGATGCAGATCCTCAGATCATCTCC 2- ACGTTGGATGAAGGAGAAGCCAAGTAGAGG 45.5 72.9 UEP- aGGAACCATGGAGCCAAGT rs12531256 1- ACGTTGGATGTTTGTGGGATTCAGCTGAAC 2- ACGTTGGATGGTTAGAAGTAAAACAGCGCC 52.5 70.3 UEP- CAGCGCCTGATTTGACC rs687265 1- ACGTTGGATGCAACAAATCTAGGAGTTG 2- ACGTTGGATGCATCATTTCTAATTGTCTTA 53.3 45.8 UEP- tgATTAGTCTATTTAGTGGCCTAT rs524715 1- ACGTTGGATGACAGTCCTTCTAACCTTCCC 2- ACGTTGGATGTAGGACTGGACCCTTAACTG 45.7 70.3 UEP- GTCAGGGTAGGCAAGAA rs652187 1- ACGTTGGATGGGAGAACAAATAAACACTG 2- ACGTTGGATGTGGTCACTTCTCTCAATTGC 55 20 UEP- ctACTCATCATTACTGTTTTTAGT rs9638747 1- ACGTTGGATGCTTAGGAATCCTCTTCTCGC 2- ACGTTGGATGACTTCCTACTTGAGAGACCC 48.3 59.1 UEP- TTGAGAGACCCCTAACATAG rs6964052 1- ACGTTGGATGTTGACAAGCTAATCCATAG 2- ACGTTGGATGTATCTGTAGCCTTCTCTGGG 55 84.2 UEP- ccgAAATGTAGACTTCCTCTCAGTTAT rs9785042 1- ACGTTGGATGGATTCTTTATCTGACATGCC 45.8 55.9 2- ACGTTGGATGGGAACAGAACAGAAAGCCCA
99 UEP- cAAGCCCAAAAATAAATCCACGCT rs12699872 1- ACGTTGGATGCCCTCTCCCTCTCAAATATA 2- ACGTTGGATGGTGGATGGAATTATTCAGA 54.2 90 UEP- GATGGAATTATTCAGAAAGTTGAG rs1562632 1- ACGTTGGATGTCTACTTGTGCCTAGATGCC 2- ACGTTGGATGACATCTTATTGCCAAGAGTG 54.2 94.8 UEP- gATTGCCAAGAGTGGCACCAGACC rs7788582 1- ACGTTGGATGCAGAAGGGATTATCAAGGTG 2- ACGTTGGATGAAGACTTTAGTCCTTTCACC 54.3 45 UEP- AAATGACCACACTAGCCAAG rs2192073 1- ACGTTGGATGTTGTCTTCTACAAGGCCTAT 2- ACGTTGGATGGGGAGGTTGAAGTGGATTAC 54.2 17.2 UEP- ccTACCCTGCTAGTGGGATTGTA rs10235024 1- ACGTTGGATGTCTAAAAAGTAGCACTTTC 2- ACGTTGGATGGTTTTGGGAGGTTATTTGAG 46.7 13.3 UEP- =TAAGGGAAGAATTAGCAATAGTTA rs12672575 1- ACGTTGGATGTTGCTCACTTGCTTGCTCTC 2- ACGTTGGATGCCTCACTTTCTGGCCAAATG 47.4 33.3 UEP- GGCAACTTACAACATGG rs10950672 1- ACGTTGGATGAGGAGATCGTCCTTGATAAC 2- ACGTTGGATGCTACTACCTCACAGAACAAG 45.8 61.7 UEP- ggTCTGGAGAAAGTTGGTAGCA rs2723548 1- ACGTTGGATGCGTCATCCTCCATTACCTTG 2- ACGTTGGATGATGGGCGAGATGAGCAGAAA 54.2 59.3 UEP- GCGAGATGAGCAGAAAACAATA rs2110013 1- ACGTTGGATGACAGCACTCTAGCCTGTGT 2- ACGTTGGATGGCTCTATTGTTTAGGATGAG 48.3 17.9 UEP- CTTCAGACAGGGTCTTG rs1051603 1- ACGTTGGATGGACCAGATACAGATGAGAAG 2- ACGTTGGATGCTGCACTCTACAATTAACGTC 45.8 1.7 UEP- ATTAACGTCTCCAAAAGTTAATA
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40. Haruta, M., Arai, Y., Sugawara, W., Watanabe, N., Honda, S., Ohshima, J., Soejima, H., Nakadate, H., Okita, H., Hata, J., Fukuzawa, M., and Kaneko, Y. Duplication of paternal IGF2 or loss of maternal IGF2 imprinting occurs in half of Wilms tumors with various structural WT1 abnormalities. Genes Chromosomes Cancer, 47: 712-27, 2008.
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105 Chapter IV
SOSTDC1, AN ANTI-PROLIFERATIVE BMP ANTAGONIST, IS POORLY
EXPRESSED IN PATIENTS WITH ADVANCED BREAST CANCER
Kimberly R. Blish, Matthew A. Triplette, Wei Du, Mark C. Willingham, Timothy E.
Kute, Charles E. Birse, Surekha R. Krishnan, Julie C. Brown, Ralph B.
D’Agostino, Frank M. Torti, and Suzy V. Torti
The following manuscript was prepared for future submission to Breast Cancer
Research. Stylistic variations are due to the requirements of the journal. Figure and manuscript preparation by K.R. Blish. Experiments performed and data gathered by K.R. Blish and M.A. Triplette (Western blots from cell lines and tissue microarray quantitation) with assistance and expertise from W. Du
(immunostaining) and J.C. Brown (cell culture). Dot blot and preparation of rhSOSTDC1 by C.E. Birse and S.R. Krishnan at Human Genome Sciences, Inc.
Collaboration and analysis of tissue microarray data accomplished with T.E. Kute and M.C. Willingham; statistical analyses by R.B. D’Agostino, Jr. F.M. Torti and
S.V. Torti served advisory and editorial roles related to experimental design, data analysis, and figure preparation.
106 Abstract
Introduction: Bone morphogenetic proteins (BMPs) are extracellular signaling molecules that accomplish important tasks in development and maintenance of normal adult tissues. Although the data are varied, multiple BMPs and receptors are associated with breast cancer, demonstrating the need for regulation of this pathway. This project focuses on the BMP antagonist sclerostin domain- containing-1 (SOSTDC1). Current knowledge concerning SOSTDC1 has focused on its expression in the murine kidney, bones, and teeth and potential roles in progression of nephropathies and there are no published studies on SOSTDC1’s expression or actions in breast cancer.
Methods & Results We analyzed the expression of SOSTDC1 protein using in vitro cell culture models of breast cancer progression, and found that less
SOSTDC1 is secreted from transformed cells. A significant decrease of
SOSTDC1 message is observed in ~90% of breast cancer patients, with the greatest loss in metastatic disease. We studied SOSTDC1 protein expression in tissue microarrays containing samples equally from recurrent and non-recurrent disease in African American and non-African American populations. Data from these tumor microarrays has shown that SOSTDC1 negatively correlates with tumor size and stage; demonstrating that less expression is associated with more advanced disease. Additionally, there are strong correlations between
SOSTDC1 expression and the expression of known breast cancer prognostic markers EGFR, IGFR1 and PTEN.
107 We made recombinant SOSTDC1 protein and report that SOSTDC1 is a functional extracellular antagonist of BMP-7 signaling in MCF7 adenocarcinoma cells. Finally, we observed that exogenous SOSTDC1 suppresses proliferation in MCF7 cells.
Conclusions: Taken together these data suggest that proper levels of secreted
SOSTDC1 are important to the homeostasis of normal cell signaling and proliferation. Restoration of SOSTDC1 to breast tumors may have future therapeutic potential.
108 Introduction
Breast cancer is a group of related neoplastic diseases, each with their own
variations in etiology, pathology, and clinical course. The majority of cases
(>90%) are epithelial tumors including: adenomas, intraductal papilloma, Paget’s
disease, ductal carcinoma in situ (DCIS) and lobular carcinoma in situ (LCIS),
infiltrating ductal carcinomas (including inflammatory disease), and infiltrating lobular carcinomas. As of 2008, there is an annual age-adjusted incidence rate
of 99.8 per 100,000 and mortality rate of 20.3 per 100,000 for all types of breast
cancer (1). While advances in academic knowledge and technologic savvy
caused a decrease in mortality rates by 2.2% from 1990-2004 (2;3); much
remains to be discovered regarding the pathology of breast tumors to develop
more efficacious treatment and prevention strategies.
One approach for identifying new therapeutic targets has been to study common
regulatory and developmental signaling pathways in cancer cells (4-8). Many of
these pathways include known oncogenes or tumor suppressor genes and affect
cell cycle, apoptosis, or proliferation regulation. Either inappropriate activation or
disintegration of these pathways can provide causal impetus for carcinogenesis.
One pathway of interest involves members of the transforming growth factor
(TGF-β) superfamily, the bone morphogenetic proteins (BMPs, 9).
After first identification as ectopic bone formation inducers in vivo (10), BMPs have been shown to be organism-wide morphogens and are expressed in many
109 tissues (11-13). The mammalian BMP family members largely function as
extracellular cell-cell communication molecules. Each is expressed in unique
patterns throughout development and into adulthood and has varying roles in the
adult organism. Changes in cellular morphology or function are induced through
the activation of intracellular R-Smad proteins (Smads-1,-5, and -8) after BMP
ligands bind hetero-tetrameric BMP receptor complexes at the cell surface.
Constitutively active serine/threonine kinases on BMP Type II receptors
phosphorylate the Type I receptors, leading to subsequent phosphorylation and
activation of the R-Smads (14). This pathway is analogous to TGF-β signaling
and also shares the use of the “common” Smad protein (15), Smad-4 in the
formation of the Smad signaling complex that then translocates to the nucleus
and causes changes in expression of genes bearing Smad binding regulatory elements (SBEs, (16). Alterations in BMP pathway signaling have been documented to affect cell survival, proliferation rates, differentiation, invasiveness, and migration; all processes that can contribute to the formation and aggressiveness of cancer (17-20).
In normal breast tissues, few BMPs are expressed at detectable levels and the expression of BMP ligands, receptors, and signaling molecules in breast cancers varies (21-24). There are many reports linking BMP activity or lack of BMP regulation to invasive breast cancer. However, the overall contributions of BMP signaling to breast carcinogenesis remain complex and at times contradictory, as the exact in vivo contributions of the BMP ligands are difficult to understand. For
110 example, BMP-2 is anti-proliferative on breast epithelial cells at low
concentrations, affecting cell cycle proteins p21, Rb, and the tumor suppressor
PTEN (21;25;26). Both BMP-2 and BMP-6 counteract proliferation induced by estrogen (27).
However, other evidence supporting the pro-cancer actions of BMP signaling
include: Overexpression of BMP-4 (28) and BMP-7 and BMP type I receptor has
been documented in tumors (22;29). Additionally, BMP-2 has been shown to be
pro-angiogenic with the ability to aid in cell survival during hypoxia (30;31).
Nuclear staining of activated phospho-Smad-1,-5,-8 is increased in primary
breast tumors, and bony metastasis (32). Overexpression of dominant-negative
TGF-β type II receptors or BMP type II receptors limits BMP signaling and inhibits
the invasive potential of breast cancer cell models (32-34).
While the collective contribution of BMPs signaling in the etiology and
progression of tumors remains complex, the fact remains that careful regulation of this pathway is necessary for normal tissue homeostasis. Thus, the extracellular regulators or antagonists of BMP signaling become important foci of
research and may have therapeutic potential (35).
This study focuses on the regulation of BMPs in breast cancer via a new
antagonist named SOSTDC1 (SclerOSTin Domain-Containing-1). Current
knowledge concerning SOSTDC1 (and its orthologs USAG-1 (36), ectodin ((37),
111 or wise (38)) focuses on its expression in the murine kidney, bones, and teeth.
SOSTDC1 knock-out mouse models show supernumerary teeth formation indicating a role for SOSTDC1 as an integrator of multiple developmental pathways (39-41). In humans, the BMP-7/SOSTDC1 relationship is being evaluated for potential roles in modulation of nephropathies (42;43). We have previously reported on the downregulation of and anti-proliferative capability of
SOSTDC1 in renal carcinomas (44). However, no published reports currently exist for the expression or activity of SOSTDC1 in breast tissue.
As SOSTDC1 is an antagonist of BMP-2, 4, and 7 (45), these findings suggest that SOSTDC1 could have a role in suppression of the cancer phenotype, we hypothesized that SOSTDC1 is a repressor of breast carcinogenesis through extracellular antagonism of BMP signaling. We tested this hypothesis through the following studies: 1) Expression of SOSTDC1 in breast tissues and whether that expression is changed during carcinogenesis; 2) Ability of SOSTDC1 to module BMP signals in breast cells; and 3) whether SOSTDC1 affects the transformed phenotype of in vitro models of breast cancer.
112 Materials & Methods
Recombinant proteins and reagents
Recombinant human BMP-7 (rhBMP-7) and noggin-Fc chimera were purchased
from R & D Systems (Minneapolis, MN). The following commercial antibodies
were obtained: anti-phospho-Smad-1,5,8 (Cell Signaling Technologies, Danvers,
MA), anti-glutaraldehyde-3-phosphate dehydrogenase (GAPDH, Research
Diagnostics International, Flanders, NJ), horseradish peroxidase (HRP)-
conjugated anti-rabbit IgG (Biorad, Hercules, CA), and goat anti-rabbit IgG-HRP
(Jackson Immunoresearch, West Grove, PA). The generation of anti-
hSOSTDC1 antiserum, the construction of rhSOSTDC1 mammalian expression
vectors and production of rhSOSTDC1 protein have been previously described
(44).
Cells and culture conditions
The mammary epithelial adenocarcinoma cell line MCF7 (ATCC HTB-22) was
maintained in DMEM/F12 media supplemented with 10% fetal bovine serum
(FBS, Gibco/Invitrogen, Carlsbad, CA) and 1% penicillin/streptomycin (P/S,
Invitrogen). The ductal carcinoma cell line T47D (HTB-133) was maintained in
RPMI 1640 media with supplements as described by the ATCC. Human
mammary epithelial cells (HMECs) were purchased from Cambrex and
maintained in MEGM media (Cambrex, MA). The HMEC stepwise-transformed cell lines were a gift from the lab of R. Weinberg and were maintained according to previously published protocol (46).
113 Detection of cell-associated and secreted SOSTDC1 in breast cells
HMECs, four lines of HMEC-transformed cells from the stepwise model of breast
cancer progression (hTERT, SV40 T antigen, and H-ras overexpressing cells),
MCF7, and T47D cell lines were all weaned to a defined media system over a period of two-weeks. All cell lines were seeded at equal density, and were maintained in FBS-free media for 72 hours until conditioned media removed and
cells harvested by scraping and lysed. Conditioned medias were filter-sterilized
and snap-frozen until concentration. 30 mLs of each conditioned media were
concentrated ~300 fold with Amicon and Microcon 10,000 kDa cutoff spin filters
(Millipore, Billerica, MA). Total protein concentrations of each cell lysate and
concentrated media samples were determined by Bradford or BCA assay
colorimetric assays. Equal amounts of cellular lysates or concentrated media
were electrophoresed via SDS-PAGE. Nitrocellulose blots were stained with
Ponceau S as a loading control and then probed with anti-human-SOSTDC1.
RNA dot blot
BD Clontech™ Cancer Profiling Array I was probed with radiolabeled SOSTDC1 per manufacturer’s instructions. Tissues in the array included matched normal and tumor tissues from 50 breast cancer patients (including three patients with samples from both primary tumor and metastatic site. Resulting signals were scanned and quantified with UnScanIt (Silk Scientific, Orem, UT).
114 Tissue microarrays and immunohistochemistry
The creation of tissue microarrays of tumor cores from 198 breast cancer
patients with recurring tumors has been previously described (3). The blocks of
arrayed tissues were sectioned and fixed on slides. Each section was
deparaffinized with xylene, rehydrated, treated with 0.3% hydrogen peroxide, and
antigen unmasking solution (AUS, Jackson Labs). After blocking, slides were
incubated with anti-SOSTDC1 primary antibody in 1% BSA-PBS, followed by
(HRP) - conjugated secondary antibody to rabbit IgG. DAB substrate was used
to indicate location of antibody binding, followed by counterstaining with
hematoxylin. Bright field digital images of each core in the analysis (n = 81) were
obtained using 20x magnification at equal exposure conditions.
Intracellular SOSTDC1 quantification and statistical analysis
The intracellular staining intensity of these images was quantified in Adobe
Photoshop as mean pixel density based on previously reported protocol (47).
Briefly, bright field digital images were adjusted identically to full RBG “Red-Blue-
Green” ranges. Random areas of cytoplasm from 10 separate cells were
selected with the “magic wand tool” and mean pixel intensity was quantitated
using the “histogram” function of total luminosity. Mean pixel intensity was
averaged for the 10 separate cells and reported as a percentage of maximal
intensity. The intensity for the control image (no primary antibody) was
subtracted as background from the percentage of intensity for its corresponding
115 SOSTDC1 image for tissue, thus obtaining an absolute intensity reading for each sample.
The resulting 81 SOSTDC1 absolute intensity values were imported into SAS statistical analysis software (Cary, NC) along with samples values for patient age, race (African-American or other), recurrence and survival info, tumor size, grade, stage, lymph node involvement, metastasis occurrence and metastasis location. SOSTDC1 staining intensity was individually correlated to each of these variables by calculation of Pearson’s coefficient. Additionally, SOSTDC1
staining intensity was compared to survival times (length in years) via
construction of Kaplan-Meier estimates. Finally, staining intensity of SOSTDC1 was correlated to staining of known biomarkers: PTEN, EGFR, and IGFR-1 previously obtained in these microarray samples (48).
Stimulation of phosphorylated Smad (pSmad) production with BMP-7
MCF7 breast cancer cells were plated in DMEM/F12 with 5% FBS and 1% P/S and allowed to attach overnight. Treatment media was plain DMEM/F12 plus some combination of rhBMP-7 (75 ng/mL), rhnoggin-Fc chimera (150 ng/mL), or rhSOSTDC1 (150 ng/mL). After 4 hours, cells were harvested directly into SDS-
PAGE loading buffer. Lysates were run on SDS-PAGE gels followed by pSmad and GAPDH detection by Western blot.
116 Proliferation experiments
MCF7 cells were transiently transfected in OptiMEM media (Gibco, Invitrogen)
with pFLAG-CMV5a (empty, Sigma) and pFLAG-CMV5a-SOSTDC1 using
LipofectAMINE™ 2000 (Invitrogen) via manufacturer’s instructions. 6 hours after transfection, FBS was added to the transfected cell cultures to a final concentration of 5%. 24 hours after transfection, cells were trypsanized and reseeded at 5,000 cells per well in an E-plate for use on the ACEA RT-CES® system (ACEA Biosciences, Inc., San Diego, CA). This system allows real-time monitoring of cultures grown in a 96-well plate format. Each type of transfected cell was re-plated in its own conditioned media. The remaining cells from each transfection were plated at 200,000 per well in a 6-well plate for timelapse bright field photography study of cell viability and morphology.
117 Results & Discussion
SOSTDC1 expression in cell culture models and normal breast samples
As SOSTDC1 levels have not previously been reported in normal breast tissues or available cell models of breast cancer, we first examined SOSTDC1 protein via immunohistochemistry and immunoblotting. Normal human mammary tissues express SOSTDC1 in both ductal and lobular epithelial cells (Figure 1).
Additionally, human mammary epithelial cells (HMECs) also express SOSTDC1 endogenously and secrete mature protein into the culture media (Figure 2).
Figure 1.
Figure 1. Immunohistochemistry of SOSTDC1 in normal and breast tumor tissues. Representative pictures shown. Rabbit-anti-human SOSTDC1 serum was used to stain paraffin sections of normal (left) and breast tumor tissue (right) from the same patient. Moderate intracellular SOSTDC1 staining is seen in the epithelia and ducts of normal tissue while the tumor tissue shows approximately 60% less SOSTDC1 in the tumor cells.
118 Loss of mature, extracellular SOSTDC1 expression in conditioned media of
breast cancer cell lines
We next examined endogenous SOSTDC1 in a panel of breast cancer cells
including a model of breast cancer progression made by step-wise stable
expression of telomerase, SV40 small and large T antigen, and h-ras in HMECs
(46). SOSTDC1 was detected at comparable levels in the whole-cell lysates in
every cell line studied (Figure 2, top panel). As cultures were harvested by scraping, it is assumed that all cell membrane-associated proteins would be in
the cellular lysate.
All known activities of SOSTDC1 occur in the extracellular space, thus the
amount of mature SOSTDC1 in the conditioned media is biologically relevant.
We found that secreted SOSTDC1 was undetectable in the supernatants from all
transformed cell lines; including HMECs expressing SV40 T antigens and ras
oncogene (Figure 2, middle panel). Interestingly, HMECs with over-expression
of telomerase still expressed some extracellular SOSTDC1 (although less than
the normal HMECs) but once another oncogene was introduced (SV40 T
antigens) no secreted SOSTDC1 was detected.
We wanted to ensure the lack of secreted SOSTDC1 was not solely attributable
to changes in the secretory pathway characteristic of cancer cells (49), so the
conditioned medias were analyzed by SDS-PAGE with two different loading
strategies. First, equal amounts of total media proteins were analyzed and
119 immunoblot only revealed the SOSTDC1 band doublet in the conditioned media of HMECs and HMEC-hTERT cells (data not shown). In the event that the secretion machinery of the transformed cells was affecting outcome, media proteins were loaded on a second gel as a function of total intracellular protein.
Ponceau S stain for this blot showed some variation in the amount of total protein in the conditioned media, presumably showing different secretion rates of the cultures (Figure 2, bottom panel). The observable amounts of SOSTDC1 in the media did not differ (Figure 2, middle panel), so we conclude the observed decrease of secreted SOSTDC1 is not due solely to changes in the overall secretion mechanism.
The explanation for the lack of free, extracellular SOSTDC1 is a subject of ongoing study in our laboratory. One possibility is that a change in the expression or secretion of proteolytic factors is affecting the rate of SOSTDC1 degradation in more transformed cells. Current experiments with protease inhibitors are addressing this. Genetic experiments or RNA studies would be necessary to determine whether mutated SOSTDC1 or alternative splice variants exist that are not efficiently secreted. Another possibility is that other regulatory molecules, such as binding partners or chaperones, are affecting the expression, sorting, or secretion of SOSTDC1. Feedback loops commonly exist where BMP signaling causes the upregulation of inhibitors (50) and we have observed changes in SOSTDC1 expression in response to BMP-7 treatment (unpublished data). Expression of the known BMP ligands that SOSTDC1 binds (BMPs -2, -4,
120 -7, -6) and BMP receptors as well as co-localization studies would start to
address these issues.
Figure 2.
Figure 2. Secreted SOSTDC1 decreases in mammary cells with increasing
transformation. A rabbit anti-human SOSTDC1 antibody was used to probe
Western blots of various normal and cancer cell lysates and culture
supernatants. Top row, cell-associated protein: 26 μg of total cellular protein for each cell lysate were loaded and immunoblotted with anti-
SOSTDC1. Recombinant human SOSTDC1 (rSOSTDC1, 20 ng) was used as a positive control for bands at 32 KDa. All normal and cancer cell lines show comparable amounts of endogenous, cell-associated SOSTDC1.
Middle panel: SOSTDC1 has sites for N-glycosylation and is usually detectable extracellularly as a doublet of bands at 32 kDa. Conditioned culture media representing equal amounts of total intracellular culture
121 protein were concentrated for SDS-PAGE- the Ponceau S stain of this blot
(bottom panel) shows some differences in total protein loaded, possibly
reflecting different secretion rates of the cultures. No secreted SOSTDC1
was detected in transformed cells or cancer cell lines. HMECs = Human
Mammary Epithelial Cells; hTERT = HMECs stably overexpressing human
telomerase; LT = hTERT cells also stably expressing the large and small
SV40 T antigens causing inactivation of p53, Rb pathways; H-ras V21- lo
and –hi = LT cells also stably expressing mutant H-ras at low and high
levels (HMERs). MCF7, T47D breast cancer cells also used in this study.
SOSTDC1 message is downregulated in breast tumors
We next asked whether downregulation of SOSTDC1 expression also occurs in
clinical samples. A commercially-available cDNA dot blot containing matched
normal and tumor samples from breast cancer patients (Cancer Profiling Array I,
Clontech) was probed using radiolabeled SOSTDC1 oligonucleotide (Figure 3A).
SOSTDC1 mRNA levels noticeably decrease in 47/53 (89%) of tumors compared
to matched normal tissue. We also observe that baseline levels of SOSTDC1
expression can vary greatly from one individual to another. SOSTDC1
expression is lowest in those patients with metastatic disease (Figure 3B). This observation is consistent with recent theories concerning the roles of BMPs in promoting invasion and metastasis in breast cancer. It has been suggested that like TGF-β (51;52), BMPs when properly expressed inhibit proliferation of normal
122 breast epithelia or early stage lesions; however, when BMPs are overexpressed
in malignant cells a pro-invasive state is achieved (32).
Figure 3.
Figure 3. Quantification of SOSTDC1 message in breast cancer patients.
A. Clontech’s Cancer Profiling Array I of 53 matched normal and tumor
tissues (both primary and distant metastases) was probed with anti-
SOSTDC1 radiolabled primers. SOSTDC1 message visibly decreases
between normal and tumor tissue in at least 47 (89%) sample pairs and
disappears almost entirely in 16 (30%) sample pairs. Lanes 1 & 3, N =
Normal tissue; Lanes 2 & 4, T = tumor tissue. B. The resulting blot was scanned and dot intensity quantitated with UnScanIt Image Quantification
123 software. Results are summarized in the graph on the right. Average staining intensity of tumors is ~60% of all normal tissues (* = p < 0.05), while metastasis show even less SOSTDC1 (** = p <0.01).
SOSTDC1 protein is lowest in patients with advanced disease
As SOSTDC1 message is downregulated in many tumors, we asked whether protein levels are also decreased. To study this as thoroughly as possible we performed immunohistochemistry (IHC) on breast cancer tissue microarrays
(TMAs). The samples on these TMAs were evenly divided between recurrence rates and African American race status, to ascertain whether differentially expressed proteins predict the imbalance in recurrence rates frequently seen in
African American populations (3). A total of 5 normal breast samples and 81 breast cancer tissues from the microarrays stained with anti-SOSTDC1 serum were included in the analysis. Average intensity of SOSTDC1 staining was compared to a variety of clinical parameters including: recurrence rate, race
(African American vs. non), tumor size, tumor stage, tumor grade, hormone receptor status, cell cycle percentage information, number of positive lymph nodes, occurrence and site of metastasis, time to recurrence, survival time, and disease-metastasis free survival.
We observed a decrease in SOSTDC1 protein from normal breast tissue to breast tumors in 3 matched pairs (Figure 1 for an example). More matched samples would need to be examined to draw further conclusions about the
124 frequency of loss of SOSTDC1 protein in the population. We found a large range of expression of SOSTDC1 in the tumors, which fell in a fairly normal distribution across the sample set (Figure 4). This agrees with the SOSTDC1 dot blot, which also showed a wide range of baseline mRNA expression levels.
Comparing SOSTDC1 to other parameters, SOSTDC1 did not correlate significantly with race (p = 0.99 , recurrence (p = 0.12) , or hormone receptor status (p = 0.62 for progesterone receptor, p = .28 for estrogen receptor). We found significant associations between: SOSTDC1 expression and tumor size, tumor stage. An association between SOSTDC1 and tumor grade approached significance (Table 1). Larger, more advanced tumors have less SOSTDC1. As there was no association with SOSTDC1 expression and race or recurrence rates, we conclude that loss of SOSTDC1 may be a negative prognostic factor across all racial groups. These data leads us to hypothesize that loss of
SOSTDC1 may allow the development of more aggressive tumors.
125 Figure 4.
14
12
10
8
6
# of tissues 4
2
0
-0 -5 0 5 0 5 0 5 0 5 0 5 0 5 0 -1 -1 -2 -2 -3 -3 -4 -4 -5 -5 -6 - 5 0 5 0 5 0 5 0 5 0 5 1 1 2 2 3 3 4 4 5 5 Range Density Values
Figure 4. SOSTDC1 protein levels by immunohistochemistry for 81 breast tumors. Frequency distribution histogram of SOSTDC1 staining intensities over all samples quantified. Staining intensity was calculated as a function
SOSTDC-1 stained pixel density minus control (no primary antibody) sample pixel density.
Relationship of SOSTDC1 to disease metastasis-free survival (DMFS)
We next compared SOSTDC1 expression to incidence of metastasis and survival times (ranging from <1 to 15 years) for the patient samples in the TMAs. We found that those patients with the highest levels of SOSTDC1 had a median survival time of 8.5 years (n = 19) compared to those with the lowest SOSTDC1 expression, median survival time of 5.4 years. However, Kaplan-Meier analysis
126 of these data did not result in a significant difference (p= 0.15). It is possible that for this analysis, the sample size was too small.
In this population, SOSTDC1 expression did not significantly correlate with overall occurrence of metastasis, site-specific metastasis or DMFS. Again, further studies in a larger group will be necessary to ascertain whether sample size is an issue here, or whether there is discordance between the downregulation of SOSTDC1 message seen in metastasis (Figure 3) and the amount of SOSTDC1 protein levels.
Table 1: Correlations Between SOSTDC1 Staining Intensity and Tumor Parameters
Stage Mean N Tumor Mean N Grade Mean N SOSTDC1 size SOSTDC1 SOSTDC1 (mm3) 1 37.3 12 0-20 36.9 18 1 43.0 4 2 35.3 41 20-50 33.2 42 2 33.0 15 3 26.5 12 >50 20.6 12 3 31.03 37 4 25.7 3 Pearson: -0.289 Pearson: -0.351 Pearson: -0.203 P = 0.012 p = 0.0025 p = 0.105
Table 1. SOSTDC1 staining of TMAs- significant correlations. Pearson’s correlation coefficient determined for average SOSTDC1 intensity versus tumor stage, size, and grade. The Pearson score and p values are shown for each comparison.
127 Relationship of SOSTDC1 to known prognostic markers of breast cancer
Through the TMA study, we evaluated SOSTDC1’s potential as a prognostic marker through comparisons to other known prognostic markers and/or proposed tumor suppressors such as IGFR-1 (insulin-like growth factor receptor-1), EGFR, and PTEN (phosphatase and tensin homolog). SOSTDC1 expression significantly correlates with expression of all of these markers, but in different manners (Table 2). SOSTDC1 expression positively correlates with PTEN and
IGFR-1 expression (higher levels of SOSTDC1 are found in samples with higher levels of these gene products); grouping SOSTDC1 with protective markers and known tumor suppressors. In contrast, SOSTDC1 has a strong negative correlation with EGFR expression. As high levels of EGFR are a known negative prognostic marker, the negative association still supports the hypothesis that high
SOSTDC1 levels are protective.
As discussed above, SOSTDC1 expression did not significantly correlate with survival. We have speculated this could be due to small sample size in this TMA staining. Lending some support to this idea, both PTEN and EGFR expression are known to be significantly associated with survival rates in breast cancer (53-
55); however, in our population, neither PTEN nor EGFR were associated significantly with survival (p=0.5597 for EGFR and .7250 for PTEN).
128
Table 2: Correlations Between SOSTDC1 Staining Intensity and Staining Intensity of Various Prognostic Markers EGFR IGFR-1 PTEN PC: -0.323 PC: 0.331 PC: 0.395 SOSTDC1 p:0.0048 p: 0.0067 p: 0.0006 N: 75 N: 66 N: 72
Table 2. Comparisons between SOSTDC1 staining intensity and other tumor markers in the TMA tissues. PC = Pearson’s Coefficient, p values, and number of observations used in each comparison are shown.
SOSTDC1 antagonizes the signaling of BMPs in breast cancer
As the effects of BMPs on breast cancer cells are diverse, we next asked whether SOSTDC1 antagonizes BMP signaling in breast cancer cells and what the functional consequences of such antagonism might involve. We established a model of BMP/SOSTDC1 activity in a breast cancer cell system. Several breast cancer cell lines were cultured and treated with varying concentrations of recombinant, human BMP-7 (rhBMP-7) ligand for 0, ½, 1, 2, 4, 6, and 10 hours. rhBMP-7 ligand was chosen both because breast cancers have been shown to overexpress BMP-7 (22;56) and SOSTDC1 binds to BMP-7 with highest affinity of BMP ligands -2, -4, -6, and -7 (45). After treatment, the presence of BMP- response Smads (R-Smads-1,-5,-8) was assessed via Western blot with anti- phospho-Smad-1-5-8. MCF7, T47D, and HMEC-Ras/HMER cells (see description in Figure 2) all responded to BMP-7 with a noticeable increase in
129 phosphorylated R-Smad between 2 and 8 hours (data not shown). MCF7 cells
showed the most robust response at 4 hours of treatment time and were used in
further experiments to test the ability of SOSTDC1 to antagonize the BMP-7-
induced rise in phospho-R Smads-1,-5,-8.
Co-treatment with rhBMP-7 and recombinant, human SOSTDC1 (rhSOSTDC1)
showed a great reduction in the amount of phosphorylated R-Smad produced
compared to BMP-7 alone (Figure 5). rhSOSTDC1 was able to suppress
phospho-Smad formation more effectively than the “classical” BMP antagonist
noggin. The efficiency of SOSTDC1’s antagonism of BMP-7 in a breast cancer
model led us to hypothesize:if breast epithelial cells lost their expression of
SOSTDC1, the proper balance of BMP signals maintaining homeostasis could be jeopardized. As BMPs have various effects on the cell viability and proliferation
rates, we explored the functional consequences of overexpression of SOSTDC1.
130 Figure 5.
Figure 5. SOSTDC1 inhibits BMP-7-initiated phosphorylation of intracellular
R-Smads-1,-5, and -8. Western blots of phosphorylated R-Smad-1,-5,-8 and
GAPDH from MCF7 breast adenocarcinoma cell lysates after treatment with recombinant human BMP-7 (75 ng/mL) for 4 hours with or without inhibitors. Cells treated with BMP-7 showed transmission of BMP signal with an increase in pSmad (lane 2 compared to lane 1 control). Cells concurrently treated with a 2-fold excess of SOSTDC1 or the known BMP
antagonist noggin showed lesser amounts of pSmad (lanes 3 and 4);
SOSTDC1 co-treatment inhibited pSmad formation by BMP-7 signaling to a
greater extent than noggin.
131 SOSTDC1 suppresses proliferation of MCF7 breast cancer cells
MCF-7 cells transiently transfected with either pFLAG-CMV5a-SOSTDC1 vector
or empty vector control were plated on an E-plate and culture growth monitored
on an ACEA biosystems RT-CES machine for 6 days. Empty vector control cells adhered to the plate within the first 10-12 hours after plating and then started to proliferate over the rest of the time period. In contrast, SOSTDC1-expressing cells showed some adherence to the plate during the first 12-hours, but then cells showed no signs of proliferation (Figure 6A). Additionally, this phenomenon of proliferation suppression was not rescued with addition of BMP-2 or BMP-7 to the culture media at the time of plating (data not shown). Transfected cells not plated in the E-plate were observed for morphogenetic changes via time lapse microscopy. Representative pictures show rounding up of SOSTDC1-MCF7 cells with occasional blebs and spiking (Figure 6B). After 3 days of culture, the cells in the time lapse experiment were harvested and viable cells counted with trypan blue. Control cultures proliferated 5.6 fold, SOSTDC1 overexpressing cultures only 1.4 fold.
This profound block of culture proliferation warrants further investigation to identify the mechanisms. As discussed previously, BMPs have published roles controlling proliferation via cell-cycle regulation, prevention of apoptosis, and changing the morphology/invasive potential of breast cancer cells. Any of these mechanisms may be involved in producing the cell phenotypes seen here. This is particularly true as SOSTDC1 binds to more than one BMP, with the potential
132 to concurrently antagonizing multiple ligands by sequestering them away from
the receptors. Additionally, studies in our lab and others with other tissues have shown that SOSTDC1 is a potential dual-pathway inhibitor, also antagonizing
Wnt pathway signaling. Exploration of these mechanisms will provide fertile research fields for future studies.
Figure 6.
133 Figure 6 (Previous Page). Cells overexpressing hSOSTDC1-FLAG show
proliferation suppression. MCF7 cells were transiently transfected with
either the pFLAG-CMV-5a-SOSTDC1 vector (SOSTDC1) or empty pFLAG
vector (EMPTY CONTROL). Culture proliferation was measured on the
ACEA RT-CES system as a function of electric resistance over time
(reported as the Cell Index, CI). Results shown are representative of three
separate experiments. A. The ACEA E-plate was prepared at time = 0 and
background reading of media only obtained. Cells were plated at 7 hours
(gold arrow) and had adhered to the plate by 10-12 hours (red arrow).
Increases in CI over the next 5 days showed steady proliferation of control
cells compared to complete suppression of proliferation in cells
overexpressing SOSTDC1. B. Phase contrast microscopy (20X) of cells at
Day 1 and Day 3 of experiment confirm lack of live cell expansion in
SOSTDC1 cells. Red arrows show a cell undergoing dying with blebbing
and apoptotic echinoid spike formation (middle and right bottom panel,
40X).
134 Conclusions
This is the first report of the distribution of the BMP antagonist SOSTDC1 in human breast tissues and its subsequent loss in many breast tumors. Loss of expression of mature, secreted SOSTDC1 was identified in breast cancer cells.
The downregulation was explored at the message and protein level in clinical samples with the use of tissue microarrays. We identified SOSTDC1 as a potential biomarker of breast cancer (negatively associated with tumor stage and size) with correlations to other known prognostic markers. Additionally, our findings that overexpression of SOSTDC1 causes proliferation suppression in an invasive breast cancer model suggests that SOSTDC1 may have tumor suppressive activities. The potential roles of SOSTDC1 uncovered by this work increase our enthusiasm to understand more about this molecule.
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141 CHAPTER V
GENERAL DISCUSSION
SOSTDC1 in Cancer
K. BLISH
142 The introductory chapter suggested that models of cancer involving key
pathways of cell viability and behavior are necessary for the development of
novel anti-neoplastic treatments. The work presented here developed models of
BMP and Wnt signaling in breast and kidney cancers. The goal was to better
understand regulation of these pathways through studies of the dual-pathway
inhibitor, SOSTDC1. The strategy for accomplishing this goal involved: looking
for loss-of-heterozygosity (LOH) and mutations at the SOSTDC1 locus, studying
SOSTDC1 expression and actions in vitro, and evaluation of SOSTDC1 expression in patient samples. In this discussion, the success of meeting these goals will be evaluated and an examination of the challenges encountered during this work will be presented.
Success in Accomplishing the Goals of this Project
Review: SOSTDC1 in Cancer, What Has Been Accomplished
This body of work has yielded new and relevant information to the understanding of SOSTDC1 as well as BMP and Wnt signaling in breast and renal tumors.
From the initial array observations showing SOSTDC1 as downregulated in a wide range of tissues and tumors, the potential tumor suppressor actions of
SOSTDC1 have been explored.
143 Current evidence shows that:
1) LOH affects SOSTDC1 in a ~16% of Wilms Tumors. This rate is comparable
to LOH seen at other Wilms tumor suppressor genes. LOH along the same
section of chromosome 7p was described for the first time in adult renal cancers.
Direct sequencing confirmed LOH and unveiled some previously unpublished single nucleotide polymorphisms (SNPs).
2) SOSTDC1 message and protein are downregulated in renal and breast tumors. Mature SOSTDC1 protein is secreted from overexpressing cells and distributes to nearby cells in a concentration gradient. SOSTDC1 expression was examined in a step-wise model of breast cancer progression (where normal
HMECs receive 4 genetically defined hits causing, in order, unlimited replicative
potential, immortality, increasing growth factor signaling, insensitivity to apoptotic
signals, and the ability to invade surrounding tissue). Mature SOSTDC1 was
secreted from normal HMECs and HMECs overexpressing telomerase, but once
immortality and full transformation is achieved, extracellular SOSTDC1 was lost
from the cultures. Additional work with cell culture kidney models (data not
shown) revealed that many renal carcinoma cell lines do not make routinely
detectable amounts of SOSTDC1 message or protein.
Functional consequences of this loss may affect BMP pathway regulation as exogenous SOSTDC1 is able to effectively inhibit BMP-7 signaling in breast and
renal cancer cells and BMP-2 signaling in breast cancer cells (pilot study data not
144 shown). Additionally, the ability of SOSTDC1 to antagonize Wnt signaling was
shown for the first time in human tissues. SOSTDC1 inhibited Wnt-3a induced
TCF/LEF promoter activation almost as well as the Wnt specific inhibitor DKK.
This is the first evidence of the dual-pathway effects of SOSTDC1 in human tissues, normal or cancerous. Finally, restoration of SOSTDC1 to a variety of breast and renal clear cell cancer lines blocked the proliferation of these cultures better than DKK-1 or noggin alone (renal) and was not rescued by BMP-2 or
BMP-7 treatment (breast).
3) Within clinical kidney tissue samples, clear cell renal carcinomas show the lowest amounts of SOSTDC1 protein. In breast samples, loss of SOSTDC1 significantly correlates with markers of more advanced disease: larger tumor volume, higher stage, and prognostic markers such as EGFR. Early studies have shown that SOSTDC1 can be detected in urine and serum (data not shown).
While these data are quite general observations about SOSTDC1, putting them together builds a model that is thus far consistent with the early hypothesis of this
work. SOSTDC1 does have extracellular actions to inhibit BMP signaling in
kidney and breast tissues and furthermore, the lost of SOSTDC1 is seen with
high penetrance in renal and breast tumors and appears to correlate with more
advanced disease. This hypothesis is broad and much remains to be determined
145 regarding the specifics of SOSTDC1’s protective mechanisms and the phenotypic consequences of SOSTDC1 loss in cancer.
Challenges Encountered in this Project
As this project started with an observation concerning (at that time) a hypothetic protein in humans with little to no data about its nature or mode of actions, the goals for this project were by necessity broad. Therefore, the roadblocks encountered during these studies were of two varieties: technical and conceptual.
Technical Challenges
Production of rhSOSTDC1: Technically, reagent development was a limiting step as the production of rhSOSTDC1 was at best a tedious process and at worst, unsuccessful. Overexpression within bacterial systems never yielded mature soluble protein and the process for protein collection from HEK293 cells yields very little amount of protein. This is not uncommon as most cystine-knot containing proteins are notoriously insoluble and hard to produce/purify. Work continues on this front. In the meantime, investigation of the production and experimental usefulness of SOSTDC1 conditioned media might be a short-term solution.
In vitro models and transient transfection issues: In addition to having proper reagents, it was important to develop appropriate cell models systems for the
146 experimental questions. For example, cells in which to test the effects of
SOSTDC1 overexpression should also express appropriate BMP and/or Wnt pathway receptors and ligands in order to respond to SOSTDC1’s proposed activities. Determining this could be time-consuming and yet not yield useful information about whether SOSTDC1 will cause a phenotypic change in those cells. For this project, kidney cell lines and breast cell lines were screened for endogenous SOSTDC1 production (Kidney data not shown, all cell lines had low to non-detectable amounts of SOSTDC1, Breast cell data in Chapter 4). Then, the choice was made to test the effects of SOSTDC1 overexpression on phenotype of these cells. If cancer cells responded to SOSTDC1, further studies of ligand, receptor, and signaling protein availability in the cell lines of interest would be undertaken. Cancer cell lines from kidney and breast were identified that did respond to overexpression of SOSTDC1, but characterization of interacting proteins from the BMP and Wnt pathways is still ongoing.
By nature, in vitro cell models are prone to additional challenges associated with gene delivery method and dosage issues. As recombinant protein was scarce, overexpression of SOSTDC1 from plasmids was a necessary step. This introduces another layer of artificial set-up to the model as overexpression via transient transfections are notoriously hard to reproduce at similar levels, have varying toxicities to the cells, and likely express the target gene at levels beyond physiologic significance. To deal with some of these disadvantages, cell-lines stably overexpressing SOSTDC1 were attempted. Overexpression of a growth
147 inhibitor, such as SOSTDC1, poses further technical difficulty, as successfully
transfected cells by definition will not replicate as quickly as non-transfected
counterparts. The use of an inducible expression system is one solution to this problem and is being developed for use in future models. Even with successful creation of stables cells lines with inducible SOSTDC1 expression, the accurate assessment of the roles of SOSTDC1 at physiologic levels is necessary via alternative approaches including identification of likely physiologic levels and treatment with rhSOSTDC1.
The need for in vivo experiments: Having a good in vitro model in which to
determine a novel protein’s actions is a beneficial tool; however, even a good
model has its conceptual drawbacks as it is still a very artificial system. This is
particularly true with studies of secreted proteins that interact in paracrine
manner. There are many examples of Wnt/BMP interactions between cell types,
including tumor and stroma (1-5). Thus, an in vivo model provides the only way to truly study the full range of effects of SOSTDC1.
The most immediate line of inquiry would be experiments comparing the in vivo
characteristics of cancer cell lines with or without overexpression of SOSTDC1,
such as a tumor flank or mammary fat pad tumorigenesis model. Such
experiments would allow for a closer approximation of the effects of SOSTDC1 in
an environment where there are multiple cell types. Effects could be observed
148 on many processes including: tumor incidence after cell injection, tumor
growth/size, angiogenesis, tumor invasiveness and metastasis.
Mouse knockouts of SOSTDC1 have been created by several groups (6-8).
Reported phenotypes of living mice include: supernumerary and malformed teeth, craniofacial abnormalities affecting the trigeminal nerve, and increased
resistance to kidney injury (9). While Kassai et al. report knockout mice are fully
fertile and viable out to 15 months, Shigetani et al. more recently report that
some homozygous knockouts die shortly after birth, and suggest that full
penetrance of the knock-out gene may be hard to obtain. This phenotype
suggests that as a solitary event, loss of SOSTDC1 is not causative for
tumorigenesis, which is not surprising considering the number of BMP and Wnt
antagonists and their overlapping functions. However, because the loss of
SOSTDC1 is a frequent event in human tumors, further investigation is
worthwhile. It remains to be determined whether loss of SOSTDC1 makes mice
more susceptible in response to other carcinogenic exposures such
environmental carcinogens or cross-breeding with other mouse models
expressing oncogenes.
Conceptual Challenges
Even when all technical issues are addressed and all desired model systems obtained, there still remain a variety of conceptual challenges. These are the issues at the heart of experimental design and finding efficient ways to address
149 these challenges raises fundamental questions about the nature of SOSTDC1.
The conceptual challenges include: 1) addressing the proof-of-principle studies,
2) considering the multiple potential levels of SOSTDC1’s regulation, 3) attending
to SOSTDC1’s unknown mechanism(s) of action, and 4) grappling with the complexities inherent to a dual-pathway antagonist orchestrating more than one signaling network in a tissue-specific manner.
1) Proof-of-Principle Studies
RNA/cDNA and protein studies have shown that SOSTDC1 expression is greatly downregulated at a high penetrance in many tumor types, particularly breast and renal. This suggests that SOSTDC1 has a protective effect in normal tissue that must be overcome for cancer to form or progress. As discussed above, current mouse knockout strains show that SOSTDC1 loss is not sufficient to cause transformation. The question remains whether SOSTDC1 loss contributes to the tumorigenic process. The main proof-of-principle experiment to address this question lies in knock-down of SOSTDC1 expression from normal cells, then testing whether these cells have greater sensitivities to oncogenic agents or
develop a more transformed phenotype. Cell knock-down SOSTDC1 models
have been attempted but successful knock-down has yet to be accomplished
(data not shown).
150 These experiments might also address a secondary question: whether
SOSTDC1 loss occurs as a cause that then predisposes cells to cancer, or
whether loss is a result of other changes. Supporting the latter argument, the cell
line step-wise model of breast cancer discussed in Chapter 4 showed a loss of
mature, secreted SOSTDC1 after the addition of SV40 T antigens to the
immortalized HMECs. The expression of SV40 large T antigen causes disruption
to p53 as well as pRb, p300, and CBP, leading to large-scale changes in genome
integrity. It is reasonable to speculate such changes could affect SOSTDC,
either by inducing mutations or affecting its regulation. However, little is known
about what regulates the expression or activity of SOSTDC. More effort on this
front would inform future experiments concerning any causal relationship
between SOSTDC1 and carcinogenesis.
2) Regulation of Expression
There is very little published data about what affects SOSTDC1 expression.
Examples of regulatory mechanisms affecting other BMP/Wnt antagonists as well as pilot data inform of possible mechanisms worth investigation. These examples are diverse and fall into genetic regulatory mechanisms, epigenetic, transcriptional (splicing), gene dosage, as well as protein regulation.
151 Genetic
The LOH studies described in Chapter 3 show that loss of chromosomal data around the SOSTDC1 locus affects 10-15% of kidney cancers. The remaining allele appears to be normal in all sequenced sections. The observed LOH includes other genes within the 2 Mbp region, so causality between changes in
SOSTDC1 genetic material and Wilms or adult RCC cannot be concluded.
However, the fact remains that as many as 90% of renal clear cell carcinomas show a reduction in SOSTDC1 message. Therefore, it is presumed that alternative mechanisms exist affecting the downregulation of SOSTDC1 message in kidney.
The first consideration when interpreting these results is the current incomplete understanding of the SOSTDC1 allele. Direct sequencing to look for significant mutations was undertaken at 5 potential exons. However, the promoters, enhancing elements, and other regulatory regions are not defined. Thus, mutations may exist in important regulatory regions for this allele that have not been defined and may not have been included in the sequencing. If such mutations exist, then loss of heterozygosity concurrent with such a mutation on the remaining allele could cause loss of SOSTDC1 expression in these 10-15% of cases where LOH occurs. This still leaves ~80% of cases showing decreased
SOSTDC1 expression to explain by other regulatory mechanisms.
152 Epigenetic
There are many examples of BMP and Wnt pathway proteins whose expression
is modulated by epigenetic mechanisms. One pertinent example is the secreted
Wnt antagonist, sFRP1. The sFRP1 locus shows hypermethylation in breast,
oral, hepatocellular, and colon cancers at an incidence of between 10-50% (10-
12). Loss of sFRP1 causes constitutive Wnt signaling upregulation and loss of
anti-proliferative and apoptotic responses in these cells (13). Analysis of the
SOSTDC1 locus reveals a potential CpG island near the 3’ end of what is termed
exon 1 (see Chapter 3 for locus map) and other potential methylation sites exist around several of the putative transcription start sites. Methylation-specific PCR in the Wilms and adult kidney cancer populations would address whether a
certain percentage of patients experiencing silencing at the SOSTDC1 allele.
Splice variants
Mutant splice variants, changes in ratios of variants, and changes in gene
dosage are all documented methods of tumor suppressor gene inactivation. For
example, in Wilms Tumor, the prototypical tumor suppressor allele, WT1 does
not often undergo LOH or other deletions of material. Instead, dysregulation of
WT1 function is accomplished by shifts in the transcript variants resulting from
alternative splicing over the two coding exons and use of two translation state
sites (14). mRNAs have been identified that show the existence of as many as 6
different transcripts from the complex SOSTDC1 locus (Aceview Project, NCBI,
(15). It remains to be determined whether these are detectable species in
153 tissues and whether these alternative transcripts produce variant proteins or play
regulatory roles.
Protein regulation
BMP/Wnt Negative Feedback Loop: It is a well-established paradigm in BMP and
Wnt regulation that ligand signaling induces upregulation of transcription of pathway antagonists as a negative feedback mechanism. Pilot data for this project has shown that treatment of SaOS-2 cells with BMP-7 increases
SOSTDC1 mRNA by 2-4 fold. Additionally, sequence analysis of the 5-exon potential SOSTDC1 locus has identified possible Smad-responsive SBE sequences; however, a more careful investigation of this is necessary, potentially including mutational studies. SOSTDC1 upregulation in response to Wnt signaling has not yet been studied. Other pathways could potentially regulate
SOSTDC1 as well; one possibility might be steroid hormone regulation as is
observed for sclerostin (16), but BMP and Wnt regulation remain the most likely
candidates.
Subcellular Localization and Modulation of Secretion from the Cell:
SOSTDC1 has a signal peptide and has been seen in the media of cell cultures
(Chapter 2, Chapter 4). It is hypothesized that all actions of SOSTDC1 occur
outside the cell; however, this has not been formally studied. First, regulation
can occur at the level of protein processing and secretion. Mutations which
affect the appropriate secretion of SOSTDC1 could exist and prevent the proper
154 localization of active molecules to the extracellular space. However, Itasaki et al recently report different roles for Xenopus wise depending on subcellular localization. When wise/SOSTDC1 is secreted, it binds to Wnt proteins and either activates or inhibits Wnt signaling through either β-catenin or the planar cell polarity pathways (17). However, if wise is maintained in the endoplasmic reticulum, it binds to LRP6 and reduces its expression on the cell surface, inhibiting Wnt signals. The mechanism for retention of wise in the ER is unclear
(18). Investigation of this phenomenon in cancer models is necessary and can be initiated with studies of exogenous protein that lacks the N-terminal signal peptide domain. Mammalian expression constructs have been prepared for mutant SOSTDC1 that expresses only residues 24-206 and would likely not get secreted from the cell.
Once SOSTDC1 is outside the cell, binding partners within the extracellular matrix that modify its dispersal provide another potential level of regulation.
Studies to examine these issues have been complicated, as little is known about the folding and partitioning of Wnt or BMP proteins (including SOSTDC1) due to their insolubility (19). However, sclerostin is known to bind heparins (20) and the use of glycoprotein scaffolding to provide concentration gradients of BMP and
Wnt proteins is a well-established phenomenon.
Ratio of SOSTDC1 to binding partners: The last issue to consider concerning
SOSTDC1 regulation is the ratio of SOSTDC1 to its binding partners. It is
155 currently unknown what happens to SOSTDC1 if there are no BMP/Wnts to bind,
or if such a scenario is possible, but the actions of the antagonists and ligands
are always interrelated. Several groups have shown that the stoichiometry of
various BMP and/or Wnt pathway members and antagonists changes the
formation of receptor complexes and subsequent downstream signaling (21-24),
at times changing the activated intracellular pathway entirely (such as from Smad
to p38/MAPK signaling) . There are also many examples of antagonists that
interfere with each other’s actions, such as noggin and sclerostin when co- expressed (25), thus its possible that the actions of SOSTDC1 are dependent upon the levels of other interacting proteins. This analysis awaits full identification of SOSTDC1s binding partners.
3) SOSTDC1’s Unknown Mechanisms of Action
The observed downregulation of SOSTDC1 in a variety of cancer tissues and cell
culture models of cancer suggests that many of its in vivo actions may be tumor suppressive in some capacity: whether anti-transformative, anti-proliferative or
anti-metastatic. Anti-proliferative activity has been established in our renal and breast cancer cell culture models. It remains to be determined precisely how the profound proliferation block of cancer cell culture is achieved. It is possible that
SOSTDC1 overexpression increases the rate of cancer cell apoptosis, or causes
a cell cycle block, or affects both processes. Furthermore, the therapeutic
potential of SOSTDC1 would need to be assessed to see if treatment with
156 recombinant protein at physiologic concentrations could obtain these anti- proliferative results.
Some of the actions may be dependent on the binding partner(s) of SOSTDC1.
Yanagita and colleagues showed that USAG-1 (rat homologue of SOSTDC1) binds to BMP-7 in vitro most tightly followed by , -2, -4, and -6 (26). Only
SOSTDC1’s interactions with BMPs-7 and -2 have been studied thus far. This is significant particularly in regards to BMP-6, which often has contradictory roles to
BMP-7,-2, and -4. Additionally, all studies of BMP/SOSTDC1 interaction have been in artificial systems, as it is currently unknown what physiologic concentrations SOSTDC1 and interacting BMPs would achieve in vivo, or even which BMP or Wnt ligands are co-expressed with SOSTDC1.
More thorough approaches are necessary to understand the interacting proteins with SOSTDC1 - such as a yeast 2-hybrid system. Rationale for undertaking a more thorough investigation of this matter comes from the example of sclerostin, as it’s actions have been in part explained by its multiple binding partners (BMPs,
LRP-5, LRP-6). Sclerostin is able to inhibit both pathways and this is an important regulatory mechanism of bone formation. Loss of BMP signaling decreases sclerostin expression. The resulting loss of sclerostin leads to an increase in canonical Wnt signaling (27).
157 Interestingly, Itasaki et al. and Ohazama et al. provide evidence that SOSTDC1’s
Xenopus ortholog binds to the Wnt co-receptors LRP-6 and LRP-4. LRP-4
(Lipoprotein related peptide-4) is homologous to the more thoroughly studied
LRP-5 and -6 Wnt co-receptors and is hypothesized to regulate sonic hedgehog
(Shh) signaling between epithelial and mesenchymal cells (28). The hypothesized main role of these co-receptors is to stabilize the Wnt-Frizzled complex. An interesting possibility raised by Itasaki and colleagues is that
SOSTDC1 is able to simultaneously bind BMPs and LRP-4. This could effectively antagonizing both pathways concurrently- depending upon the expression levels of BMP binding partners and LRP-4- as it would sequester
BMPs away from the cognate receptor complexes while disrupting the Wnt-
Frizzled interaction. The biologic consequences of SOSTDC1’s ability to interact in this manner remain to be determined in various human tissues and disease processes.
Early evidence points to SOSTDC1 having a greater effect on proliferation of renal clear cell cultures than either DKK1 (Wnt only antagonist) or noggin (BMP only antagonist). Whether this effect is due to a synergistic effect of concurrent antagonism, sequential antagonism, or another phenomenon altogether will be a subject of future investigation. Experiments will be necessary in the renal and breast cells to determine with which proteins SOSTDC1 is interacting, and whether both pathways are active in a particular tumor or cancer model. One general starting point could be to check for SOSTDC1 association with
158 expressed BMPs, receptors, LRPs, and Wnts via fluorescent labeling and
confocal microscopy. Pathway activation could be established via staining for
phospho-Smad or active-β-catenin in clinical samples or use of luciferase-based
reporter assays in cell culture based models. Assuming the active intracellular
messangers exist, mutants or knockdowns of LRP proteins, disheveled, BMP
receptors, Smads, or other pathway players shown to interact with SOSTDC1
would be necessary to being characterizing each pathway’s contribution to the
overall phenotype.
When considering the possible mechanisms of action, it is necessary to take into
account the great potential for pathway cross-talk. The phenotypic effects of
SOSTDC1 may not be due to any primary action of the molecule itself, but a
result of expression level changes of molecules regulated by Wnt and/or BMP
signaling. This is not only inherent in the nature of SOSTDC1 as a dual-pathway
antagonist, but it is also compounded given the breadth of cellular changes
affected by BMP and Wnt signaling (29). The strong associations between
SOSTDC1 and EGFR, IGFR-1, and PTEN expression in breast cancer tumors
hints at the intertwined nature of these pathways. For example, inhibition of Wnt signaling can transactivate EGFR (30). Microarray expression experiments in cells treated plus or minus SOSTDC1 might begin to identify which pathways are most critical.
159 4) Tissue Specificity: The Complexity of Multiple Pathway Involvement
In the previous section, the point was discussed that the mechanisms of
SOSTDC1 actions is likely dependent upon its binding partners and the result of modulations in both Wnt and BMP pathways. As SOSTDC1 may have the potential to concurrently inhibit both pathways, this introduces complexity into the model of SOSTDC1’s actions in cancer. This complexity is illustrated when you compare the differences in the models of SOSTDC1 in kidney cancers versus breast cancers.
For example, the kidney is the predominant location for expression of BMP-7 in the adult and BMP-7 is the main BMP ligand expressed in normal and disease states. Additionally, Yanagita, et al. report that SOSTDC1 is the BMP antagonist expressed at the highest levels in the kidney (26). In agreement, data in this project (Chapter 2) showed fairly consistent levels of SOSTDC1 message and protein staining in normal kidney samples, particularly in the distal tubules.
There was also a very penetrant significant decrease in SOSTDC1 in renal cell carcinoma samples. Taken together, these data suggests that in the kidney, the
BMP7/SOSTDC1 axis is the primary BMP pathway control system and it is the ratio between these two proteins that is important in the modulation of disease.
While data does not exist for any pro-carcinogenic actions of BMP-7 on the kidney itself, BMP-7 does play a role in mediating TGF-β signaling, which is
160 connected with kidney tumors as it balances the epithelial-mesenchymal transition step of dedifferentiation during transformation (4).
The connections between Wnt signaling and kidney cancer are newly expanded with the discovery of WTX (31) and identification of the anti-proliferative properties of sFRP (32). SOSTDC1 appears to have preferential antagonism of
Wnt ligands as Wnt3a antagonism has been observed in this work (Chapter 2), while previous studies show no antagonism of Wnt1 (26). The significance of these observations remains to be determined as little is known about the effects of individual Wnt proteins in kidney tumors.
In contrast to the kidney, breast tissue expresses a many BMPs and their expression and activities change in diverse ways throughout the spectrum of transformation (discussed in Chapter 4). The model of the BMP-7/SOSTDC1 axis of control would not fit here; instead, more investigation is needed into the relationship between the network of BMPs and Wnts expressed. Results of these studies found widely varying basal expression levels of both SOSTDC1 message and protein in normal tissues. Additionally, SOSTDC1 levels changed in varying amounts from normal to tumor tissues and did not correlate with a specific pathologic type of breast cancer. This suggests that a more complex network of SOSTDC1 regulation might exist in breast tissue. SOSTDC1 regulation of Wnt signaling in breast tissue is yet to be explored. This is likely a
161 promising area of future investigation as there are many reports of changes in
Wnt signaling that correlate with the progression of breast cancer.
Overall Conclusions
Simplification of carcinogenesis models by focusing on extracellular regulation of
BMP and Wnt signaling pathways led to the identification of SOSTDC1 as a novel dual-pathway antagonist. Loss of heterozygosity has been shown to affect
10-15% of kidney cancers, the striking downregulation of SOSTDC1 in breast and kidney tumors has been more carefully characterized, the extracellular antagonistic capabilities of SOSTDC1 in cell culture models established, and
SOSTDC1’s potential to block proliferation identified.
These findings suggest that SOSTDC1 is an important regulator of both Wnt and
BMP pathways and that loss of SOSTDC1’s activities may contribute to more advanced disease. SOSTDC1’s actions on multiple pathways increases its attractiveness as a potential anti-cancer therapeutic. Future investigations of this potential should occur by: greater characterization of the SOSTDC1 locus, study of SOSTDC1’s actions on in vivo models, further exploration of SOSTDC1 as a prognostic biomarker, and biochemical identification of the mechanism behind proliferation suppression.
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166 SCHOLASTIC VITA
KIMBERLY ROSE BLISH
BORN: June 18, 1981, Winston-Salem, North Carolina
UNDERGRADUATE Eastern Nazarene College STUDY: Quincy, Massachusetts B.S., Chemistry, summa cum laude 2002
GRADUATE Wake Forest University Winston-Salem, North Carolina Ph.D. 2009
SCHOLASTIC AND PROFESSIONAL EXPERIENCE: MD/PhD Graduate Fellowship, 2002 -2009
Teaching and Laboratory Assistant, Eastern Nazarene College, 1999-2002.
Summer undergraduate research project: QSAR Modeling to Predict Blood-Brain Barrier Partitioning, 2001.
HONORS AND AWARDS:
Predoctoral Traineeship Award: BC043274, “A Novel Bone Morphogenetic Protein Antagonist as a Breast Cancer Growth Suppressor.” Breast Cancer Research Program, CDMRP, Department of Defense, 2005.
1st Place Student Presentation, WFU Cancer Biology Department, 2005 & 2008.
Senior Award in Biochemistry- American Institute of Chemists, 2002.
James Norris-Richard Flack Summer Undergraduate Research, Scholarship. Northeastern Section of the American Chemical Society, 2001.
National Merit Scholarship Award, 1998.
PROFESSIONAL SOCIETIES:
2007-Present American Association for Cancer Research (AACR)
2004-06 American Physician Scientist Association (APSA) Wake Forest University Institutional Representative.
2002-04 American Medical Student Association (AMSA)
167 2002-03 American Institute of Chemists (AIC)
PUBLICATIONS: Articles: Blish, KR, Willingham MC, Du W, Birse CE, Krishnan SR, Brown JC, Wang W, Hawkins GA, Garvin AJ, D'Agostino RB, Torti SV, Torti FM. A Novel, Human Bone Morphogenetic Protein Antagonist is Down regulated in Renal Cancer. Mol. Biol. Cell, 2008 Feb; 19(2):457-464 E-published: 2007 Nov 21.
Blish, K.R.; Ibdah, J.A. Maternal heterozygosity for a mitochondrial trifunctional protein mutation as a cause for liver disease in pregnancy. Med. Hypotheses. 2005. 64(1): 96- 100.
Rose, K.; Hall, LH. E-State Modeling of Fish Toxicity Independent of 3D Structure Information. SAR QSAR Environ. Res. 2003. 14 (2): 113-129
Rose, K.; Hall, LH; Kier, LB. Modeling blood-brain barrier partitioning using the electrotopological state. J. Chem. Inf. Comput. Sci. 2002. May – June; 42(3): 651-66.
Abstracts: June 2008: Blish KR, Triplette, MA, Willingham MC, Kute TE, Du W, Birse CE, Krishnan, SR, Russell GB, Brown JC, Torti FM, and Torti SV. A Bone Morphogenetic Protein Antagonist as a Breast Cancer Growth Suppressor. Poster for DOD Era of Hope Meeting. Baltimore Convention Center, Baltimore, MD, June 26, 2008.
April 2007: Annual Meeting of the American Association for Cancer Research (AACR), Los Angeles, CA. Kimberly R. Blish, Mark C. Willingham, Wei Du, Charles E. Birse, Surekha R. Krishnan, Julie C. Brown, Wei Wang, Gregory A. Hawkins, A. Julian Garvin, Ralph B. D'Agostino Jr., Frank M. Torti, Suzy V. Torti. The human bone morphogenetic protein antagonist BARC is downregulated in renal cancers In: American Association for Cancer Research Annual Meeting: Proceedings; 2007 Apr 14-18; Los Angeles, CA. Philadelphia (PA): AACR; 2007. Abstract #3785.
April 2006: BARC: A Novel, Human BMP Antagonist Downregulated in Breast and Renal Cancers. K.R. Blish, M.C. Willingham, W. Du, C. Birse, J.C. Brown, M.A. Triplette, A.J. Garvin, F.M. Torti, S.V. Torti. Poster for ASCI/AAP Joint Meeting. The Fairmont Hotel, Chicago, Illinois, April 29, 2006.
January 2004:SCGF in Breast Cancer Growth and Detection. K.R. Blish, J. Brown, M. Willingham, C. Birse, F.M. Torti, and S.V. Torti. Poster for the Breast Cancer Center of Excellence Retreat. Comprehensive Cancer Center of Wake Forest University. January 15, 2004. Winston-Salem, NC.
May 2003 Rose, K.; Zhao, Y.; Ibdah, JA. Liver Disease in Pregnancy Associated with Maternal Heterozygosity for A Mitochondrial Trifunctional Protein Mutation and A Normal Fetal Genotype. For presentation at Digestive Disease Week, the joint conference of the AASLD, AGA, ASGE, and SSAT. May 17-22, 2003. Orange County Convention Center, Orlando, Florida.
168