NOVEL INSIGHTS INTO BONE MORPHOGENETIC PROTEIN (BMP) AND MAMMALIAN TARGET OF RAPAMYCIN (mTOR) SIGNALING AXIS IN PROSTATE CANCER.
by
REEMA SAID WAHDAN-ALASWAD
Submitted in partial fulfillment of the requirements
For the degree of Doctor of Philosophy
Dissertation Advisor: David Danielpour, Ph. D.
Department of Pharmacology
CASE WESTERN RESERVE UNIVERSITY
August 2011 CASE WESTERN RESERVE UNIVERSITY
SCHOOL OF GRADUATE STUDIES
We hereby approve the thesis/dissertation of
___Reema Said Wahdan-Alaswad_____ candidate for the _ Ph. D. ______degree *.
(signed)______Noa Noy, Ph. D.______
(chair of the committee)
______David Danielpour, Ph. D. ______
______Bing-Cheng Wang, Ph. D. ______
______Yu-Chung Yang, Ph. D.______
______Johannes von Lintig, Ph. D.______
______
(date) _December 1, 2010______
*We also certify that written approval has been obtained for any
proprietary material contained therein.
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To my parents Said & Urayb, husband Mazen, daughter Lamya, and Siblings
Ruba, Rana and Thair.
2
Table of Contents
List of Tables ...... 5
List of Figures ...... 6
Acknowledgements ...... 10
Abbreviations ...... 12
Abstract ...... 15
Chapter 1 Introduction ...... 15
1.1. Prostate Cancer Overview/ Prostate Anatomy ...... 16 1.1.1 Prostate Zones and Lobes ...... 17
1.1.2 Prostate Cancer Etiology ...... 17
1.1.3 Prostate Cancer Risk Factors ...... 17
1.1.4 Treatment ...... 18
1.1.5 Prostate Cancer Screening, TNM Staging & Gleason Score ...... 19
1.1.6 Progression of Prostate Cancer, Oncogenes, or Tumor Suppressor Genes ...... 20
1.2. TGF-β superfamily signaling in prostate cancer ...... 21 1.2.1 The TGF-β superfamily regulatory activity and receptors activation...... 22
1.2.2 Jekyll and Hyde function of TGF-β-Superfamily during prostate carcinogenesis...... 23
1.2.3 Alterations of BMP pathway in human cancers ...... 24
1.2.4 Implications of BMP in advanced prostate cancer ...... 26
1.3. Smads: critical mediators of TGF-β signals ...... 27 1.3.1 Main features of Smad proteins ...... 27
1.3.3 Smad Transcriptional Activation and BMP Response Elements ...... 28
1.4. TGF-β and BMP Cross-talk with Major Pathways ...... 29 1.4.1 BMP and the MAPK pathway ...... 30
1.4.2 BMP mediated crosstalk with AR ...... 31
1.4.3 BMP Crosstalk with IGF-I Signaling Pathway ...... 32
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Figures ...... 39 Chapter 2: Insulin-Like Growth Factor I Suppresses Bone Morphogenetic Protein
Signaling in Prostate Cancer Cells by Activating mTOR Signaling ...... 71
ABSTRACT ...... 71 INTRODUCTION ...... 72 METHODS ...... 74 RESULTS ...... 83 DISCUSSION ...... 90 FIGURES ...... 93 TABLES ...... 125 Chapter 3 Smads 1 and 5 but not Smad8 are Activated by Rapamycin and
Promote Cytostatic/Cell Death Responses in Prostate Cancer Cells...... 129
ABSTRACT ...... 129 INTRODUCTION ...... 129 METHODS ...... 130 RESULTS ...... 137 Chapter 4 Summary, Discussion and Future Directions ...... 183
4.1 Summary ...... 183 Chapter 2: IGF-I suppresses BMP4-induced cell death through a PI3K/Akt/mTOR dependent
mechanism...... 183
Chapter 3: Rapamycin (an mTOR inhibitor) reverses mTOR-mediated inhibition of BMP
signaling, thus leading to enhanced BMP-mediated activation of Smad(s) 1/5/8, Id1
transcriptional regulation, and cell death in vitro and in vivo ...... 185
4.2 Discussion and Future Directions for Chapter 2 ...... 187 4.2.1 BMPRI, BMPRII, Smad1, Smad5, and Smad8-mediated interactions with mTOR, Rictor
and Raptor in LNCaP cells...... 187
4.2.2 BMP4-mediated apoptosis and survivin in prostate epithelial cell lines...... 188
4.2.3 IGF-I and BMP-induced apoptosis in prostate epithelial cells...... 189
4.3 Future Directions for Chapter 3 ...... 190 4.3.1 FKBP12-mediated inhibition of BMP signaling...... 190
4.3.2 Androgen Receptor and BMP-mediated Smad1/5/8 activation and Id1 promoter activation.191
4.3.3 Smad1, Smad5, and/or Smad8 as prognostic markers for prostate cancer...... 193
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List of Tables
Table 2.1. List of BMP4 regulated genes specifically altered by IGF-I…………...127
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List of Figures Figure 1.1. Prostate Anatomy...... 39
Figure 1.2. Zone of the Prostate ...... 41
Figure 1.3. Anatomical staging of Prostate Cancer...... 43
Figure 1.4. Prostate Cancer Staging System...... 45
Figure 1.5. Human Prostate Cancer Progression from Normal Epithelium to Metastasis...... 47
Figure 1.6. TGF-β Superfamily Signaling...... 49
Figure 1.7. TGF-β Superfamily role in Human Cancers...... 51
Figure 1.8. TGF-β Superfamily Ligands ...... 53
Figure 1.9. TGF-β Pathway and Human Disease...... 55
Figure 1.10. BMP mediated Signaling ...... 57
Figure 1.11. Dual Role of TGF-β Mediated Signaling ...... 59
Figure 1.12. Mutation or Alterations in BMPRII...... 61
Figure 1.13. TGF-β/BMP-mediated Smad signaling ...... 63
Figure 1.14. Smad Molecular Structure...... 65
Figure 1.15. BMP and MAPK Crosstalk...... 67
Figure 1.16. TGF-β signaling and IGF-I cross talk...... 69
Figure 2.1. Biological activity of TGF-β superfamily ligands on prostate epithelial cell lines...... 93
Figure 2.2. TGF-β superfamily-induced activation of Smads in PC3 cells...... 95
Figure 2.3. LR3–IGF-I blocks BMP4-induced cell death in nontumorigenic (NRP-152 and DP-153) and tumorigenic (LNCaP and VCaP) prostate epithelial cancer cell lines...... 97
Figure 2.4. LR3-IGF-I blocks BMP4-induced cell death in NRP-152 cells...... 99 6
Figure 2.5. LR3-IGF-I increases G2/M progression but has no effect on G1 or S phase cell cycle progression in NRP-152 cells...... 101
Figure 2.6. LR3-IGF-I inhibits BMP-induced cell death in NRP-152 and total adherent cells in a number of rat prostate epithelial cell lines (NRP-152, DP-153, RWPE-1) and human prostate epithelial cancer cell line (PC3)...... 103
Figure 2.7. LR3–IGF-I abrogates BMP4-induced activation of Smad1/5/8, and Id1, Id2, and Id3 expression...... 105
Figure 2.8. LR3-IGF-I blocks BMP4-mediated Smad1/Smad5/Smad8 phosphorylation in NRP-152 cells...... 107
Figure 2.9. LR3-IGF-I inhibits BMP4-mediated Id1 promoter activation in LNCaP cell line ...... 109
Figure 2.10. LR3–IGF-I inhibits BMP4-mediated responses through a PI3K/Akt/mTOR-dependent mechanism...... 111
Figure 2.11. LR3-IGF-I inhibits activation of BMP-mediated responses through a PI3K or Akt-dependent mechanism...... 113
Figure 2.12. Raptor, Rictor, and mTOR mediate the IGF-I suppression of BMP-induced Id1 promoter expression in NRP-152 prostate epithelial cells...... 115
Figure 2.13. mTOR mediated IGF-I suppression of BMP-induced Smad 1,5,8 activation in NRP-152 prostate epithelial cells...... 117
Figure 2.14. IGF-I–mediated inhibition of BMP-induced gene microarray analysis and in vivo examination of mTOR-mediated inhibition of Smad1/5/8 in advanced human prostate adenocarcinoma...... 119
Figure 2.15. Differential expression analysis of BMP4, BMP6, and BMP8B mRNAs in human prostate samples...... 121
Figure 2.16. Loss of Id1 induces growth arrest in prostate cancer cell line LNCaP...... 123
Figure 3.1. Rapamycin-mediated cell death and Smad activation in prostate cancer cell lines...... 149
Figure 3.2. Characterization of rapamycin induced phosphorylation and activation of Smad1/5/8...... 151
Figure 3.3. Rapamycin-mediated phospho-Smad1/5/8 and Id1 luciferase activity is time and dose-dependent in mediating cell death of human and rat prostate
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epithelial cell lines (PC3 and NRP-152)...... 153
Figure 3.4. Rapamycin-mediated cell death is enhanced in the presence of BMP4 as examined in human and rat prostate epithelial cell lines...... 155
Figure 3.5. mTOR inhibition in the presence of BMP4 enhance apoptosis in PC3 cells...... 157
Figure 3.6. Silencing Smad1 and Smad5 repress rapamycin-induced Smad activation, Id1 promoter activity and cell death...... 159
Figure 3.7. Silencing Smad1, Smad5, and/or Smad1/5 cells enhance rapamycin-mediated cell death...... 161
Figure 3.8. Silencing Smad1, Smad5, and/or Smad1/5 cells enhance rapamycin-mediated Id1 promoter activation...... 163
Figure 3.9. Overexpression of Smad1 and Smad5 enhance rapamycin-induced Smad activation, Id1 promoter activity and cell death in LNCaP cells...... 165
Figure 3.10. Overexpression of Smad1 and Smad5 enhance rapamycin-mediated Smad activation and Id1 promoter activity and cell death in PC3 cells...... 167
Figure 3.11. Rapamycin-induced Smad1/5/8 activation, Id1 promoter activity, and cell death requires BMPRI...... 169
Figure 3.12. Rapamycin-induced Id1 promoter activity and expression requires BMPRI...... 171
Figure 3.13. Silencing Id1 enhanced rapamycin-mediated cell death in LNCaP cells...... 173
Figure 3.14. Rictor, Raptor and mTOR block rapamycin-mediated Id1 promoter expression in NRP-152 prostate epithelial cells requires BMPRI...... 175
Figure 3.15. Rapamycin and everolimus (RAD001) enhances phospho-Smad1/5/8 expression in vivo in PC3 Xenografts in tumors of patients with newly diagnosed localized prostate cancer...... 177
Figure 3.16. Smad1/5/8 expression is enhanced in RAD001 treatment compared to non-treated controls in vivo...... 179
Figure 3.17. Smad1/5/8 and Phospho-S6 expression in variable stages of human prostate cancer progression in vivo...... 181
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Figure 4.1. Smad1, Smad5, and Smad8-Flag tagged constructs in pDC516 vector plasmid...... 195
Figure 4.2. BMPRIIA (iso1 and iso2), ActRIIB, MISRII-Flag tagged constructs...... 197
Figure 4.3. Overexpression of BMPRII (Δ1-172aa) blocks BMP-induced Id1 promoter activation and apoptosis in NRP-152 cells...... 199
Figure 4.4. BMP-inhibits survivin in PC3 cells...... 201
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Acknowledgements I am humbled and indebted to the many people who guided me to a career in
science that I enjoy. For all the training, mentorship, and friendship that I have gained
throughout my journey, I would like to express my extreme gratitude to all. Without your aid and support to push me on to achieving all that I could, I would not be the scientist
that I am today.
I would like to start by thanking my thesis advisor, Dr. David Danielpour for his
guidance, mentorship, and support over the past few years. Your careful attention to
detail has helped shape my ability to execute my experiments with precision. My
laboratory skills have tremendously improved since joining your lab in 2005. Skills that
have enhanced my career include careful training, critical scientific thinking, independent workmanship, and expansion of collaborative interactions with other scientists.
Second, I would like to thank my thesis committee: Dr. Noa Noy, Dr. Yu-Chung
Yang, Dr. Bing-Cheng Wang, and Johannes Von Lintig for serving on my dissertation committee and providing endless inquisitive questions that have helped shape my projects. With your dedication and patience with me, I am indebted to you all and look at
you as my mentors that I aspire to become in the future. In addition, I would like to thank
the Department of Pharmacology for a strong foundation in scientific training and the
Case Comprehensive Cancer Center for the training environment and research grant
support.
Third, I am extremely appreciative for the support and guidance I have received
from my colleagues and friends in the Danielpour laboratory. Dr. Kyung Song, who has 10
helped me on a day to day basis and allowed me to bounce ideas and experimental techniques that have aided my projects. Without your assistance and guidance I wouldn’t have made it this far. Next, I would like to thank present and past members of the
Danielpour lab: Tracy Krebs, Dr. Jiayi Yang, Dr. Hui Wang, and Dorjee Shola. You all have been and force of positive energy, always close friendships, and there to help me in a time of need. All members have aided my research and help guide me during my training, for this I thank you all. I would also like to thank my dearest friends: Dr.
Vivian Gama, Dr. Ndiya Ogba, Dr. Jose Gomez, Dr. Melissa Srougi, Dr. Kevin Eng, Eric
Lam, and Tonibelle Gatbonton.
I would like to thank Case Comprehensive Cancer Center Research Oncology
Training Grant and National Research Service Award for grant support during the course of my training.
Last, but most importantly I would like to thank my parents, siblings, and husband for all their support, encouragement, and dedication to my training. I love you and I am indebted to you all.
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Abbreviations ACTRII: Activin-receptor type II
ADT: androgen deprivation therapy
ALK: Activin receptor-like kinase
AR: androgen receptor
BMP: bone morphogenetic proteins
BMPRIA/B: BMP-receptor type IA or BMP-receptor type IB
CA: constitutively active
Co-Smad: co-mediator Smad
DHT: dihydrotestosterone
DMEM/F12: Dulbecco’s modified Eagle’s medium/Ham’s F-12
DN: dominant negative
ERK: extracellular signal-regulated kinase
FBS: fetal bovine serum
HLH: helix-loop-helix
Id: inhibitor of differentiation or inhibitor of DNA proteins
IGF-I: Insulin-like growth factor
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IHC: Immunohistochemistry
KD: Knock-down
KO: knockout
MAPK: mitogen-activated protein kinase
MISRII: anti-Mullerian hormone receptor, type II mTOR: mammalian target of rapamycin
PI3K: Phosphatidylinositol-3-kinase
PSA: prostate-specific antigen
Rb: retinoblastoma protein
R. L. A.: relative luciferase activity
RNAi: RNA interference
R-Smad: receptor-regulated Smad
RT-PCR: reverse transcription-polymerase chain reaction
RT-q-PCR; Real-time PCR
SBE: Smad-binding element shRNA: small hairpin RNA
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SMADs: drosophila protein: mothers against decapentaplegic (MAD) and c. elegan protein SMA.
TGF-β: transforming growth factor-β
TβRI: TGF-β type I receptor
TβRII: TGF-β type II receptor
3TP-lux: a TGF-β-responsive reporter containing the promoter region of
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BMP/Smad Signaling in Growth Control of Prostate Epithelial Cells
Abstract by
REEMA SAID WAHDAN-ALASWAD
Bone Morphogenetic Proteins (BMPs) are pleiotropic cytokines that play integral
roles in embryogenesis, osteogenesis, and organogenesis. BMPs belong to the transforming growth factor-β (TGF-β) superfamily and are reported to function as tumor suppressors in early preneoplastic lesions of the prostate and tumor promoters in late stage prostate adenocarcinoma. Moreover, BMPs have been recently shown to be pivotal in controlling prostate tumorigenesis, and loss of BMP receptor function has been correlated to a higher Gleason grade in prostate cancer patients. During advanced prostate cancer, reports have shown that the IGF-I/PI3K/Akt/mTOR pathway is hyperactive in 50% of patients examined. In this light, we provide evidence that support that the IGF-I signaling axis inhibits BMP4-induced apoptosis, Smad-mediated gene expression, and BMP specific downstream targets. Suppression of the BMP4 signaling by IGF-I was reversed by direct genetic manipulation using enforced expression of wt-
PTEN or DN-PI3K, use of chemical inhibitors against PI3K/Akt/mTOR, or small hairpin
RNA-mediated silencing of mTORC1/2 subunits Raptor or Rictor. Our results support that IGF-I blocks BMP-induced transcription of Id1, Id2, and Id3, which are all downstream targets of BMP, through a PI3K/Akt/mTOR-dependent mechanism. Using various rat and human prostate epithelial cell lines as well as human prostate pathological specimens, we provide the first evidence that mTOR mediates inhibition of BMP-induced
Smad1/5/8 activation. Deregulation of mTOR-mediated inhibition of BMP signaling
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pathway may be crucial to halting progression of prostate cancer formation.
Furthermore, we report that direct inhibition of mTOR by rapamycin or rapalogs enhances BMP-mediated Smad1 and Smad5 activation in human prostate cancer cell lines and tissue. Utilization of lentivirus-based silencing and retroviral-based overexpression enabled us to show that Smad1 and Smad5 mediate rapamycin-induced cell death and activation of Id1. On the other hand, we showed that Smad8 represses rapamycin’s action in human prostate cell lines. All in all, the studies described here provide novel implications in the BMP and mTOR signaling axis in prostate cancer and new potential targets for the therapeutic intervention of this malignancy.
Chapter 1 Introduction
1.1. Prostate Cancer Overview/ Prostate Anatomy The prostate is gland that is about the size of a walnut that is located proximal to the rectum and under the urinary bladder (Fig. 1.1). The prostate plays a central role in secreting fluid that provides proper nourishment and protection for sperm cells in the semen. Prostate growth is supported by the secretion of androgen, testosterone, which is generated in the testicles. Testosterone is converted to dihydrotestosterone (DHT) though interaction with 5-alpha-reductase enzyme. DHT primarily is involved in prostate growth. Increase in prostate size results in a condition called benign prostatic hyperplasia
(BPH). According to the National Institutes of Health (NIH), BPH affects over 50% of men over the age of 60 and as many as 90% of men over the age of 70. Although BPH can be a serious medical issue, it is not cancerous and is resolved with surgery (in serious cases) or pharmacological agents.
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1.1.1 Prostate Zones and Lobes The prostate is sectioned into distinct anatomic regions according to their
function. The main zones of the prostate are central (CZ), peripheral (PZ), and
transitional (TZ) (Fig. 1.2). The prostate is divided into distinct lobes: anterior lobe,
median lobe, lateral lobe, and posterior lobe. The anterior lobe is the portion of the
prostate in the front of the prostate gland lying directly in front of the urethra and is
devoid of glandular cells. The median lobe is the arched-shaped part of the gland that is
located between two ejaculatory ducts near the urethra. The lateral lobe (both right and
left lobes) are primarily made of glandular tissue and are posterior to the urethra. Last,
the posterior lobe is the most proximal portion of the prostate that is located behind the
urethra.
1.1.2 Prostate Cancer Etiology In the United States, prostate cancer is the most common cancer excluding skin
cancer in males, and is only second to lung cancer to cause death [1]. When examining
the heterogeneous types of cells found in the prostate, over 99% of prostate cancer
develops primarily from gland cells. Mutations in the prostate gland cells result in
cancerous lesions that form prostatic intraepithelial neoplasia (PIN) which then gives rise
to localized invasive cancers to metastatic, and finally hormone refractory disease. When
prostate cancer becomes hormone refractory, patients typically do not respond to hormone therapy and quickly succumb to the disease.
1.1.3 Prostate Cancer Risk Factors The main risks other than being a male for prostate cancer include age, race, family history, and diet [2]. Interestingly, age is the highest risk factor for prostate
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cancer. The risk of prostate cancer increases after the age of 50 in men with little to no family history. About 80% of men over the age of 80 have prostate cancer. Next, men who have a clear family history (only accounts for 7-11% of all cases) of prostate cancer are considered to be at a higher risk than those with no family history, and are encouraged to be screened at the age of 40 [2]. African American men have a 60% higher risk of prostate cancer compared to white American men. Last, many experts speculate that high saturated fat diets also are a contributing factor to prostate cancer development. Europe and North America yield high-saturated fat diets when compared to Africa and Asia, and the incidence of prostate cancer is much higher in Easter
European nations and America than other nations (reviewed in [3]).
1.1.4 Treatment There are a number of standard treatment options for prostate cancer including; expectant management, surgery (prostatectomy), external beam radiation therapy, bracytherapy, and hormone therapy. 1) Expectant management: refers to monitoring patient conditions without invasive treatments or until symptoms are altered. 2) Surgery: is typically the one form of eradication of prostate cancer. Prostate will be removed
(radical prostatectomy) with no radiation of chemotherapy, or adjuvant therapy. 3)
External Beam Radiation Therapy (EBRT): typically includes radiation therapy (RT) and intensity modulated radiation therapy (IMRT). Radiation therapy utilizes X-ray and other forms of radiation to kill cancer cells and minimize risk to normal cells. 4)
Brachytherapy: involves implantation of small radioactive devices (the size of a rice grain) into the prostate to destroy cancerous lesions. 5) Hormone therapy: prostate cancer in stages II-III can be treated directly with surgery or with hormone therapy. Hormone
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therapy dramatically reduces the supply of androgens that directly inhibits growth of
prostate gland cells. In addition to the pre-described therapy option, advanced forms of
treatment include chemotherapy and immunotherapy.
1.1.5 Prostate Cancer Screening, TNM Staging & Gleason Score Prognostic factors that affect a patient’s response to treatment include; prostate
cancer screening, TNM Staging System, and Gleason score. Screening for prostate cancer
involves a complete digital rectal exam (DRE) whereby the physician is able to
accurately detect whether a tumor has formed, how large the tumor may be, and whether
the tumor is located on both lobes of the prostate. In addition to a DRE, a physician also
measures a baseline of Prostate-Specific Antigen (PSA) test. The PSA test measures the
blood levels of kallikrein3 (Kal3) which is a secreted protein biomarker that is released
into the blood. PSA is produced primarily by the prostate. PSA levels in the blood of prostate cancer patients are higher than in normal patients, where levels of ≥ 4 ng/ml result in further examination of the prostate with biopsy and pathological examination.
Physicians also use TNM system to stage a cancer as proposed by the American Joint
Committee on Cancer (Fig. 1.3). TNM system examines size of tumor, lymph node swelling, and any form of metastasis that could have formed. There are four stages (I-IV)
attributed to TNM staging (Fig. 1.4): 1) Stage I contain a small tumor node that is
confined to the prostate and typically involve benign prostatic hypertrophy (BPH). 2)
Stage II involves a more pronounced lump that can be detected via digital rectal exam
(DRE) with a higher Gleason score. 3) Stage III the prostate tumor node has spread throughout the transitional zone and can be felt on the anterior or posterior portion of the
prostate via DRE. 4) Stage IV prostate cancer cells extravasate into the nearby structures
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or spread to lymph nodes or other organs such as bladder, bones, liver, lungs, or brain. In addition to the TNM scoring system, physicians also rely on the Gleason Grading System
(2-10) which is based on the tissue and cellular content from biopsies. This pathophysiological grading system relies heavily on pathologist to examine cellular content and tissue distribution to confirm whether prostatic epithelial cells are intact, lumen of the prostate is clearly identified, and there is no damage or tumor tissue found within the prostate capsule. Tumors with a higher Gleason score depict metastatic capabilities of prostate cancer cell growth.
1.1.6 Progression of Prostate Cancer, Oncogenes, or Tumor Suppressor Genes In addition to understanding the pathophysiological staging system, there are a number of oncogenes and tumor suppressor genes that have been identified and mutated in prostate cancer (Fig. 1.5). Oncogenes play an important role in the regulation of cellular differentiation, proliferation, and apoptosis in the prostate. Oncogenes that may be involved in the progression of localized to metastatic prostate cancer include, c-Myc,
BCL-2, ERBB2, AR, among others (Fig. 1.5). Mutations, overexpression or amplification of these oncogene are prevalent in metastatic lesions and recurrent tumors [4].
In prostate cancer, a number of tumor suppressor genes have been identified on chromosome 8. These tumor suppressor genes include, NKX3.1, PTK2B, MSR1, and
N33 [5]. Loss of heterozygosity of chromosome 8p appears to be an early event attributing to the cancer development in the prostate resulting in the formation of PINs lesions [5]. Additional tumor suppressor genes include TP523, PTEN, p16 (CDK2A), p27 (CDKN1B) [6]. Loss of heterozygosity and point mutations of these tumor suppressor genes lead to the loss of function in prostate cancer and may play a role in the 20
acquisition of metastatic potential. In addition to these tumor suppressor genes, loss of
transforming growth factor-β (TGF-β) or bone morphogenetic protein (BMP) is
commonly observed during the progression of prostate cancer. The exact mechanism of
such loss is not fully understood, but will be examined in the next sections.
1.2. TGF-β superfamily signaling in prostate cancer The transforming growth factor-β (TGF-β) superfamily members have been well
documented for their unique roles as endogenous cytokines that regulate cell growth,
differentiation, adhesion, proliferation, and cell death [7-12] (Fig. 1.6). This superfamily
is comprised of multifunctional cytokines including TGF-βs [9, 13], Activins/Inhibins
[14, 15], Bone Morphogenetic Proteins (BMPs) [16-18], Anti-Müllerian Hormone
(AMH/MIS) [19], Nodal, Cripto, among others. The TGF-β superfamily exists as tumor
suppressors, functioning as checkpoints to properly direct cells toward cell cycle arrest or
apoptosis during early tumorigenesis [20-22] (Fig. 1.7). On the other hand, the function
of the TGF-β superfamily may transform to being oncogenic promoting tumor progression, adverting cell death, and increasing cell growth and metastasis [20, 22].
Changes in signal intensity and connectivity of these pathways may underlie the complex transition of TGF-β superfamily’s role as a tumor suppressor to oncogene during carcinogenesis [23]. BMP’s have been well documented for their role in embryogenesis, endochondrial bone formation and resorption, and organogenesis [9, 16-18, 22, 24, 25].
Although much of our understanding of BMP’s role in cancer is limited, further
clarification of BMP-mediated tumor suppressive function will be elaborated in the
following sections.
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1.2.1 The TGF-β superfamily regulatory activity and receptors activation. There are two major types of membrane-bound receptors for TGF-β superfamily ligands, namely type I and type II receptors. The receptors contain an extracellular ligand binding domain, a single membrane-spanning domain, and an intracellular domain containing a specific serine/threonine kinase region. To date, there are five mammalian type II receptors: TGF-β type-II (TβR-II) [13, 26], Activin receptor type-II (ActR-II)
[15], Activin receptor type-IIB (ActR-IIB) [14, 27], Anti-mullerian hormone or MIS type-II (AMHR/MIS) [19], and BMP type-II receptor (BMPR-II) [16]. In addition, there are seven type-I receptors in mammals known as activin receptor-like kinases (ALK-1 to
-7) [28-31]. Within the TGF-β superfamily ligands, TGF-β (TGF-β1, TGF-β2, TGF-β3) and Activin are able to bind to type-II receptor and transphosphorylates the type-I receptor at the juxtamembrane intracellular position known as the GS box (Fig. 1.8-Fig.
1.10). On the other hand, BMP ligands (1-20 different isoforms) are able to bind to the type I receptors that then dimerize with type-II receptors, which in turn transphosphorylate the type-I receptors at the GS domain [7, 29, 32-36]. The GS box is a region of conserved glycine and serine residues preceding the receptor kinase domain.
Once the type I receptor is phosphorylated, it in turn transphosphorylates a set of intracellular substrate signaling proteins called Smad(s). Specific Smad proteins that are phosphorylated by type-I receptors are referred to as receptor-regulated Smads or (R-
Smads). Two main branches of the TGF-β superfamily mediate phosphorylation of specific R-Smads: TGF-β/Activin/Nodal activate Smad2/Smad3, whereas BMP/MIS activate Smad1/Smad5/Smad8. Phosphorylation occurs predominantly on two serine residues with conserved SS(M/V)S motif at Ser465 and Ser467 at the C-terminus of the
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R-Smads [34, 36]. The R-Smads form a complex with co-Smad (Smad4) (either heterodimers (Smad1/5/8-Smad4) or heterotrimers (two Smad1/5/8 to one Smad4)), which then translocate to the nucleus and activate a number of different target genes and regulate gene expression and biological functions [20]. In addition, inhibitory Smads
(Smad6 and Smad7), act as negative regulators of the TGF-β superfamily pathway by directly interfering with receptor/R-Smad activation.
The specificity for Smad signaling is determined by the type-I receptor, rather than the type-II receptor [7]. BMP type II receptors are able to bind exclusively with
BMP ligands, including BMP-2,-3, -4, -5, -6, -7, -15, GDF-5, -9 etc. ALK-2 (ActRI-A),
ALK-3 (BMPR-IA), and ALK-6 (BMPR-IB) have been identified as BMP type I receptors [14, 37], that are activated by BMPRII [32, 33]. Promiscuity between the BMP ligands and the BMP receptors has contributed to the difficulty of understanding the biological functions of BMP-signaling in prostate cancer.
1.2.2 Jekyll and Hyde function of TGF-β-Superfamily during prostate carcinogenesis. Original discovery of TGF-β began during the early 1980’s in Michael Sporn’s group at the National Cancer Institute. They showed that all “transforming” activity from sarcoma growth factors (SGF) was lost when SGF extracts were fractionated through gel filtration columns [38, 39]. Two fractions arose from these crude extracts: transforming growth factor-α and transforming growth factor-β [40]. Soon after, alternate TGF-β superfamily members were identified. In this report I have mainly focused on Bone
Morphogenetic Proteins (BMPs). BMP-mediated signaling has been reported to have dual functions, one as a tumor suppressor in normal prostate epithelium and the other as
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tumor promoter in late stage prostate cancer [41-43] (Fig. 1.11). In addition to the dual role BMPs may play during carcinogenesis, mutational inactivation of BMP signaling pathway is linked to the pathogenesis of a number of sporadic and inherited human cancers [44, 45]. During tumor progression, tumor cells typically acquire resistance to growth-inhibitory effects of BMP due to defects in the BMP-signaling pathway, such as loss of BMPRI or BMPRII. Specifically, inactivation of either BMPRIA (ALK3) or
Smad4 may cause familial juvenile polyposis syndrome [46-49]. Expression of BMPRII,
BMPR-IA and BMPR-IB were examined in normal, benign, and well-differentiated prostate cancer specimens at the IHC or mRNA level, where BMP receptors were loss in late stage human prostate cancer [50, 51].
A number of mechanisms have been proposed to explain the basis for the switch of BMP from a tumor suppressor to a tumor promoter. Loss of BMP receptors in late stage cancers is a poor prognostic indicator, and such a loss may be gradual as the cancer progresses from stage I to stage IV [51, 52]. In addition to loss of BMPRs, hyperactivation of oncogenic pathways, such as IGF-I/PI3K/Akt/mTOR or Ras/MAPK, could also result in suppression of BMP-mediated growth suppression in human prostate cancer [51-53]. Moreover, alterations in Smads, via inhibition of phosphorylation could also disrupt BMP-mediated tumor suppressive capabilities.
1.2.3 Alterations of BMP pathway in human cancers Disruption in tumor suppressive capabilities of BMP is prompted by either alterations or modifications to components of the BMP signaling pathway. Modulations in BMP signaling pathway is marked by either alterations in receptors (BMPRIA,
24
BMPRIB, ACVRI, or BMPRII), Smads 1, 5, or 8, Smad4, or Smad6 [7, 9, 16, 24, 52,
54]. Genetic or epigenetic modifications are examined below:
A. BMP- Receptors Mutations in BMPRII (BMPR2) are found to be largely somatic, which may occur from
microsatellite instability (MSI). Defective missmatch repair mechanisms yield high
incidence in MSI, leading to subsequent mutations in BMPRII. A number of mutations
that may arise in BMPRII include non-sense mutations yielding a truncated receptor
devoid in trans-membrane and intracellular domains that elicit down-stream activation of
Smads or BMP-specific target genes in a number of human cancers [55, 56] (Fig. 1.12).
Moreover, BMPRII mutations have been shown to cause primary pulmonary
hypertension (PPH) [57]. BMP receptors (BMPRIA, BMPRIB, and BMPRII) are
expressed in colonic epithelial cells lines [58], and may prompt formation of polyps.
Moreover, several frameshift mutations were observed in the BMPRII gene [59], where
about 7% of gastric cancers and 13% of colorectal cancer had high MSI, suggesting that
BMPRII mutations might contribute to cancer pathogenesis via inactivation of BMPRII
resulting in abrogation in of BMP signaling [60].
Alterations or mutations in BMP receptor type I (BMPRIA, BMPRIB, or
ACVRI), also known as ALK2, ALK3 and ALK6, expression via IHC analysis have been
observed to be lost in a number of late stage human cancers [61, 62]. Mutations of
BMPRIs have been observed in osteoclast/osteoblast function (reviewed in [63]).
SMAD4 Germline and somatic mutations in SMAD4 have been linked to colon and
pancreatic cancers [64]. Further, inactivation of Smad4 has also been linked to familial 25
juvenile polyposis syndrome [46, 48]. Moreover, sporadic mutations of Smad4 have been reported in a number of human tumor types, including prostate, colon, pancreas, breast, among others [65, 66].
1.2.4 Implications of BMP in advanced prostate cancer BMP signaling pathway has been implicated in prostate cancer progression, in particular during metastasis to the bone [67]. Loss of BMPRIA, BMPRIB and BMPRII expression has been examined in human prostate cancer patient biopsy samples and associated with poor prognosis [50, 51, 55, 67]. BMPs play a prominent role as a tumor suppressor by inducing apoptosis in a number of human prostate epithelial cell lines [51].
Knock-down of BMPRII inhibited the growth of prostate cancer cell line (PC3M) in a xenograft murine model system [51]. Further, levels of BMP-2 and BMP-6 are reported to be higher in advanced metastatic stage prostate cancer [68, 69]. BMP-2, BMP-4,
BMP-7 and BMP-9 are expressed in normal prostate tissues and have altered or reduced expression in advanced stage prostate cancer [67, 70-73]. Expression of BMP-7 was reported to be lower in human prostate epithelial cells and localized prostate cancer tissue after hormone therapy, whereas treatment with dihydrotestosterone resulted in an increase in BMP-7 [71]. Loss of BMP-7 has been linked to a more aggressive phenotype in the prostate [73]. On the other hand, overexpression of BMP-9 blocks growth of prostate cancer cells [74]. Expression levels of BMPs and mRNA levels in the prostate may differ from their expression levels in the bone. All in all, BMPs play a critical role in the development and progression of prostate cancer, and a differentiated role in advanced prostate cancer that has metastasized to the bone.
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1.3. Smads: critical mediators of TGF-β signals The Smad proteins are identified as Mad protein in Drosophila and Sma Proteins in C. elegans [75, 76]. To date, there are eight Smad proteins that have been identified and characterized in mouse and human genomes (Smad1-8), four in Drosophila, and three in C. elegans (Sma1-3). Smad proteins serve as principle substrates that are involved in signal transduction from cell membrane to the nucleus for the TGF-β/BMP signaling pathways (Fig. 1.13). Smads are divided into three different classes based on the biological function and structure. The first group is known as receptor-regulated
Smads (R-Smads) (Smad1, Smad2, Smad3, Smad5, Smad8). Smad1, Smad5, and Smad8 are activated by BMP/Anti-Muellerian signaling pathway and Smad2 and Smad3, are activated by TGF-β/Activin/Nodal signaling pathway. R-Smads are activated through phosphorylation by type-I receptors that are activated by heterodimerization with type-II receptors. Once R-Smads are activated they couple with co-Smad (Smad4), which is a common mediator that directly interacts with R-Smads. Last, the third class involve inhibitory Smads (I-Smads) (Smad6 and SSmad7) which serve as a decoy interfering with R-Smads, Smad-Smad interaction, or Smad-receptor interactions. Smad6 blocks
BMP-mediated signaling, whereas Smad7 repressed TGF-β mediated signaling.
1.3.1 Main features of Smad proteins Smad proteins share amino acid sequence homology; they contain an MH1 domain, a linker region, and an MH2 domain and are about 500 amino acids in length.
N-terminal domain (also known as Mad-homology 1 or MH1 domain) and the C-terminal domain with two globular domains that are linked by a variable linker domain (Fig. 1.14).
The MH1 is highly conserved in all Smads (Smad1, 2, 3, 4, 5, and 8) but not in Smad6 or
27
Smad7. Similarly, the MH2 domain is quite conserved in all Smad proteins. The crystal structure of the MH1 and MH2 domains provides crucial information regarding the ability of Smad proteins to interact with the DNA. The MH1 domain is able to interact with the DNA through a β-hairpin structure and tightly bound by a zinc atom. All
Smads, with the exception of Smad2 bind to the DNA, where Smad2 has an extra insert sequence in its MH1 domain disrupting its binding to the DNA. The MH1 domain contains a linker region, which acts as a docking site for proteins such as Smurf, which is
Smad ubiquitination-related protein, phosphorylation site for a number of protein kinases, and ubiquitin ligases. In Smad4, the middle linker also serves as phosphorylation site for nuclear export signal (NES). I-Smads lack middle linker region. Further, the MH2 domain has a number of protein-interacting sites that propagate signal transduction. For example, R-Smads have a Ser-X-Ser motif on the C-terminal end of the MH1 complex that is phosphorylated by active receptor complexes. The Ser-X-Ser motif further allows this region to further interact with the active receptor. The MH2 domain is involved in
Smad-receptor interaction, heteromeric and homomeric Smad complex formation.
Within the crystal structure of MH2 domain, there are a set of hydrophobic regions, also known as “hydrophobic pockets” which contain the site for multiple interactions such as with nucleoporins, DNA-binding cofactors, etc (reviewed in [75]).
1.3.3 Smad Transcriptional Activation and BMP Response Elements Once Smad1/5/8-Smad4 form a complex they are able to translocate to the nucleus, bind to DNA through the MH1 domain, and regulate a number of different target genes involved in transcription. The MH1 is able to achieve high specificity within the
DNA-binding region, because it contains a number of BMP response elements (BRE)
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that enable the binding of Smad1/5/8 to the DNA-binding region encoded upstream from
the transcriptional start site. The BRE includes a GC-rich sequence, such as 5’-
GGCGCC-3’ [77-80]. Katagiri et. al., 2002 previously identified a ~29 bp GC-rich
elements that were later identified as BMP-response elements (BRE) located in the 5’-
flanking region of human Id1 gene. Inhibitor of DNA binding/differentiation (Id-1) is a
basic helix-loop-helix protein that has been reported to be upregulated by BMP-2, -4, -6,
and -7 [81]. Id1 lack DNA binding domain, however, they can form heterodimers with
alternate basic helix-loop-helix family of transcription factors thus inhibiting transcriptional activity [82]. Further, Smad1/Smad4 complex was shown to recognize the
BRE in response to BMP-2 [81].
1.4. TGF-β and BMP Cross-talk with Major Pathways The TGF-β/BMP signaling pathways control a wide array of events, including
differentiation, proliferation, apoptosis, immune function, tumor invasion/metastasis, and
extra cellular matrix (ECM) formation (reviewed in [53]). Much of these intricate
processes arise from the ability of TGF-β/BMP signaling to be able to cross-talk with
alternate intracellular and extracellular signals (Fig. 1.6). TGF-β/BMP cross-talks with
other signaling mediators, such as insulin-like growth factor-I (IGF-I)/ phosphoinositide
3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway,
mitogen-activated protein kinases (MAPKs), Notch, Jak-STAT, Rho-like GTPases
pathways, among others (reviewed in [53, 83]). The activation of these receptor-
mediated downstream pathways may be either Smad-dependent or Smad-independent.
The interplay between TGF-β/BMP signaling with other pathways provides wide
diversity and complexity of the biological functions that have been identified in a number
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of studies. In this thesis, we will focus on BMP-mediated cross-talk with a number of
key signaling pathways that have generated much inquisition in the past decade or so.
1.4.1 BMP and the MAPK pathway BMP pathway has been shown to interact with Ras/MAP kinase pathway, where
mitogenic signals through the receptor serine/tyrosine kinase and Ras have been shown to
act cooperatively as well as antagonistically in oncogenesis [84] (Fig. 1.15). A number
of extracellular stimuli can activate the cascade of serial phosphorylation and activation
of MAP kinase kinase kinase (MAPKKK) to MAP kinase kinase (MAPKK) to MAP
kinase (MAPK). MAPKs are able to activate ERK1/2, JNK1/2/3, and p38. Components
of MAPKs are evolutionarily conserved and essential regulators of a number of cellular
events. In recent reports, Smad1 has been shown to be directly phosphorylated by ERK at a number of MAPK motif sites in the linker region, resulting in deactivation of Smad activity [84-87]. The BMP and Ras/MAPK pathways have been shown to directly cross- talk with one another during development in Xenopus, where Smad1 was shown to inhibit neural development and differentiation [86]. Localized expression of Smad1 in Xenopus leads to differentiation toward a pre-ventral-like phenotype. Similarly, Aubin et. al. 2004, generated a Smad1 deficient mutant mouse model which directly lacks MAPK consensus motifs located in the linker region [88]. Generation of these mutant mice provided evidence that MAPK suppresses Smad1 through the middle linker region. Deletion of the
MAPK consensus motifs located in Smad1 linker region caused Smad1 protein to be resistant to MAPK-mediated phosphorylation. MAPK inhibition of R-Smad middle
linker region, typically results in loss of membrane potential which can lead to alterations
in cytoskeleton arrangement and modify cell adhesion [84]. Erk1/2 has been shown to
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directly phosphorylate the middle linker of Smad1 and Smad5, resulting in inhibition of
Smad1 or Smad5 activation and translocation to the nucleus. In the prostate, Erk has
been shown to phosphorylate the linker region of Smad1, which subsequently allows
Smad1 to interact with androgen receptor (AR) in the presence of BMP [89]. The ability
for Smad1 to bind to AR further blocks androgen-stimulated prostate cell growth by
BMP. Ras can further decrease Smad4 stability, as MEK/Erk preferentially
phosphorylate Smad4 and promote proteasomal degradation. Similarly, Erk, JNK and
p38 are able to control transcriptional regulation of Smad6 and regulate BMP mediated
signaling [90].
1.4.2 BMP mediated crosstalk with AR Cooperation among Smads and nuclear receptors has been proposed, with
particular interests in specific cross-talk between androgen receptor (AR) and Smad3
[91]. Smad3 was shown to directly modulate AR activity in response to TGF-β [91-93].
Alternatively, Smad1 was reported to bind to AR and inhibit its function [89]. In addition, alternate reports have demonstrated that the expression of BMPR-IB, not
BMPR-IA or BMPRII, was up-regulated by androgen and in an androgen sensitive prostate cancer cell line, LNCaP. BMPR-IB mRNA levels were significantly lower in prostate tissues after androgen withdrawal therapy [55]. Further, BMP-2 and BMP-4 have been reported to inhibit growth of androgen-sensitive (AR positive) LNCaP cells but not the androgen-insensitive PC3 cell line [55]. Moreover, BMP-mediated Smad1 activity was able to inhibit growth through BMP-induced interaction among Smad1 and
AR [89].
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1.4.3 BMP Crosstalk with IGF-I Signaling Pathway Insulin-like growth factor-I (IGF-I) is a critical survival factor, known to control
growth of both normal and malignant cells in a number of different tissues, including the
prostate [83, 94] (Fig. 1.16). IGF-I has been reported to be dysregulated during tumor
progression and metastasis in a number of different human cancers, including prostate.
IGF-I has also been known to be hyperactive in about 50% of prostate cancer patients. In
advanced prostate adenocarcinoma, redundant signal transduction among IGF-I and
downstream signaling molecules appear to increase during tumor progression. IGF-I is
able to activate tyrosine kinase receptor (IGF-IR), which relays a cascade of signals
transduction through phosphatidylinositol-3 kinase (PI3K)/Akt/mammalian target of
rapamycin (mTOR). Dysregulation of this pathway results in aberration of proliferation,
resistance to apoptosis, and alteration in cell metabolism, which are major hallmarks of
transformed cells. Correlative studies have linked high serum IGF-I levels and the risk of
prostate adenocarcinoma, whereby overexpression of IGF-I ligand in the prostate basal
epithelial layer in transgenic mice yielded a similar phenotype as human prostate
adenocarcinoma [95-97]. The molecular mechanisms that influence IGF-IR up-regulation
may involve the loss of tumor suppressive functions of PTEN and/or loss of either TβRII or BMPRII loss of receptor activation. Overall, inhibition of IGF-I pathway may deter the activation of this anti-apoptotic signal and induce cell death by TGF-β/BMP.
Examination of IGF-I downstream targets may unleash signaling mechanism involved in
IGF-I mediated inhibition of TGF-β/BMP signaling pathway responses in prostate cells.
Detailed analysis of PI3K/Akt/mTOR mediated interactions with BMP signaling pathway
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will allow us to understand IGF-I mediated inhibition of BMP signaling pathway, and
shed light on possible therapeutic intervention for prostate cancer.
A. PI3K and Akt signaling pathway PI3K is often activated by RAS or directly by specific tyrosine receptor kinases
(TRKs), growth factors, or cytokines such as interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-6,
platelet growth factor (PDGF), insulin-like growth factor (IGF-I), IGF-2, IGF-3,
epidermal growth factor (EGF), colony stimulating factor (CSF), among others [98].
PI3K, specifically, is activated in a number of different human cancers, and contributes to
cell cycle progression, alterations in differentiation, aberration in apoptosis, and increase
in metabolic capabilities of cancer cells. Hyperactivation of PI3K has been linked to
tumor progression and cell transformation in breast, ovarian, renal, and brain carcinomas
(reviewed in [99]). Active PI3K is able to phosphorylate inositol lipids at the 3’ position
of the inositol ring, allowing the product PI3-phosphate [PI(3)P], and PI3,4-bisphosphate
[PI(3,4,)P2] and PI3,4,5-triphosphate [PI(3,4,5)P3]. These lipid products are able to
induce a number of cellular responses, such as cell proliferation, survival, cell adhesion,
motility, angiogenesis, and cytoskeletal organization. Once PI3K is active, a myriad of
downstream signals are able to induce protein kinase B (PKB), also known as Akt,
which has been implicated in a wide array of cellular functions including cell
transformation, cell proliferation, apoptosis, angiogenesis, and tumor growth [100, 101].
Akt activation is dependent on PI(3,4)P2 which is required for Akt to be anchored in the membrane to PDK1. PDK1 is able to phosphorylate Akt at Thr308 [102]. A number of
Akt substrates exist, such as GSK3, protein BAD, forkhead family of transcription factors, endothelial nitric oxide (eNOS), mTOR, BRCA1, and 6-phosphofructo-2-kinase,
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pamong others. Akt is also able to indirectly activate mTOR via tuberous sclerosis (TSC), which activates a number of downstream targets involved in translation of mRNA, cell cycle, and apoptosis [98, 103].
B. PTEN and BMP role in Prostate Cancer Phosphatase and Tensin homolog (PTEN/MMAC1/TEP1), an inhibitor of PI3K, is commonly lost or mutated in human prostate adenocarcinoma [104, 105]. Loss of tumor suppressor phosphatase and tensin homolog (PTEN) aberrantly activates
PI3K/Akt/mTOR signaling in up to 50% human prostate cancer [105, 106]. Transgenic mice with functional loss of PTEN have been confirmed to induce development of spontaneous tumor nodes in the prostate which subsequently leads to the activation of
Akt and mTOR, both critical components in the oncogenic function in prostate adenocarcinoma [106-109]. Germline mutations of PTEN develop Bannayan Zoanna syndrome, which is the underlying cause for hamartoma and Cowden disease. PTEN has been well characterized to act as a tumor suppressor by arresting cell growth by antagonizing protein tyrosine kinases. PTEN also functions to regulate tumor cell invasion and metastasis through interacting with focal adhesions. PTEN regulates PI3K pathway by dephosphorylating phosphatidylinositol 3,4,5-triphosphate at the D3 position, whereas mutations in PTEN are unable to convert PI(3,4,5)P3 to PI(2,3)P2. A number of mouse models confirm that loss of heterozygosity of PTEN along with activation of Akt, mTOR and P70S6K promotes neoplasia in prostate cancer [110, 111].
C. mTOR Signaling in human Cancers The mammalian target of rapamycin (mTOR) is a 298 kDa serine-threonine kinase downstream of PI3K/Akt signaling that plays a vital role in initiating signals to
34
control cell growth, survival, mRNA transcription, protein synthesis, proliferation, metabolism, membrane trafficking, cytoskeletal organization, and angiogenesis [112].
Activation of mTOR is mediated through insulin-like growth factor (IGF-I)/PI3K/Akt
[102, 113-115], where hyperactivation of mTOR has been reported in a number of human cancers due to either loss of tumor suppressor genes or deregulated activation of oncogenes [94, 116-118]. Further, mTOR activation involves the functional loss of
PTEN via amplification or mutation of the PI3K catalytic subunit (p110α) or regulatory subunit (p85α), amplification of either AKT-associated isoenzymes AKT1 and 2, and inactivation or mutation of the regulatory proteins such as tuberous sclerosis 1 (TSC1) or
TSC2 [119-126].
mTOR functions as a macromolecular complexes called mTOR complex 1
(mTORC1) and mTOR complex 2 (mTOR2) that are either rapamycin sensitive or insensitive. mTORC1 contains an mTOR catalytic subunit that complexes with raptor,
PRAS40, and mLST8; whereas mTORC2 complex contains mTOR, rictor, mLST8 mSIN1, and PROTOR [127-130]. mTORC1 is activated through activation of Akt- mediated phosphorylation of PRAS40 (proline-rich substrate of Akt) and tuberous sclerosis complex (TSC1) and TSC2 [131-133]. Raptor and GβL (an alternate protein) have been proposed to negatively regulate mTOR through strong association between raptor and mTOR [134-137]. Last, mTORC2 contains rictor which is able to function as the PDK2 activity to phosphorylate Akt on Ser473 [116]. mTORC2 controls actin cytoskeleton as well as AKT/PKB actions [138]. Along with the PI3K/Akt signaling pathway, mTOR plays a prominent role as a key mediator in cancer signaling networks
[102, 103, 120, 126, 130]. Also alterations in mTOR dependent-Akt signaling cascade 35
can result in the development of prostate intraepithelial neoplasia (PIN) lesions which
mark the initiation of prostate cancer formation [139].
Aberrant signaling regulating mTOR catalytic activity contributes to cancer progression and metastasis in a number of in vitro and in vivo models. The rationale behind using mTOR inhibitors, such as rapamycin to tightly binding to an intracellular protein FKBP12, where the complex then binds to mTOR juxtaposed to its catalytic kinase domain at the FKBP-12-rapamycin-binding (FRB) domain [132]. In the clinic, mTOR-targeted therapy is used to efficiently inhibit abnormal cell proliferation, cell metabolism, and tumor angiogenesis in a number of different solid tumors [140].
Utilization of rapamycin and rapamycin-like analogs has shown much promise in advanced stage renal cell carcinoma, endometrial cancers, mantle cell lymphoma, among others. Rapamycin, Everolimus (RAD-001), and Temsirolimus (Torisel) have been confirmed to have cytostatic activity and show clear promise for the therapeutic intervention of prostate cancer [94, 141, 142]. The mechanism of rapamycin-anti-tumor activity is yet to be defined. Using mTOR inhibitors, our lab reported the cross talk interplayed between the mTOR and TGF-β signaling pathway [143, 144], where we observed direct mediated inhibition of TGF-β signaling pathways. Now our current work has highlighted mTOR-mediated inhibition of BMP signaling (chapter 3).
D. IGF-I mediated inhibition of TGF-β pathway Previously, our lab reported that IGF-I was able to block TGF-β mediated responses, such as transcription, apoptosis, and Smad3 activation in the well characterized NRP-152 non-tumorigenic rat prostate epithelial cell line developed in our lab [143, 145, 146]. These studies demonstrated that IGF-I was able to block TGF-β- 36
induced transcriptional activation of plasminogen activator inhibitor1 (PAI1) promoter construct (3TP-luciferase). To examine the biological effects of IGF-I on prostate epithelial cells, we used an analogue of IGF-I, (LR3-IGF-I), shown to bind poorly to
IGF-I binding proteins (IGFBPs). This analogue is 500-fold more active then IGF-I in blocking TGF-β-induced apoptosis and blocks cell death by >95% [143]. LR3-IGF-I was also shown to repress TGF-β-mediated activation of Smad3, but not Smad2 or Smad4 in
NRP-152 cells. The mechanism of IGF-I mediated inhibition was thought to be prompted through Akt, which suppressed TGF-β responses by directly binding to Smad3.
Not only was Akt able to block Smad3 activation, but Akt-mediated suppression also involved mTOR [8, 143]. Much of our current knowledge has focused on the role of
IGF-I mediated inhibition of TGF-β1-induced apoptosis. To date, I have unraveled novel observations that IGF-I-mediated inhibition of the BMP signaling pathway, by suppressing Smad1/5/8 activity, Id1 promoter activity, and apoptosis in prostate epithelial cell lines through a PI3K/Akt/mTOR dependent mechanism. Moreover, rapamycin was shown to block mTOR mediated inhibition of BMP signaling by enhancing Smad1 and
Smad5 expression in vitro and in vivo. Therapeutically, this study will provide a clear assessment of the molecular mechanisms of rapamycin’s action in prostate cancer.
37
38
Figures
Figure 1.1. Prostate Anatomy. The prostate is the size of a walnut that is located between the bladder and the penis. The prostate is proximal to the rectum. The urethra runs through the middle of the prostate, from the bladder to the penis, allowing urine output flow out. (Image adapted from www.cancer.gov/cancertopics/wyntk/prostate, Created by the National Cancer Institute).
39
(Figure 1.1)
40
Figure 1.2. Zone of the Prostate The Three zones of the prostate are sectioned into the peripheral zone (PZ), Central zone
(CZ), and transition zone (TZ) (Adapted from www.prostateuk.org/prostate/aboutprostate.htm).
41
(Figure 1.2)
42
Figure 1.3. Anatomical staging of Prostate Cancer. The TNM system examines the location and size of prostate tumor. T=local tumor growth, N=the lymph nodes, M=distant metastases (Adapted from edoc.hu- berlin.de/.../HTML/chapter1.html)
43
(Figure 1.3)
44
Figure 1.4. Prostate Cancer Staging System. Stage I: In stage I, the prostate lesions known as prostatic intraepithelial neoplasia (PINs) are found in the prostate. Stage I is smaller than a gram and may be hard to detect microscopically or DRE. Stage II. The tumor has grown within the confines of the prostate, but has not expanded beyond. Stage III. Prostate cancer cells have spread outside the prostate gland to tissues around the prostate, but not the lymph nodes. Stage
IV. Cancer cells have metastasized to the lymph nodes or to nearby organs and tissues away from the prostate such as bone, lungs, liver, or brain. Image adapted from NCI cancer information page (http://www.cancer.gov/cancertopics/pdq/treatment/prostate).
45
(Figure 1.4)
46
Figure 1.5. Human Prostate Cancer Progression from Normal Epithelium to Metastasis. The schematic view of human prostate duct. Stages of progression of prostate cancer are
shown along with the molecular processes and genes/pathways that are likely to be
disrupted during each state. (Adapted from Abate-Shen & Corey , Genes and
Development 2000).
47
(Figure 1.5)
48
Figure 1.6. TGF-β Superfamily Signaling.
Signaling is initiated once ligand binds to either Ser/Thr Type I or Type II receptor,
which oligomerizes and initiated phosphorylation of cytoplasmic signaling molecules
Smads (Smad2/3 for TGF-β pathway or Smad1/5/8 for the BMP pathway). The C-
terminal phosphorylation of Smads by activated receptors results in R-Smads to bind to
co-Smad (Smad4). The R-Smad/Smad4 complex translocate to the nucleus, bind to
specific Smad Response Elements (SRE) or BMP Response Elements (BRE) which
induce different set of target genes that activate a myriad of different diverse biological
functions. Last, I-Smads (Smad6 or Smad7) serve a decoy to block receptor mediated
Smad activation or play a role as a negative feed-back loop. Image adapted from
(www.cellsignal.com).
49
(Figure 1.6)
50
Figure 1.7. TGF-β Superfamily role in Human Cancers. TGF-β superfamily signaling pathway is able to play a prominent role at the G1 stage to inhibit cell proliferation, induce differentiation, and promote apoptosis. However, dysregulations in the pathway may result in the transformation of a cell to a cancerous cell, which give rise to the pathways role as a tumor promoter. Mutations, loss of receptor functions, or alternate consequences may allow previous TGF-β/BMP sensitive cells to become resistant to TGF-β/BMP, resulting in uncontrolled proliferation. Further, increased production of TGF-β/BMP may also affect surrounding stromal cells, immune cells, and endothelial cells and smooth muscle cells to induce angiogenesis and increase the invasiveness of the tumor. Picture adapted from (Blobe GC et al NEJM 2000).
51
(Figure 1.7)
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Figure 1.8. TGF-β Superfamily Ligands TGF-β superfamily is comprised of TGF-β ligand (TGF-β1, TGF-β2, TGF-β3), Bone
morphogenetic proteins (BMPs) (BMP1-20), Activin (Activin A, Activin B, Activin AB),
Nodal, Cripto, Growth and differentiation factors (GDFs), Anti-mullerian hormone
(AMH). (Picture adapted from Li L & Xie T Annu rev Cell Dev. 2005).
53
(Figure 1.8)
54
Figure 1.9. TGF-β Pathway and Human Disease. Image adapted form (Gopal Sapkota) http://www.ppu.mrc.ac.uk/research/profiles/10/img/Figure1.gif)
55
(Figure 1.9)
56
Figure 1.10. BMP mediated Signaling
BMP signaling is activated once BMP ligand (1-20 different ligands) are able to bind to
BMPRI which then dimerizes with BMPRII allowing heterodimeric complex to
transphosphorylate BMPRI and initiate activation of cytoplasmic Smads (Smad1, Smad5,
or Smad8). C-terminal phosphorylation of Smad1,5,8 then couple to co-Smad (Smad4), translocate to the nucleus and bind to BRE and activate a number of BMP-target genes.
Inhibitory Smad (Smad6) can block R-Smads. BMP can also cross-talk with MAPK,
JNK, NF-κB. Picture adapted from (www.bioscience.org/2009/v14/af/3293/figures.htm)
57
(Figure 1.10)
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Figure 1.11. Dual Role of TGF-β Mediated Signaling Dual Role of TGF-β Signaling Pathway. TGF-β can act as a tumor promoter and tumor suppressor. There are two models proposed for TGF-β-mediated signaling in human cancers. Model 1 TGF-β invokes a tumor suppressive function in epithelial cells, whereas it acts as a tumor suppressor in stromal cells. In model 2, TGF-β is able to convert from a
tumor suppressor to a tumor promoter through fundamental alterations in the response of epithelial-derived cancer cells to TGF-β, whereby epithelial to mesenchymal (EMT) transition take full force. (Image adapted from Rebecca L Elliott, Gerard C Blobe, J
Clinical Oncology 2005).
59
(Figure 1.11)
Model 1 TGF‐β Tumor Tumor Suppressor Promoter
Epithelial Cells Stromal Cells •hTERT repression •Induction of angiogenesis •Induction of apoptosis •Immunosuppressant •Growth inhibition •Promotion of metastasis
Model 2 TGF‐β
Epithelial Cells Tumor Suppressor Stromal Cells •Induction of angiogenesis •Promotes differentiation EMT •Induction of apoptosis •Promotion of metastasis •Growth inhibition •Induces migration •Smad‐dependant? ? •Increases invasion •Smad‐independent?
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Figure 1.12. Mutation or Alterations in BMPRII.
BMP ligands bind to BMPRII, which in turn mediates signaling that result in apoptosis
and inhibition of proliferation. Modifications in the cystine residues within the ligand
binding domain or kinase domain of BMPRII results in alterations in downstream
signaling. Non-cystine mutations within the kinase domain will enable the signal to
reach cell surface, but abrogates activation of Smads. Missense mutations in the
cytoplasmic tail of BMPRII are able to traffic to the cell surface and signal via Smads,
but may be defective in MAPK signaling. (Image adapted from Rudarakanchana, N. et al. Hum Mol Genet 2002).
61
(Figure 1.12)
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Figure 1.13. TGF-β/BMP-mediated Smad signaling
TGF-β superfamily signaling is initiated one ligand binds to the serine/threonine kinases
(receptor type I and II), which form a heterotetrameric receptor complex. Active receptors then mediated phosphorylation of the GS domain in SXS motif in the C- terminus or R-Smad. R-Smads couple to Smad4 which then translocate to the nucleus and recognize specific DNA-response elements. DNA-binding cofactors and corepressors that activate to repress transcriptional activation. (Image adapted from
Massague Genes and Development 2005; Crystal structure from Shi et al 1997; Huse et al
1999; Wu et al 2000).
63
(Figure 1.13)
.
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Figure 1.14. Smad Molecular Structure.
Regulatory Smads (R-Smads) have a conserved MH1 and MH2 domains which interact with DNA-specific sequences (CAGAC) located at the β-hairpin region, type I receptor binds to the L3 loop, and the α-helix region. The MH1 and MH2 domains are connected with a linker region, which contains MAPK/Erk consensus sequences that regulate Smad activation. Last, receptor-mediated activation of R-Smads occurs at the C-term at the
SSXS motif. (Adapted from Massague 2005; Shi et al 1997; Chen YG et al. 2000).
65
(Figure 1.14)
66
Figure 1.15. BMP and MAPK Crosstalk.
Components of Smad1, Smad5, and Smad8 middle linker region known to contain
MAPK consensus regions which may be phosphorylated and alter Smad1/5/8 function.
Cross-talk between MAPK and BMP results in inactivation of Smad function. (Image adapted from Petra Knaus lab).
67
(Figure 1.15)
68
Figure 1.16. TGF-β signaling and IGF-I cross talk.
IGF-I mediated cross talk with TGF-β signaling pathway. (Adapted from D Danielpour and K. Song Cytokine & Growth Factor Review, 2006).
69
(Figure 1.16)
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Chapter 2: Insulin-Like Growth Factor I Suppresses Bone Morphogenetic Protein Signaling in Prostate Cancer Cells by Activating mTOR Signaling
The work described in this chapter has been accepted in Cancer Research (2010)
Reema S. Wahdan-Alaswad1,2, Kyung Song1, Tracy L. Krebs1, Dorjee T.N. Shola1,4, Jose A. Gomez2, Shigemi Matsuyama2,3, and David Danielpour1,2,5
ABSTRACT Insulin-like growth factor (IGF) I and bone morphogenetic proteins (BMP) are
critical regulators of prostate tumor cell growth. In this report, we offer evidence that a
critical support of IGF-I in prostate cancer is mediated by its ability to suppress BMP4-
induced apoptosis and Smad-mediated gene expression. Suppression of BMP4 signaling by IGF-I was reversed by chemical inhibitors of phosphoinositide 3-kinase (PI3K), Akt, or mTOR; by enforced expression of wild-type PTEN or dominant-negative PI3K; or by small hairpin RNA–mediated silencing of mTORC1/2 subunits Raptor or Rictor.
Similarly, IGF-I suppressed BMP4-induced transcription of the Id1, Id2, and Id3 genes that are crucially involved in prostate tumor progression through PI3K-dependent and mTORC1/2-dependent mechanisms. Immunohistochemical analysis of normal human prostate tissue or human prostate tumors offered in vivo support for our model that IGF-
I–mediated activation of mTOR suppresses phosphorylation of the BMP-activated Smad transcription factors. Our results offer the first evidence that IGF-I signaling through mTORC1/2 is a key homeostatic regulator of BMP4 function in prostate epithelial cells, acting at two levels to repress both the proapoptotic and pro-oncogenic signals of BMP-
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activated Smads. We suggest that deregulation of this homeostatic control may be pivotal to the development and progression of prostate cancer, providing important implications and new potential targets for the therapeutic intervention of this malignancy.
INTRODUCTION Bone morphogenetic proteins (BMP) are multifunctional cytokines belonging to the transforming growth factor (TGF)-β superfamily that play critical roles in osteogenesis, organogenesis, and embryogenesis, in which they control cell differentiation, proliferation, migration, and apoptosis (1–6). BMP signaling is initiated by the association of a BMP ligand (any 1 of ≥14 isoforms) with two transmembrane serine/ threonine receptor kinases: BMP receptors II and I (typically BMP receptors IA and IB), the latter of which directly phosphorylate the transcription factors Smads 1, 5, and 8 [9, 17, 18,Chen, 2004 #172, 25]. The phosphorylated Smads then couple to Smad4 and translocate to the nucleus, where they modulate the transcription of numerous genes in part by binding to BMP response elements. Whereas BMPs function as tumor suppressors in early-stage prostate cancer, they are reported to also promote progression of advanced/hormone refractory prostate cancer [41-43]. However, the mechanisms underlying this functional dichotomy are poorly understood but likely involve the combined action of multiple gene changes.
Insulin-like growth factor (IGF)-I is a well-known survival factor for both normal and malignant cells in many tissues, including the prostate [147, 148], although IGF-I has been shown to also be critical in controlling the differentiation of many tissues through mechanisms that remain underexplored [149-152]. The survival function of IGF-I seems to be predominantly through a signal transduction cascade involving phosphoinositide 3-
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kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR; refs. [101, 148, 153]).
Numerous studies collectively suggest that enhanced IGF-I signaling is critical for the
development and progression of prostate cancer [148]. Importantly, correlative studies
have linked high plasma IGF-I levels and prostate cancer risk [154]. Moreover,
transgenic mice overexpressing IGF-I in the prostate basal epithelial layer develop
prostate cancer [155], strongly implicating high IGF-I levels in the etiology of prostate
cancer. Significantly, functional loss of PTEN, which induces the development of
prostate cancer in knockout mice, leads to activation of Akt, a critical component of the survival and oncogenic function of IGF-I [94, 148].
Recent studies show that IGF-I can inhibit TGF-β transcriptional activity through
selective suppression of Smad3 activation through a PI3K/Akt-dependent mechanism
[143]. Further work has implicated mTOR in such regulation [144]; however, the
mechanism of how mTOR intercepts TGF-β signaling remains to be defined. With the
use of rat and human prostate epithelial cell lines, we provide the first evidence that IGF-I suppresses BMP4-induced cell death; activation of Smads 1, 5, and/or 8; as well as induced expression of BMP4 target genes through a mechanism dependent on the PI3K,
Akt, mTOR, Raptor, and Rictor signaling pathway. Particularly intriguing is our
observation that this IGF-I signaling pathway clearly represses the ability of BMP4 to
induce expression of inhibitor of differentiation/DNA binding (Id)1, Id2, and Id3,
proteins whose overexpression promotes growth and progression of prostate cancer [156-
158]. Our results support that the ability of mTOR to repress BMP signaling is part of an
important homeostatic switch that is deregulated in prostate cancer.
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METHODS Recombinant human BMP4 and TGF-β1; anti-Id1 antibody (R&D Systems, Inc.),
Stemfactor Recombinant human BMP4 (Stemgent); LY294002 and rapamycin (BioMol),
perifosine (Selleck Chemicals LLC); anti–phospho-Smad3 antibody (P-Smad1/3/5/8);
anti–phospho-Smad1/5/8 antibody (P-Smad1/5/8), anti–phospho-Smad2 (Cell Signaling); anti-Smad2 antibody (Transduction Laboratories); anti-Smad3 and anti-Smad1 (Santa
Cruz Biotechnology, Inc.); IGF-I and LR3-IGF-I (GroPep); DMEM/Ham's F-12 (1:1);
characterized fetal bovine serum (FBS) (HyClone Inc.); insulin (BioSource
International); cholera toxin and dexamethasone (Sigma); pCEP4-PTEN (Dr. Ramon
Parsons); DN-PI3K (pSG5-p85αΔSH2) and CA-PI3K (pSG5-p110αCAAX; gift from Dr.
Downward), and DN-Akt1 (pUSE-Myc-Akt1K179M; Upstate Biotechnology, Inc.) were
used.
Cell culture
The LNCaP, PC3, RWPE-1, VCaP, and DU145 cell lines were obtained from American
Type Culture Collection (ATCC) and maintained in either DMEM/Ham's F-12
containing 5% to 10% FBS or keratinocyte medium (RWPE-1). All above cell lines were
authenticated by ATCC with the use of various tests, including DNA profiling,
cytogenetic analyses, flow cytometry, and immunohistochemistry, and used in our
experiments within 20 passages (60 doublings) of receipt. The NRP-152 and DP-153 cell
lines were developed in our laboratory and maintained in GM2.1 and GM2, respectively,
as previously described [144]. The NRP-152 and DP-153 cell lines were authenticated
by karyotype and isozyme analysis, and used within 20 passages of authentication. All
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above cell lines were confirmed to be free of myoplasma contamination by the
MycoAlert Mycoplasma Detection kit (Cambrex Bio Science).
Cell viability assay
Cell viability was assessed by trypan blue exclusion under phase-contrast microscopy as
before [159]. Cells were plated in 12-well dishes in low serum conditions at a density of
30,000-50,000 cells/1 ml/well as previously described [144, 160]. Cells were pre-treated
with LR3-IGF-I (10 nmol/L) or vehicle control 24 h prior to BMP4 (5 ng/ml) or control
treatment for 24-72 h. After 72 h, cells were detached by a 5 min treatment with Tryspin-
EDTA, titrated with an equal volume of DMEM/F12 + 5% FBS. Cells were spun at 500g
for 5 min, resuspended in DMEM/F12 + 5% FBS, and 10 μl of the resulting cell
suspension was combined with 10 μl Trypan Blue dye solution (0.4% [w/v] in normal
saline) and placed on a hemocytometer. The fraction of viable cells (dye excluded) was
enumerated from a total of 300 cells.
Hoechst 33258 staining
Cells were plated in six-well dishes at a density of 3 × 104 to 5 × 104 cells per well in 2
mL of DMEM/Ham's F-12, 1% FBS, and 15 mmol/L HEPES (pH 7.4; for LNCaP, PC3,
DU145), or in GM3.1 (for NRP-152, DP-153). Cells were treated with vehicle or LR3–
IGF-I (10 nmol/L) 24 hours before BMP4 (5 ng/mL) addition. After 24 to 48 hours, cells were stained with 10 μg/mL Hoechst 33258 (Sigma), and apoptotic cells were counted with the use of fluorescent microscopy. Three hundred cells were analyzed in triplicate
[161].
Flow cytometry
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Detached cells (1.5 × 106) were washed once with PBS, fixed with 90% methanol,
sequentially incubated with 0.1 mg/mL of RNase A followed by 50 μg/mL of propidium
iodide, and then analyzed with an EPICS XL-MCL flow cytometer. Sub-G1 cells, which
have <2n DNA content, are considered to be apoptotic.
Cell number assay
Cells (3 × 104 to 5 × 104 cells/1 mL) were seeded in 12-well dishes in medium described
in Hoechst staining assay. The next day, cells were pretreated with ± LR3–IGF-I (10
nmol/L) for 24 hours before ± BMP4 (5 ng/mL) treatment for up to 72 hours. Adherent
cells were detached by trypsinization and enumerated with a Coulter Electronics counter.
Id1 promoter assay
Cells were plated overnight at a density of either 1.0 × 105 cells/1 mL/well or 2.0 × 105
cells/2 mL/well in 12-well or 6-well dishes, respectively, transfected as before [143, 144,
158] with the human Id1 promoter construct (pGL2-Id-1; 1-2 μg) and 20 ng of cytomegalovirus-renilla reporter constructs. Transfection reagents were washed off 3 hours later, and cells were allowed to recover overnight in the low serum conditions and then pretreated with LY294002 (10 μmol/L), perifosine (5 μmol/L), rapamycin (200 nmol/L), or vehicle 2 hours before ± LR3–IGF-I (10 nmol/L, 24 h) followed by ± BMP4
(5 ng/mL, 24 h). Luciferase activity was measured with the Promega Dual-Luciferase
Assay kit and ML300 Microtiter Plate Luminometer.
Western blot analysis.
Following treatment, cells were washed with phosphate-buffered saline and lysed at 4°C with radioimmune precipitation assay buffer (phosphate-buffered saline, 1% Nonidet P-
40, 0.1% SDS, 0.5% sodium deoxycholate) supplemented with 1 mM sodium 76
orthovanadate, Roche’s complete mini protease inhibitor mixtureTM, 1 mM
phenylmethylsulfonyl fluoride, 1 mM EDTA, 1 mM β-glycerophosphate and 2.5 mM sodium pyrophosphate. Lysates were clarified at 14,500 rpm for 20 min (4°C), and quantified by the BCA protein assay (Pierce). Fifty μg protein lysates were boiled for 5 min in SDS-PAGE loading buffer containing 5% 2-mercaptoethanol, electrophoresed
through 4–12% NuPAGE BisTris gel (Invitrogen) and transferred to nitrocellulose
membranes. Membranes were blocked for 1 h in TBS (10 mM Tris-HCl, pH 8.0, and 150
mM NaCl) containing 5% nonfat dry milk and 0.05% thimerosal, and incubated with the
indicated primary antibodies followed by the appropriate horseradish peroxidase-
conjugated secondary antibody (1:5,000) (Jackson Immunoresearch Laboratory). All
antibodies used are described in the Materials section.
Reverse transcriptase-polymerase chain reaction (RT-PCR).
Total RNA was extracted using PureLink™ RNA Mini Kit (Invitrogen) and reverse- transcribed using M-MLV Reverse Transcriptase (Promega). Taq Polymerase Master
Mix (Promega) was used for the PCR amplification of rat or human Id1, Id2 and Id3 (see
supplemental information for PCR primer sets). PCR amplifications were: 25 to 35
cycles of 95°C for 15s, 55°C for 30 s, and 72°C for 1.5 min, and products were by
separated by electrophoresis through TAE-1% agarose gels and evaluated using a Biorad
Molecular Imager Gel Doc XR+ System.
PCR primers.
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The PCR primers were custom made by Operon Biotechnologies, Inc. They included rat
Id1 sequences (Forward): 5’-CCAGCTGTTCGCTGAAGG-3’ and (Reverse) 5’-
CGCCAAGGCACTGATCTC-3’, rat Id2 (Forward): 5’-
CCTTCAGTCCGGTGAGGTC-3’ and (Reverse) 5’-
TGCTGTCATTCGACATAAGCTC-3’, rat Id3 5’-CAACATGAAGGCGCTGAG-3’ and
(R) 5’-CTCAGAAGGGAAGTGGCAAA-3’, human Id-1 sequences (Forward): 5’-
AAGAATCATGAAAGTCGCCAGTGGC-3’ and (Reverse) 5’-
ACACAAGATGCGATCGTCCGC-3’, human Id2 sequences (Forward): 5’-
AAAGCCTTCAGTCCCGTGAGGT-3’ and (Reverse) 5’-
GCCACACAGTGCTTTGCTGTCATTT-3’, Human Id3 sequences (Forward): 5’-
ACCTCTGGACTCACTCCCCAG-3’ and (Reverse) 5’-
GTGGCAAAAGCTCCTTTTGTCG-3’. Human Id1 gene promoter region (29655143-
29656805) located on chromosome 20 was PCR amplified from genomic DNA of HEK-
293 cells using forward (5’-AAGCCATTCTCCTGTCTCGAGCTCCCGA-3’) and reverse (5’-AAACTCGAGGTGGAAGCCCGAAGCAGAT-3’) primers. This 1663 bp
DNA fragment upstream of the transcriptional initiation site of the Id-1 gene was subsequently cloned into Sac I and Xho I site of pGL3-Basic vector (Promega). The vector construction was confirmed by restriction enzyme digestion and sequencing
(Biotic Solutions).
Adenovirus.
Adenovirus vectors that modulate the expression of DN-PI3K (Admax DN-PI3K ) or
DN-Akt1 (Admax DN-Akt1) were previously described [143]. The ability of viral
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preparations to deliver the desired functional protein was evaluated by overnight
infection of NRP-152 cells followed by western blot analysis of cell lysates and, in
certain cases, by direct immunostaining of the infected cells. To assay viral titers,
aliquots of virus stocks were diluted 100-fold in lysis solution (0.1% sodium
dodecylsulfate, 10 mM Tris–HCl (pH 7.4), 1 mM EDTA) and incubated for 10 min at
50ºC. The optical density of the samples at 260 nm was used to calculate the virus
content using the relationship of 5x1012 virus particles/ml/OD260 unit. Cells were treated
with (1:200) dilution of virus for 24 h prior to other treatments.
Lentivirus based silencing strategy shmTOR, shRaptor, shRictor, and sh-Id1. The production of inducible gene silencing shRNA expression lentiviruses was previously
described [162]. The generation and production of stable gene silencing, multiplicity of
infection of lentiviruses harboring shRNA-lacZ, shmTOR, shRaptor, and shRictor were
used for NRP-152 cells (to derive NRP-152-tTR-shRNA cells). The targeting sequences
for these shRNA constructs were: LacZ 5’-GTGACCAGCGAATACCTGT-3’(Qin et al.,
2003), mTOR 5’- CCCAGCCTTTGTCATGCCT -3’, Raptor (#1) 5’-
GGAGAATGAAGGATCGGAT -3’ and (#2) 5’- AGAATGAAGGATCGGATGA -3’,
Rictor (#1) 5’- ATGTAGAATTAGAGCGAAT -3’and (#2) 5’-
TATTAAATGAGGCGAAAGA -3’ (Designed by Dharmacon RNAi Technology and
oligos purchased from Integrated DNA Technologies, Coralville IA). Similarly, we
established stably silenced cell lines that did not require doxycyclin-inducible gene
silencing strategy. NRP-152 cells were infected with lentivirus that contain shRNA-
LacZ, shmTOR, shRictor, shRaptor, and shRictor/Raptor which were designed as
described above. Last, we silenced inhibitor of DNA binding 1, dominant negative helix- 79
look-helix protein (Id1) using Mission© shRNA Plasmid DNA (NM_002165) (Sigma-
Aldrich, St. Louis, MO). Four shRNA plasmid DNA against different sequences of Id1 were purchased: shId1 #1 (TRCN: 0000019029), sh-Id1 #2( TRCN: 0000019031), shId1
#3 (TRCN: 0000274033), shId1 #4 (TRCN: 0000274034). The parental vector (pLKO.1) allows for transient transfection for stable selection via puromycin resistance. Each target sequence was compared to either parental vector (pLKO.1) or (pLKO.1-siGFP) to ensure proper knock down of Id1.
Real Time quantitative reverse transcriptase-PCR (RT-qPCR). cDNA was created using the M-MLV Reverse Transcriptase (Promega) on 1 ug of total
RNA. We used predesigned gene specific primer and probe set using Taqman Gene
Expression Assay (Applied Biosystems, Inc., Foster City, CA) for Id1. The assay was performed according to the manufacturer’s protocol on a 7500 Fast Real Time PCR
System (Applied Biosystems,Inc., Foster City, CA).
Immunohistochemistry and Tissue Microarray (TMA).
We obtained PR8011 tissue microarray (TMA) which included 80 cases/80 cores of a variety of prostate disease spectrum, including TNM and pathology grade. Slides were either unstained or stained with H&E. In total, the array provided 32 cases of adenocarcinoma, 2 metastatic tissues, 26 hyperplasia, 6 chronic inflammation, 6 adjacent normal tissue, 8 normal. In addition to PR8011 we also purchased BC19111 which contained an additional 6 cases of normal prostate tissue in triplicates, 3 cases of each prostate tumor and hyperplasia in duplicates. TMA were processed by
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immunohistochemsitry (IHC), where the sections were deparaffinized in xylene followed
by graded ethanol hydration into water. The sections underwent antigen retrieval by
immersing the tissue sections in 0.1M citrate buffer (pH 6.0) in a steamer for about 10-
15min. Endogenous peroxidase activity was quenched by incubating the slide with hydrogen peroxide for 10min followed by triplicate for 3 min washes in 0.01 M TBST
(pH 7.4). Nonspecific labeling was blocked using 10% goat serum (Vector Laboratories)
for 30 min. After washing sections in triplicate with 0.01M TBST for 3 min each, tissues
were then incubated with anti-phospho-Smad-1,5,8 antibody (Cell signaling Inc.,
Danvers, MA, Cat.#9511) at (1:100) dilution or Phospho-S6 Ribosomal Protein (Ser
235/236) antibody (Cell signaling Inc., Danvers, MA, Cat #2211) at (1:100) dilution or
normal rabbit IgG used as a negative control were incubated overnight at 4ºC. Sections
were then treated with biotinylated goat anti-rabbit IgG for 1 h at RT followed by
treatment with Vectastain Elite ABC reagent for 30 min, and according to manufactures
guidelines with triplicate wash with 0.01M TBST for 3 min between and after each step
(Vectastain Elite ABC kit, Vector laboratories, Burlingame, CA). Chromogen intensity
staining was performed using DAKO Liquid DAB+ Chromogen solution (Cat#K346711-
2) for 10 min, followed by triplicate rinse in 0.01M TBST (pH 7.4). Slides were then
counterstained with modified Harris hematoxylin (Richard Allan Scientific, Kalamazoo,
MI) for 35-45 sec followed by 10 dips in 0.2% Aluminum hydroxide (Fisher Scientific,
Pitsburg, PA). Last, slides were dehydrated in graded ethanol and cleared in fresh
xylene, mounted using Permount (Fisher Scientific, Pittsburg, PA). All slides were
reviewed independently by two investigators (R.W and K.S) who were blinded to the
clinical and pathological data and reconfirmed by a second set of evaluations by one
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investigator (D.S) blinded to the interpretations of the first set of evaluations, as well as clinical and pathological data. Immunostaining results were validated by the consensus of all reviewers.
Gene Expression Microarray Analysis.
Total RNA (10 µg) was isolated and purified from NRP-152 cells treated with either
vehicle, LR3-IGF-I (10nmol/L) 24 h prior to +/- BMP4 (5 ng/ml) for a total of 48 h using
Purelink™ RNA mini kit (Invitrogen, Carlsbad, CA) according to manufacturer’s
instructions. Samples were submitted to Case Comprehensive Cancer Center Gene
Expression and Genotyping Core Facility (www.gegf.net ) to be analyzed by Affymetrix
Rat Gene 1.0 ST Array microarrays containing 33,297 probe set IDs for known genes.
RNA quality was assessed and high quality RNA ( i.e., RNA integrity number was > 7.0) was used in this experiment in accordance with affymetrix GeneChip© Whole Transcript
(WT) sense target labeling assay protocol. rRNA reduction, first round doublestrand- cDNA synthesis, ss-cDNA fragment, and labeling were performed in accordance with
Affymetrix GeneChip© WT sense target labeling assay manual. Affymetrix Rat Exon
1.0 ST microarray were hybridized overnight in accordance to manual guidelines.
Affymetrix Data Analysis.
Signal intensity estimate and P Value for each of the four processed samples were then
processed to determine fold change ratios between Vehicle: BMP, Vehicle: LR3-IGF-I, and Vehicle: BMP4+LR3-IGF-I. Fold changes over control that were -1.5
deemed not altered in the comparisons by the Affymetrix algorithm and were eliminated
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from the gene list. All samples greater than or equal to 1.5 or less than or equal to -1.5
were accepted in our analysis set.
RESULTS Responsiveness of prostatic epithelial cell lines to the TGF-β superfamily ligands.
Previous work from our laboratory showed that epithelial cell lines (NRP-152, NRP-154)
derived from the preneoplastic prostate of the Lobund/Wistar rat are exquisitely sensitive
to the induction of apoptosis by TGF-β [163]. We examined the general responsiveness
of NRP-152 cells versus a metastasis-derived PTEN-null human prostate cell line, PC3,
to various members of the TGF-β superfamily [TGF-β1, Activin (A, B, or AB), BMP4,
Müllerian inhibiting substance, Nodal, or Cripto] by their ability to phosphorylate various
Smads, as assessed by western blot with the use of various phospho-Smad antibodies
(Fig. 2.1A, Fig. 2.2). Due to lack of complete isoform specificity of the antibodies available for phospho-Smads 1, 3, 5, and 8, we used an anti-phospho-Smad1/5/3/8
(antibody 1), which recognizes two specific bands [phospho-Smads 1, 5, and 8 (top), and phospho-Smad3 (bottom)], and an anti–phospho-Smad1/5/8 specific antibody (antibody
2). In both cell lines TGF-β1 and Activin B specifically activated Smads 2 and 3, but not
Smads 1, 5, or 8; and BMP4 specifically activated Smads 1, 5, and/or 8 (for simplicity, designated Smad1/5/8), but not Smads 2 or 3. We were unable to detect activation of
Smads by Müllerian inhibiting substance, Nodal, or Cripto in either cell line under these conditions. NRP-152 and PC3 cells are thus the most sensitive to TGF-β1 and BMP4 (at the indicated concentrations) among the TGF-β superfamily ligands examined.
We next assessed the ability of BMP4 to affect growth of a panel of nontumorigenic (NRP-152, DP-153) and tumorigenic (LNCaP, PC3, DU145) prostate
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epithelial cell lines (see Materials and Methods; Fig. 2.1B). All the above cell lines were
to various degrees growth suppressed by BMP4, with greater cytostatic activity occurring in the nontumorigenic (NRP-152, DP-153) and androgen-responsive tumorigenic
(LNCaP) cell lines than in the androgen refractory tumor lines (PC3 and DU145). Thus,
BMP4 seems to be more cytostatic on premalignant or early-stage prostate cancer cells than on late-stage ones.
IGF-I reverses growth suppression of prostate epithelial cells by BMP4.
Based on various published reports and our results in Fig. 2-1B, we speculated that the cytostatic activity of BMP4 was lost during prostate carcinogenesis by the activation of
IGF-I signaling, similar to our previous report on the repression of TGF-β responses by
IGF-I [143]. In a time course experiment during which 72 hours of BMP4 (10 ng/mL) treatment caused a 65% loss in NRP-152 cell number (Fig. 2.3A), that such cell death was effectively repressed by pretreatment with 2 to 10 nmol/L LR3-IGF-I (Fig. 2.3B), an
analogue that shares similar affinity to the IGF-I receptor but is essentially unable to bind
to IGF-I binding proteins. We next characterized the ability of LR3-IGF-I to suppress the
cytostatic activity of BMP4 on NRP-152 cells by measuring changes in apoptosis by
BMP4 in the presence or absence of LR3-IGF-I in three different assays. In the first
method, NRP-152 cells were pretreated with ± LR3-IGF-I (10 nmol/L) for 24 hours
followed by ± BMP4 (5 ng/mL) for 24 to 72 hours, and apoptosis was identified by
nuclear condensation and fragmentation under fluorescent (white arrows) microscopy
following Hoechst 33258 staining (Fig. 2.3C, Fig. 2.4). BMP4 caused markedly increased
numbers of apoptotic nuclei (40% of cells) over control, whereas cells pretreated with
LR3–IGF-I significantly blocked BMP4-induced apoptosis (8% of cells). These results
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were consistent with changes in cell viability (trypan blue exclusion) and apoptotic
fraction (sub-G1 by flow cytometric analysis) at 72 hours (Fig. 2.3C). The sub-G1
fraction of the cells showed that BMP4 induced apoptosis in 11% of the cells compared
with vehicle control (4%). LR3-IGF-I treatment brought the percent sub-G1 fraction in
each group to equal to or less than that of vehicle only (3%). There was no significant
change in the fraction of cells in G1, but there was an increase in the fraction in G2-M
(Fig. 2.5). Together, these studies confirm LR3-IGF-I effectively blocks the ability of
BMP4 to induce apoptosis of NRP-152 cells. We also examined the effect of LR3–IGF-I
on the cytostatic effect of BMP4 in other prostate cell lines, including LNCaP, PC3,
RWPE-1, VCaP, and DP-153 cells. LR3-IGF-I reversed the ability of BMP4 to suppress growth or induce cell death, as shown morphologically and by enumerating cells with the
use of a Coulter counter (Fig. 2.3D, Fig. 2.6). These results support the universality of
IGF-I receptor signaling on reversing the cytostatic activity of BMP4 on prostate
epithelial cells.
Effect of IGF-I on activation of Smads by BMP4
To explore the mechanism by which IGF-I intercepts BMP signaling, we assessed
the ability of LR3-IGF-I to affect BMP4-induced activation of Smad1/5/8 in NRP-152
cells (Fig. 2-7A). We pretreated these cells with ± LR3–IGF-I (2 or 10 nmol/L) or insulin
(1 μmol/L) for 24 hours, stimulated them with BMP4 (10 ng/mL) for 4 hours, and then analyzed levels of phospho-Smad1/5/8 by western blot as in Fig. 2.8. NRP-152 cells
treated with BMP4 showed robust activation of Smad1/5/8, which was suppressed by 10 nmol/L LR3–IGF-I or 1 μmol/L insulin. Similar results were observed in RWPE-1 and 85
DU-145 human prostate epithelial cell lines (Fig. 2.7C). To define how rapidly IGF-I
suppresses BMP4-induced activation of Smad1/5/8, we pretreated NRP-152 cells with
LR3–IGF-I for various times before 4 hours of treatment with BMP4 (Fig. 2.7A).
Phosphorylation of Smad1/5/8 by BMP4 was suppressed as early as 1 hour pretreatment
with LR3–IGF-I, with no change in levels of total Smad1/5/8.
IGF-I represses transcriptional activation of Id1 by BMP4
Given that Id proteins are transcriptionally induced by BMPs through Smad1/5/8, and
IGF-I blocks this activation, we hypothesized that IGF-I suppresses the expression of the
helix-loop-helix Id proteins, a well-known Smad-dependent transcriptional target of BMP
[81]. BMP4 rapidly (<4 h) induced Id1 protein levels in NRP-152 cells, and such
induction was significantly repressed by 1 hour of IGF-I pretreatment (Fig. 2.7A),
reflecting the general pattern of Smad phosphorylation. Additionally, we showed that 1-
hour pretreatment with LR3–IGF-I also reversed BMP4-induced (5 ng/mL, 4 h) Id1
promoter activity in both NRP-152 and LNCaP cells transiently transfected with a pGL2-
Id1 promoter construct containing a number of BREs [ref. [81]; Fig. 2.7A (bottom), Fig.
2.9). Semiquantitative RT-PCR was used to assess the ability of LR3–IGF-I to suppress
BMP4-induced levels of Id1, Id2, and Id3 mRNAs in NRP-152 cells (Fig. 2-7B). BMP4-
induced expression of all three Id mRNAs within 4, and 4-to-24 hours of pretreatment with LR3–IGF-I suppressed such induction. A similar response was observed in the
LNCaP cell line for Id1 mRNA (Fig. 2.7B). However, for reasons not clear, the
suppression of Id1 mRNA levels (Fig. 2.7B) was delayed relative to suppression of Id1
protein levels (Fig. 2.7A). Real-time quantitative PCR confirmed our semiquantitative
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RT-PCR data that IGF-I effectively blocked BMP-induced Id1 mRNA expression (Fig.
2.7D). Overall, these data suggest that IGF-I blocks BMP4-mediated expression of Id1,
Id2, and Id3 in prostate epithelial cells through a transcriptional mechanism involving suppression of the phosphorylation of Smad1/5/8.
Role of the PI3K/Akt/mTOR pathway in mediating IGF-I suppression of BMP responses
The PI3K/Akt pathway, which is generally hyperactivated in prostate cancer, is believed to play a prominent role in the survival function of IGF-I. We thus hypothesized that
IGF-I inhibits BMP responses through a PI3K-dependent mechanism. To test this hypothesis, we cotransfected NRP-152 cells with Id1-luciferase construct along with constitutive active PI3K (CA-PI3K), dominant-negative PI3K (DN-PI3K), or empty vector control (pSG5), and then added ±10 nmol/L LR3–IGF-I for 2 hours followed by
BMP4 (5 ng/mL) for 24 hours before luciferase assay (Fig. 2.10A, left). As anticipated,
CA-PI3K suppressed BMP-induced Id1-luciferase reporter activity, whereas DN-PI3K
reversed LR3–IGF-I inhibition of this BMP response. A highly selective inhibitor of
PI3K, LY294002, reversed the suppressive action of LR3–IGF-I on BMP4-induced Id1
promoter activity (Fig. 2.10A, right). Similar results were obtained with the Akt inhibitor
perifosine (Fig. 2.10B) or the mTOR inhibitor rapamycin (Fig. 2.10C). These results
strongly suggest that the IGF-I suppression occurs downstream of Akt and mTOR.
Overall, the above results suggest that the PI3K/Akt/ mTOR mediate the ability of IGF-I
to suppress the activation of Smad1/5/8 by BMP4 and, hence, activation of the Id1
promoter. To confirm our model, we examined the effect of LY294002, rapamycin, or
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perifosine on the ability of LR3–IGF-I to suppress BMP-induced Smad activation under
conditions as in Fig. 2.10B and C, except that cells were treated with BMP4 for 4 hours
and harvested for western blot analysis (Fig. 2.10D and data not shown). Clearly,
LY294002, perifosine, and rapamycin each reversed the ability of LR3–IGF-I to suppress the activation of Smads by BMP4. We also used adenoviral-mediated gene delivery to
efficiently overexpressed DN-PI3K or DN-Akt in NRP-152 cells. As expected,
overexpression of either DN-PI3K or DN-Akt enhanced BMP-induced phospho-
Smad1/5/8 levels (Fig. 2.11), suggesting basal levels of PI3K and Akt suppress BMP
signaling.
Silencing expression of mTOR, raptor, or rictor reverses the ability of IGF-I to
inhibit BMP signaling
We further investigated the roles of each of the two mTOR complexes (mTORC1 and mTORC2) in BMP4 signaling by efficiently and stably silencing mTOR as well as a critical component of mTORC1 (Raptor) and mTORC2 (Rictor) complexes. For this we used specific small hairpin RNA interference delivered by a doxycline-inducible lentiviral transduction system, as previously described [162], which knock down mTOR,
Raptor, and Rictor in NRP-152 cells by >95% (Fig. 2.12A). The stably silenced cell lines were treated with LR3–IGF-I before BMP4 addition and analyzed as before for levels of total and phospho-Smad1/5/8. Silencing mTOR, Raptor, or Rictor reversed the ability of
IGF-I to inhibit BMP4-induced phosphorylation of Smad1/5/8 (Fig. 2.12B and C; Fig.
2.13) and the suppressive action of IGF-I on BMP-induced Id1 promoter activity (Fig.
2.12D). Consistent with these results, overexpression of mTOR, Raptor, and Rictor in 88
NRP-152 cells suppressed BMP-induced Id1 promoter activity (data not shown). Taken together, our results suggest that both mTORC1 and mTORC2 play a role critical in mediating the suppression of BMP responses by IGF-I in prostate epithelial cells.
IGF-I represses numerous BMP-regulated genes
We examined the global effect of IGF-I on gene expression by BMP4 in NRP-152 cells with the use of microarray analysis with Affymetrix Rat Gene 1.0 ST Array microarrays containing 33,297 probe set IDs for known genes. The fold-change of each treatment set was compared with vehicle control. The total number of probe sets altered for each treatment is as follows (in brackets are number of changes ≥1.5-fold): BMP4 (521), IGF-I
(503), and BMP4+IGF-I (1,583). This analysis revealed that expression of 89 of the 235
BMP4-regulated (38%) genes was specifically altered by IGF-I in a manner that could not be accounted for by the effects of IGF-I alone (Table 2-1). Twenty of these genes were grouped to specific biological responses with the use of Pathway Studio 5.0
software to determine pathway and molecular interaction analysis for each of the
identified treatment groups (Fig. 2-14A). These data suggest that IGF-I represses the
ability of BMP to modulate the expression of a number of genes involved in tumor
growth as well as tumor suppression. Hyperactivation of mTOR in advance human prostate cancer correlates with loss of phosphoSmad1/5/8 expression To test our hypothesis that hyperactivation of mTOR represses the ability of BMP to phosphorylate
Smad1/5/8, we conducted an immunohistochemical analysis of phospho-Smad1/5/8 and a key downstream target or mTOR (phospho-S6) with the use of matched cores from a human prostate tissue microarray (PR8011 series) obtained from US BioMax, Inc. (Fig.
89
2.14B). The H-score [percent positive stained cells × intensity of staining (0-3)] of 34 cores representative of advanced localized prostate adenocarcinoma (27 stages II-IV) and
7 normal hyperplasia yielded a statistically significant inverse correlation between the levels of phospho-Smad1/5/8 and that of phospho-S6 (R2 = 0.4271; P < 0.0001; Fig. 2-
14C). This represents a significant in vivo test of our model that activation of mTOR reverses the activation of Smad1/5/8 by BMP.
DISCUSSION Here we report the first evidence that IGF-I signaling through a PI3K/Akt/mTOR pathway intercepts BMP responses by suppressing the c-terminal phosphorylation of
Smad1/5/8. Silencing either Raptor or Rictor alone reversed this IGF-I repression, indicating critical and nonredundant roles for mTORC1 and mTORC2 [102, 129] in such regulation, the mechanism of which awaits further investigation. BMPs are recognized to have both tumor-suppressor and tumor-promoting functions in the prostate, although the mechanisms mediating such opposing functions remain poorly defined [89, 164].
Whereas various BMPs have been detected in both normal and tumor prostate tissues,
BMP4 seems to be a predominant form expressed in the normal prostate relative to tumor tissue (ref. [165]; Fig. 2.15), in which it is shown to function as a repressor of prostate ductal budding and branching morphogenesis [166]. Evidence also support that response to BMPs is altered during prostate tumor development/progression [51]. Consistent with this, BMP4 induces the apoptosis of nontumorigenic prostate epithelial cell lines (NRP-
152 and DP-153), more so than tumorigenic ones (LNCaP, PC3; Fig. 2.3; Fig. 2.6), correlating with the PTEN-negative status of the latter cell lines. Immunohistochemical
90
analysis reveals that phosho-Smad1/5/8 is high in hyperplastic prostate tissues but lost in advanced localized prostate cancer, correlating with activation of mTOR or phospho-S6
(Fig. 2-14C), consistent with our in vitro data. Functional loss of PTEN, which promotes
hyperactivation of the PI3K/Akt/mTOR pathway, is well accepted to be involved in the
development and progression of the majority of prostate cancers [167] but through an
incompletely understood mechanism. Our data suggest that PI3K/Akt/mTOR plays an
important role in loss of the tumor-suppressive function of BMP4 (apoptosis/growth
arrest) in prostate cancer. Microarray expression profiling showed that IGF-I represses
BMP4 to regulate expression of 38% of the BMP4 target genes; at least two of these
BMP4-inducible ones (IGF-BP5 and Gadd45α; Fig. 2-14A) have been shown to be
associated with the control of apoptosis and growth arrest [168, 169]. Thus, the
oncogenic function of PI3K/Akt/mTOR may partly occur through intercepting the
cytostatic functions BMP4 (through suppressing activation of Smad1/5/8; ref. [105].
However, IGF-I/PI3K/Akt/mTOR pathway also represses the induction of Id1, Id3, and
other tumor promoting proteins (Figs. 2.12, Fig. 2.14, and Fig. 2.16), suggesting this
pathway also maintains homeostasis by repressing the oncogenic functions of BMP4.
Thus, Ids and other oncogenic mediators of BMP are potential new co-targets of mTOR
therapeutics.
Prostate cancer cells typically progress from a state of androgen dependence
toward that of hormone independence (castrate resistance) through mechanisms under
rigorous investigation [24, 170, 171]. Whereas advanced prostate cancer cells are
resistant to androgens, recent studies suggest they are dependent on the androgen
receptor, which is considered to become constitutively activated during tumor
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progression [167, 171]. A number of models have been proposed for the mechanisms by
which androgen receptor signaling is activated in the absence of exogenous androgens
[172]. Recently, BMP receptor signaling has been reported to suppress androgen
receptor activity through a Smad1- dependent and mitogen-activated protein kinase–
dependent mechanism involving the phosphorylation of the middle linker of Smad1 [89].
The modified Smad1 then associates with the androgen receptor and suppresses gene transcription by the androgen receptor. Through this mechanism, basal levels of autocrine
BMP activity [55] may help maintain the androgen-dependent phenotype of prostate tumors. Akt/mTOR signaling can significantly enhance androgen receptor activity, thus promoting “androgen independence” through mechanisms that are not clear [173]. Our findings suggest that this may occur through reversing the suppressive activity of the
BMP/Smad1/5/8 pathway on the androgen receptor. On the other hand, enhanced
androgen receptor activity has been shown to activate mTOR [174], and results from our
current study suggest that the suppressive activity of mTOR on BMP may serve to further
enhance the activity of the androgen receptor. This positive feedback/signal amplification
loop is likely to contribute to castration-resistant prostate cancer. In the normal or
preneoplastic prostate tissue, this positive feedback loop is likely to be kept in check
through the induction of BMP7 and BMP receptor II by androgens [55, 175]. Taken
together, our study here provides further insight on the potential mechanism by which
prostate cancer cells progress toward androgen independence, with the ultimate goal of
aiding the therapeutic management of hormone refractory prostate cancer.
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FIGURES Figure 2.1. Biological activity of TGF-β superfamily ligands on prostate epithelial cell lines.
A, NRP-152 cells were treated with ± TGF-β1 (0-10 ng/mL), Activin (A, B, AB; 10 ng/mL), BMP4 (0-10 ng/mL), or Müllerian inhibiting substance (10 ng/mL), Nodal (10 ng/mL), and Cripto (10 ng/mL) for 24 hours and analyzed for Smad activation by western blot with the use of antibodies against the two c-terminal serines of phospho-Smads 1, 3,
5, 8 (Ab #1), phospho-Smad1/5/8 (Ab #2), and phospho-Smad2. B, NRP-152, DP-153,
LNCaP, PC3, and DU145 cells treated with BMP4 (0-20 ng/mL) for 72 hours, and total adherent cells were enumerated with a Coulter Electronics counter. Values, averages of triplicate determinations ± SE.
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(Figure 2.1)
94
Figure 2.2. TGF-β superfamily-induced activation of Smads in PC3 cells.
PC3 cells were treated for 24 h ± TGF-β1 (0-10 ng/ml), Activin (A, B, AB) (10 ng/ml),
BMP4 (0-10 ng/ml), or MIS (10 ng/ml), Nodal (10 ng/ml), Cripto (10 ng/ml) and assayed for total and C-terminal phosphorylated Smads by western blot analysis.
95
(Figure 2.2)
PC3
(ng/ml) 00.31 3 100101010 00.31 3 100101010 TGF-β1 - ++++-- - - - ++++-- - -BMP4 Activin ------ABAB ------MN CMIS/Nodal/Cripto P-Smad 1/5/8 50kDa P-Smad3 P-Smad2 50kDa P-Smad 1/5/8 50kDa Smad2 50kDa Smad3 50kDa β-actin
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Figure 2.3. LR3–IGF-I blocks BMP4-induced cell death in nontumorigenic (NRP- 152 and DP-153) and tumorigenic (LNCaP and VCaP) prostate epithelial cancer cell lines.
A, NRP-152 cells were treated with ± BMP4 (10 ng/mL) for 24 to 72 hours, and total adherent cells were measured with a Coulter counter. B, NRP-152 cells were treated with
±2 nmol/L or 10 nmol/L LR3–IGF-I for 24 hours followed by ± BMP4 (10 ng/mL) for 72
hours, after which total adherent cells were counted with a Coulter Electronics counter.
C, NRP-152 cells were treated with ±10 nmol/L LR3–IGF-I for 24 hours followed by ±5 ng/mL BMP4 for an additional 72 hours and stained with Hoechst dye (left), trypan Blue
(middle); right, flow cytometry. D, NRP-152, DP-153, LNCaP, and VCaP cells were treated as C and the cells measured with a Coulter Electronics counter or examined by phase-contrast microscopy (×200) for changes in morphology. Columns (A-D), average of triplicate determinants or three independent experiments ± SE. *, P < 0.001.
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(Figure 2.3)
98
Figure 2.4. LR3-IGF-I blocks BMP4-induced cell death in NRP-152 cells.
NRP-152 cells were treated with ± 2 nmol/L or 10 nmol/L LR3-IGF-I for 24 h followed
by ± BMP4 (5 ng/ml) for 72 h, stained with Hoechst dye, and examined under
fluorescence and phase contrast microscopy at 200X. Data is representative of two
independent experiments.
99
(Figure 2.4)
Phase Hoechst Merge Control BMP4 -IGF-I 3 LR -I IGF - 3 BMP4+ LR
100
Figure 2.5. LR3-IGF-I increases G2/M progression but has no effect on G1 or S phase cell cycle progression in NRP-152 cells.
NRP-152 cells were treated with ±10 nmol/L LR3-IGF-I for 24 h followed by ± 5 ng/ml
BMP4 for an additional 72 h. Cells were then detached from plastic dishes, stained with
PI and subject to cell cycle analysis by flow cytometry.
101
(Figure 2.5)
100
75 Vehicle BMP4 50
% Cells in G1 in Cells % 25
0 Control LR3-IGF-I 12.5 15.0 12.5 10.0 10.0 7.5 7.5 5.0 5.0 % Cells in G2/M in Cells %
% Cells in S Phase in Cells % 2.5 2.5
0.0 0.0 3 Control 3 Control LR -IGF-I LR -IGF-I
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Figure 2.6. LR3-IGF-I inhibits BMP-induced cell death in NRP-152 and total adherent cells in a number of rat prostate epithelial cell lines (NRP-152, DP-153, RWPE-1) and human prostate epithelial cancer cell line (PC3).
NRP-152, DP-153, RWPE-1 and PC3 cells were pretreated with ±LR3-IGF-I (10 nmol/L)
for 24 h followed by ±BMP4 (5 ng/ml) for 72 h and examined by phase-contrast
microscopy (200X) for changes in morphology (A) and cell number using a Coulter
Electronic counter (B). Columns, average of triplicate determinants; error bar, ±S.E.
Data are representative of three independent experiments.
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(Figure 2.6)
LR3-IGF-I A. Control BMP4 LR3-IGF-I + BMP4 DP-153 NRP-152
PC3 200x
RWPE-1 PC3 B. 150 ) ) 40
4 Vehicle
4 Vehicle BMP4 P<0.001 BMP4 125 P<0.01 30 100
75 20
50 10 25 AdherentCells 10 (x Adherent Cells (x 10 0 0 Control LR3-IGF-I Control LR3-IGF-I
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.FIGURE 2.7. LR3–IGF-I ABROGATES BMP4-INDUCED ACTIVATION OF SMAD1/5/8, AND ID1, ID2, AND ID3 EXPRESSION.
A, NRP-152 cells were treated with ± LR3–IGF-I (10 nmol/L) for 24 hours followed by ±
BMP4 (10 ng/mL) for 4 hours, and cell lysates were analyzed by western blot (top), or
NRP-152 cells were cotransfected with 25 ng of cytomegalovirus (CMV)-renilla reporter
construct and 1 μg of Id1-luciferase reporter element 24 hours before ± LR3–IGF-I (10
nmol/L, 24 h), and then treated with ± BMP4 (10 ng/mL, 4 h). Dual-luciferase activity
was then assayed, and relative values of firefly luciferase were normalized to renilla
luciferase (bottom). Columns, average of triplicate determinations; bar, ± SE. B,
expression of Id1, Id2, and Id3 mRNAs in NRP-152 (B) or LNCaP (C) cells treated with
± LR3–IGF-I (10 nmol/L) for 24 hours followed by ± BMP4 (10 ng/mL) for 4 hours. C,
RWPE-1 and DU-145 were treated as specified in 3A, and cell lysates were analyzed by western blot for phospho-Smad1/5/8 activation and Id1 expression. D, real-time quantitative PCR examined expression of Id1 mRNA in NRP-152 cells ± LR3–IGF-I (10 nmol/L) for 24 hours followed by ± BMP4 (10 ng/mL) for a total of 48 hours; bottom, semiquantitative PCR. Data are representative of three independent experiments.
105
(Figure 2.7)
106
Figure 2.8. LR3-IGF-I blocks BMP4-mediated Smad1/Smad5/Smad8 phosphorylation in NRP-152 cells.
NRP-152 cells were treated with ± LR3-IGF-I (2 or 10 nmol/L) or insulin (1 μmol/L) followed by BMP4 (10 ng/ml) treatment, and whole-cell lysates (50 μg protein) were subjected to western blot analysis. Data are representative of two independent experiments. Columns, average of triplicate determinants; bar, S.E.
107
(Figure 2.8)
- + -++- + - BMP4 (10 ng/ml) - - 22-- 1010 LR3-IGF-I (nM) - -- -++- - Insulin (1 µM)
50kDa P-Smad 1/5/8 (Ab#1)
50kDa Smad 1,5,8
β-actin
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Figure 2.9. LR3-IGF-I inhibits BMP4-mediated Id1 promoter activation in LNCaP cell line
A, LNCaP cells were co-transfected with 25 ng of CMV-Renilla reporter construct and 2
μg of Id1-luciferase reporter element 24 h prior to ± LR3-IGF-I (10 nmol/L). After 24 h cells were treated ± BMP4 (5 ng/ml) for 24 h and assayed for luciferase activity using a
Dual luciferase. B, LNCaP cells were transfected with Id1-luciferase reporter element as described in A, and 24 h later cells were incubated ± rapamycin (200 nmol/L) for 2 h followed by the addition of ± LR3-IGF-I (10 nmol/L). Twenty-four h later cell received ±
BMP4 (5 ng/ml) and dual luciferase activity was measured after 24 h. Firefly luciferase activity was as expressed relative values normalized to Renilla control.
109
(Figure 2.9)
A. LNCaP 4 Vehicle BMP4 P<0.001
3
2
1 (Id-1 promoter/cmv-renilla) (Id-1 Relative Luciferase Activity Luciferase Relative
0 Control LR3-IGF-I
LNCaP B. Vehicle Rapamycin P<0.001 20
P<0.01 10 (Id-1 promoter/cmv-renilla) Relative Luciferase Activity Luciferase Relative 0 BMP4 (5 ng/ml) -++ - -++ - LR3-IGF-I (10 nM) -+- + -+- + Rapamycin (200 nM) --- - +++ +
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Figure 2.10. LR3–IGF-I inhibits BMP4-mediated responses through a PI3K/Akt/mTOR-dependent mechanism.
A (left), NRP-152 cells were transfected with 0.8 μg of expression constructs for control
(pSG5), DN-PI3K, or CA-PI3K, and co-transfected with Id1-luciferase as described above for 24 hours followed by treatment with ± LR3–IGF-I (10 nmol/L) or vehicle for
24 hours before ± BMP4 (5 ng/mL); luciferase activity was measured after 24 hours. A
(right), NRP-152 cells were cotransfected with 20 ng of CMV-renilla reporter and 1 μg of
Id1-luciferase constructs; 24 hours later cells were incubated with ±LY294002 (10
μmol/L) for 2 hours, followed by ± LR3–IGF-I (10 nmol/L) or vehicle for 24 hours. Cells
were then treated with ± BMP4 (5 ng/mL) and luciferase activity measured after 24
hours. B and C, NRP-152 cells were transfected with Id1-luciferase reporter element as
described in (B) and then incubated with either ± perifosine (10 nmol/L) or ± rapamycin
(200 nmol/L) for 2 hours, followed by ± LR3–IGF-I (10 nmol/L) for 24 hours. Cells were then treated with ± BMP4 and assayed for luciferase 2 hours later. D, NRP-152 cells were pretreated with 10 μmol/L LY294002 or 200 nmol/L rapamycin for 2 hours followed by ± LR3–IGF-I (10 nmol/L) or vehicle for 24 hours, and then treated with ±
BMP4 (5 ng/mL) for 4 hours. Western blot analysis was conducted for P-Smad1/ 5/8 (Ab
#1 or Ab #2) or total Smad1/5/8 expression. Data are representative of two or three
independent experiments. Columns, average of triplicate determinants; bar, ± SE.
111
(Figure 2.10)
112
Figure 2.11. LR3-IGF-I inhibits activation of BMP-mediated responses through a PI3K or Akt-dependent mechanism.
NRP-152 cells were infected with Admax control (Ad. Control), Admax-DN-PI3K (Ad.
DN-PI3K), or Admax-DN-Akt1 (Ad. DN-Akt1) and incubated overnight ± 10 nmol/L
LR3-IGF-I, followed by ± 5 ng/ml of BMP4 for 4 h. Data presented is representative of two independent experiments.
113
(Figure 2.11)
-+ -+BMP4 (5 ng/ml) -+ -+ BMP4 (5 ng/ml)
P-Smad 1/5/8 P-Smad 1/5/8 50kDa (Ab#1) 50kDa (Ab#1) P-Smad 1/5/8 P-Smad 1/5/8 50kDa (Ab#2) 50kDa (Ab#2) β-actin β-actin 35kDa 35kDa
114
Figure 2.12. Raptor, Rictor, and mTOR mediate the IGF-I suppression of BMP- induced Id1 promoter expression in NRP-152 prostate epithelial cells.
A, Raptor, Rictor, and mTOR were effectively silenced individually as indicated at the
protein level in NRP-152 cells. B and C, NRP-152-tTR-sh-LacZ, NRP-152-tTR-sh-
Raptor or stably silenced NRP-152-sh-Raptor, and NRP-152-sh-mTOR cells were treated
with LR3–IGF-I (10 nmol/L) 24 hours before BMP4 (5 ng/mL) for an additional 4 hours;
cells were then lysed for western blot analysis of phospho-Smads and/or total Smads. D,
NRP-152-tTR-sh-LacZ (Sh-LacZ), NRP-152-tTR-sh-mTOR (Sh-mTOR), NRP-152-tTR-
sh-Raptor (Sh-Raptor), and NRP-152-tTR-sh-Rictor (Sh-Rictor) stably silenced cells
were transfected with Id1 promoter construct 24 hours before treatment with LR3–IGF-I
(10 nmol/L). After 2 hours cells were treated with BMP4 (5 ng/mL) or vehicle, and luciferase activity was measured 24 hours later. Columns, average of triplicate determinants; bar, ± SE.
115
(Figure 2.12)
116
Figure 2.13. mTOR mediated IGF-I suppression of BMP-induced Smad 1,5,8 activation in NRP-152 prostate epithelial cells.
NRP-152-shmTOR was stably silenced cells were treated with LR3-IGF-I (10 nmol/L) 24
h prior to BMP4 (5 ng/mL) for an additional 4 h, and cells were then lysed for western
blot analysis of phospho-Smads 1,5,8, Total Smads 1,5, 8, Phospho-S6, Total S6, and β- actin. Experiment is representative of three individual experiments.
117
(Figure 2.13)
NRP-152 NRP-152 shLacZ shmTOR BMP4 (5 ng/ml) -+ -+ -+ -+ LR3-IGF-I (10 nM) -- ++ -- ++ PS158 TS158 PS6
TS6 β-actin
118
Figure 2.14. IGF-I–mediated inhibition of BMP-induced gene microarray analysis and in vivo examination of mTOR-mediated inhibition of Smad1/5/8 in advanced human prostate adenocarcinoma.
A, microarray analysis of NRP-152 cells treated with vehicle control, LR3–IGF-I (10
nmol/L, 24 h) ± BMP4 (5 ng/mL) for a total of 48 hours and analyzed to determine fold-
change relative to control; biological process was identified with Pathway Studio 5.0. B,
Immunohistochemistry of normal prostate hyperplasia (top) or advanced prostate
adenocarcinoma stage III stained with H&E, phospho-Smad1/5/8, or phospho-S6. C,
matched human prostate cancer cores (34 total cores). Left, H-score plotted (R2 = 0.431
and P < 0.0001); bar chart, sequential core expression of P-Smad1/5/8 or P-S6. D, a schematic model of IGF-I regulation of BMP signaling and its implication in prostate
cancer.
119
(Figure 2.14)
120
Figure 2.15. Differential expression analysis of BMP4, BMP6, and BMP8B mRNAs in human prostate samples.
BMP-4, -6, -8B mRNA expression profiles of human prostate tissues were mined from
Oncomine Research (www.oncomine.org). Sample descriptions were the following.
Class1: benign prostate (6 cases); class 2: prostate carcinoma (7 cases); class 3: hormone- refractory metastatic prostate carcinoma (6 cases). Correlation was -0.788 with a P value of 6.2E-5. Data link: (http://www.ncbi.nlm.nih.gov/projects/geo/query/ acc.cgi?acc=GSE3325) [176].
121
(Figure 2.15)
BMP4 BMP6 BMP8B
N=6 N=6 N=6 N=7 N=7 N=6 N=7 N=6
N=6
122
Figure 2.16. Loss of Id1 induces growth arrest in prostate cancer cell line LNCaP.
A, Four pLKO.1-sh-Id1 (#1- #4) silencing constructs were shown to silence Id1 in
LNCaP and DU145 cells compared to parental vector and vector positive control
(pLKO.1 and pLKO.1-si- GFP). B, LNCaP-sh-GFP or LNCaP sh-Id1 #1 were transiently silenced and treated with LR3- IGF-I (10 nmol/L) 4 h prior to BMP4 (5 ng/mL) for an additional 68 h, and cells were then examined using phase-contrast microscope at 200X for changes in morphology. C, LNCaP or DU145 infected with shGFP or sh-Id1 #1 virus were treated as above and total adherent cells were counted using a Coulter Electronic counter. Columns are averages of triplicate determinants; bar, ±S.E.
123
(Figure 2.16)
A. LNCaP DU145
Id-1 Id-1
β-actin β-actin
B. BMP4 + Vehicle BMP4 LR3-IGF-I LR3-IGF-I PLKO.1-Sh-GFP
x200 PLKO.1-Sh-ID-1#1
125 DU145-PLKO.1 shGFP DU145-PLKO.1-shID-1#1 C. LNCaP-PLKO.1 shGFP 125 ) LNCaP-PLKO.1-shID-1#1 4 100 * ) 4 $ 100 75 **** 75 **** 50 50 Adherent Cells (x 10 25
Adherent Cells (x 10 25
0 0 BMP4 (5 ng/ml) - +-+ -+- + BMP4 (5 ng/ml) - +-+ -+- + LR3-IGF-I (10 nM) - ---++ ++ LR3-IGF-I (10 nM) - ---++ ++ $:P<0.01 *:P<0.001 *:P<0.001
124
TABLES
Table 2.1. List of BMP4 regulated genes specifically altered by IGF-I.
Details of Gene Expression Profiling are included in supplementary methods.
125
(Table 2.1a)
126
(Table 2.1b)
127
(Table 2.1c)
128
Chapter 3 Smads 1 and 5 but not Smad8 are Activated by Rapamycin and Promote Cytostatic/Cell Death Responses in Prostate Cancer Cells.
This chapter has been submitted to Cancer Research
Reema S. Wahdan-Alaswad*, Kyung Song, Dorjee T.N. Shola, Jorge A. Garcia, and David Danielpour
ABSTRACT While hyper-activated TOR is believed to be pivotal to the progression of prostate cancer, the mechanism by which mTOR promotes tumor growth is not well understood. Here we provide the first direct evidence supporting that inhibition of mTOR by rapamycin or the rapalog everolimus promotes the activation of Smads 1 and 5 in human prostate cancer cells and tissues. Lentivirus-based silencing and retroviral-based overexpression approaches were used to demonstrate that Smads 1 and 5 mediate while Smad8 represses rapamycin-induced cell death and expression of the BMP transcriptional target Id1 in human prostate cancer cell lines. Moreover, such Smad1- and Smad5-mediated rapamycin responses were blocked by the BMP type I receptor kinase inhibitor LDN-
193189 and enhanced upon silencing mTOR, raptor, or rictor. Immunohistochemical analysis show increased levels of phospho-Smad1/5/8 concomitant with suppression of phospho-S6 and survivin levels in PC3 human prostate cancer xenografts in athymic mice administered rapamycin (i.p., 5 mg/kg/day, 2 to 6 days) versus vehicle control.
Moreover, we show that compared to untreated prostate tumor tissue, levels of phospho-
Smad1,5,8 were significantly elevated in the prostate tumor tissue of high-risk prostate cancer patients who received 8 weeks of everolimus as part of a neoadjuvant clinical trial
129
prior to undergoing local definitive therapy by radical prostatectomy. Taken together, our data implicate Smads 1, 5 and 8 as potential prognostic markers and therapeutic targets for rapalog-based therapy of prostate cancer.
INTRODUCTION
The mammalian target of rapamycin (mTOR) is a 298 kDa serine-threonine kinase plays critical roles in the regulation of growth, survival, protein synthesis, metabolism, and angiogenesis. mTOR is activated predominantly through the Akt signaling pathway [114], and is hyperactivated in many human cancers through either loss of phosphatase and tensin homolog (PTEN) or activation of oncogenes such as PI3K and Ras [94, 116, 118]. Loss of the tumor suppressor PTEN occurs in up to 50% of human prostate cancer [106], and has been implicated in the development of prostate intraepithelial neoplasia (PIN) [139], and castration refractory prostate cancer [177].
Rapamycin and rapamycin-like analogs (rapalogs) are direct inhibitors of the mTOR complex1 (mTORC1), which contains raptor and regulates protein synthesis and cell growth through phosphorylating S6K and 4E-BP1[136]. However, rapamycin does not directly inhibit mTOR complex 2 (mTORC2), which contains rictor and is a key activator of Akt [178]. Rapalogs inhibit mTORC1 through forming a complex with the immunophilin FKBP12 at a site juxtaposed to mTOR’s kinase domain [132, 179].
Rapalogs, such as everolimus (afinitor®, Novartis, New Jersey) and temsirolimus
(Torisel®, Pfizer, Inc., New York), are now a standard of care in advanced renal cell cancer patients [180, 181], and their optimal use in treating other solid tumors continues
[94, 182-184]. Despite some encouraging results, rapalogs have had limited therapeutic
130
success as single agents [113, 185], a phenomenon attributed to several mechanisms
including the activation of mitogen signaling through IRS-1by rapalogs [186] and
activation Akt through mTORC2 [187]. Notwithstanding major recent advances in the
mTOR field, strategies to fully capitalize on the therapeutic potential of rapalogs are limited by our incomplete understanding of the mechanisms by which mTOR promotes
tumor growth.
Our laboratory provided the first evidence that insulin-like growth factor-I (IGF-
I) inhibits transforming growth factor-beta (TGF-β)-induced Smad3 phosphorylation and
TGF-β responses through a PI3K/Akt/mTOR signaling pathway [143, 144]. We recently
reported that IGF-I intercepts signaling by another member of the TGF-β superfamily,
namely bone morphogenetic protein (BMP) in human and rat prostate epithelial cell lines
through blocking the activation of the transcription factors Smads 1, 5 and/or 8 [188]. In
the current study we provide evidence that the cytostatic/apoptotic activity of rapamycin
in prostate carcinoma cells is partly mediated through its ability to activate BMP
signaling. Intriguingly, Smad8 represses rapamycin-induced activation of Smads and
growth suppression. Analysis of PC3 human prostate carcinoma xenographs in mice
treated with rapamycin and human prostate cancer tissues from high-risk prostate cancer
patients treated with everolimus as a phase II neoadjuvant trial confirms the in vivo
relevance of our in vitro findings, and implicate Smads 1, 5, and 8 as potential prognostic markers for rapalog-based therapeutics .
METHODS
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Materials. Recombinant human BMP4, anti-Id1 antibody (AF4377) (R&D Systems, Inc.,
Minneapolis, MN), Stemfactor™ Recombinant human BMP4 (cat#03-007) and LDN-
193189 (Stemgent, Cambrige, MA); rapamycin, Temsirolimus, (LC labs, Woburn, MA); anti-phospho-Smad3 antibody (p-Smad1/3/5/8, Cat.#9514); anti-phospho-Smad-1,5,8 antibody (p-Smad1/5/8, Cat.#9511), anti-phospho-Smad2 (Cat.#3101), anti-Cyclin D1
(Cat.#2926) (Cell Signaling, Beverly, MA); anti-Smad2 antibody (Cat.#66220)
(Transduction Laboratories, San Diego, CA); anti-Smad3 (sc-8332), anti-survivin (sc-
10811), anti-Smad1 (sc-7965), anti-Cyclin D2 (sc-593) (Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA); DMEM/F-12 (1:1); characterized fetal bovine serum (FBS) (HyClone
Inc., Logan, UT); insulin (BioSource International, Camarillo, CA); cholera toxin and
dexamethasone (Sigma, St. Louis, MO) and HTS-466284 (EMD Chemicals, Gibbstown,
NJ).
Cell culture. The LNCaP, PC3, and DU145 cell lines were obtained from ATCC
(Rockville, Maryland) and maintained in either DMEM/F12. The above cell lines were
authenticated by ATCC (by DNA profiling, cytogenetic analysis, flow cytometry and
immununohistochemistry) and used in experiments within 20 passages. C4-2 and C4-2B
were fromDrs. Karen Knudsen and Bingcheng Wang, respectively, and used within 15
passages; they were authenticated by morphology, expression of androgen receptor and
prostate specific antigen (by Western blot), and androgen-independent growth. The
NRP-152 cell line was generated in our laboratory and maintained in GM2.1 as described
[144, 146].
132
Western analysis. Cell lysates were prepared and analyzed by Western blot as previously described [143, 189].
Id1 luciferase reporter assay.
Cells were transfected, treated and assayed similarly as before [189].
Reverse transcriptase-polymerase chain reaction (RT-PCR) and RTqPCR. Previously described [189].
Rapamycin xenograph study in vivo. PC3 cells (3x106 in 0.2 ml DMEM: Matrigel [1:1, v/v]) were implanted s.c. on opposing flanks of (6-7 week old) Ncr:NU athymic male mice, and tumors were allowed to reach an average of about 25 mm2 (L x W). Groups of
5 mice were administrated rapamycin (5 mg/kg, i.p.) or vehicle as described [190] daily for 2 or 6 days. Tumors were fixed in formalin, embedded in paraffin, and subjected to
IHC analysis. All experiments performed and euthanasia protocols are detailed in our institutional IACUC protocol #2008-0067.
Randomized phase II study of two different doses of RAD001 as a neo-adjuvant therapy in patients with newly diagnosed clinically localized prostate cancer.
Eligible patients were randomized to receive either 5.0 mg or 10 mg everolimus orally every day continuously for 8 weeks. One week after the completion of treatment, all men underwent radical prostatectomy with bilateral pelvic lymph node dissection. Pre- and post-treatment prostate tumor tissue samples were fixed in formalin, embedded in paraffin, and stained for phospho-Smad1/5/8 or phospho-S6 by IHC. .
133
Cell viability assay. Cell viability was assessed by Trypan Blue exclusion under phase-
contrast microscopy as before [159]. Cells were plated in 12-well dishes in low serum
conditions at a density of 30,000-50,000 cells/1ml/well as previously described [144,
160]. In brief, cells were pre-treated with rapamycin (200 nmol/L) or vehicle control 2 h
prior to addition of BMP4 (5 ng/ml) or control treatment for 24-72 h. Cells were
detached via trypsinization after 72 h, neutralized with DMEM/F12 + 5% FBS, spun at
500g for 5 min and cell pellet was resuspended in 100 µL DMEM/F12 + 5% FBS. Ten µl
of the cell suspension was combined with 10 µl of Trypan Blue dye solution and
examined with a hemocytometer. Viable cells (dye excluded) were counted from a total
of 300 cells examined.
Hoechst 33258 staining.
Briefly, cells were plated in 6 well dishes in at a density of 3-5x104 cells/well in 2 ml of
DMEM/F12, 1% FBS, 15 mM HEPES (pH 7.4) (for LNCaP, C4-2, C4-2B, PC3, and
DU145) or in GM3.1 (for NRP-152, NRP-154, and DP-153). Cells were treated with
vehicle or rapamycin (200 nmol/L) 2 h prior to BMP4 (5 ng/ml) addition. After 24 cells
were stained with 10 μg/ml Hoechst 33258 (Sigma) and apoptotic cells were counted
using fluorescent microscopy at each time point. Three hundred cells were analyzed in
triplicate [161].
Flow Cytometry. Cells were treated as previously described [189].
Cell number assay. 3-5 x 104 cells/ml were seeded in 12-well dishes in medium described in Hoechst staining assay. The following day, cells were pre-treated
134
±rapamycin (200 nmol/L) 2 h prior to ± BMP4 (5 ng/ml) treatment for up to 72 h.
Trypsinization was used to detach adherent cells and enumerated with a Coulter
Electronics counter.
PCR primers. The PCR primers were purchased from Operon Biotechnologies, Inc. A full description of all primers used was described previously [189].
MTT Assay. Cell viability was also assessed by use of 3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyl tetrazolium bromide (MTT) which is cleaved at the tetrazolium ring by
mitochondrial dehydrogenases, yielding purple insoluble formazan crystals. Cells were
seeded in 96-well plates at 0.5-1 x 104 cells per well in 100 µl of culture media 24 h prior
to treatment. Next day, cells were treated with 0-1000 nM rapamycin or DMSO control
for an additional 72 h. Cultures were then treated with 10 µl of 5mg/ml MTT in RPMI-
1640 without phenol red (Sigma-Aldrich), incubated at 37°C for 3–4 hours in a
humidified CO2 incubator, and colored product was solubilized by extraction with 100 µl
of isoproponal-0.1 N HCl.. Colored product was quantified within 1 h spectrophotometer
absorbance at 570 nm – 690 nm (background substration) with a Tecan-mini microplate
reader.
Retrovirus. Smad1, Smad5, and Smad8 with Flag tag was PCR amplified and subcloned
into the proretroviral construct pLPCX (BD Biosciences Clontech, Palo Alto, California,
USA). Replication-defective infectious retrovirus was generated by transfecting the
proretroviral plasmids into A-BOSC cells as previously described [191]. Exponentially
growing LNCaP or PC3 cells were transduced with pLPCX control retrovirus or pLPCX-
Smad1, -Smad5, or –Smad8 and were maintained under puromyocin selection (2 μg/ml) 135
for one week. pLPCx-control/-Smad1/-Smad5/-Smad8 viral preparations to deliver
desired functional protein was evaluated by overnight infection (followed by 48 h
recovery in DMEM/F12 medium containing 5% FBS) in either LNCaP or PC3 cells.
Lentivirus. The generation of stable shRNA expression lentiviruses harboring shLacZ, shSmad1, shSmad5, shSmad1/5, shSmad8 were used in LNCaP or PC3 cells (to drive
LNCaP-shRNA or PC3-shRNA cells). The targeting sequences for these shRNA
constructs were: shLacZ 5’-GTGACCAGCGAATACCTGT-3’[192], shSmad1#1: 5’-
GGAAACAGGGCGATGAAGA -3’, shSmad1#2: 5’-CGGAATTCCACTATTGAAA-
3’, shSmad5#1: 5’-GAGCTAAAGCCGTTGGATA -3’, shSmad5 #2: 5’-
TCAGATGGGTCAAGATAAT-3’, shSmad8#1: 5’-GCAAGGAGATGAAGAGGAA-
3’, shSmad8#2: 5’-GCTTTGAAGTCGTGTATGA -3’ (Designed by Dharmacon RNAi
Technology and oligomers were purchased from Integrated DNA Technologies,
Coralville IA).
Real Time quantitative reverse transcriptase-PCR (RT-qPCR). cDNA was designed as described previously [189] using 1 µg of total RNA. Predesigned gene specific probe sets for Id1were made for Taqman Gene Expression Assay (Applied Biosystems, Inc.
Foster City, CA) .. RT-PCR was performed as specified in the manufacturer’s protocol of the 7500 Fast Real Time PCR System (Applied Biosystems, Inc., Foster City, CA).
Immunohistochemistry and Tissue Microarray (TMA). PR8011 tissue microarray
(TMA) included 22 cases/cores of patients with prostate adenocarcinoma, including
136
TNM, and pathology grade. Slides were processed using IHC standard protocol as described [189]. Sections were either stained with anti-phospho-Smad-1,5,8 antibody
(Cell signaling Inc., Danvers, MA, Cat.#9511) at (1:100) dilution, Phospho-S6
Ribosomal Protein (Ser 235/236) antibody (Cell signaling Inc., Danvers, MA, Cat #2211) at (1:100) dilution, survivin (sc-10811) at (1:400) or normal rabbit IgG used as a negative control. All slides were reviewed independently by two investigators (R.W and
K.S) who were blinded to the clinical and pathological data and reconfirmed by a second set of evaluations by one investigator (D.S) blinded to the interpretations of the first set of evaluations, as well as clinical and pathological data. Immunostaining results were validated by the consensus of all reviewers.
RESULTS
Rapamycin activates Smads in human prostate cancer cells.
We recently reported that IGF-I inhibits BMP4-induced phosphorylation of Smads 1,5 and/or 8 by activating mTOR in prostate epithelial cell lines [188], suggesting that BMP signaling responses may be involved in the mechanism by which rapalogs repress growth of prostate carcinoma cells. To test this hypothesis, we first evaluated the response of a panel of prostate epithelial cell lines (LNCaP, C4-2, C4-2B, PC3, DU145) for cytostatic activity (by MTT and cell number assay (Fig. 3.1a&b) and activation of Smads (by
Western blot with c-terminal phospho-specific antibodies) by rapamycin (Fig. 3.1c).
Rapamycin had cytostatic activity and enhanced the phosphorylation of Smads 1, 5 and/or 8 in all those cell lines. However, in that assay we were unable to distinguish between the phosphorylation of Smads 1, 5 or 8, as they co-migrate and isoform-specific
137
phospho-antibodies for those Smads are not available. Smad2 was not phosphorylated
and Smad3 appeared to be phosphorylated only in PC3 cells, which was the only cell line in this group with detectable expression of Smad3. Levels of total Smads were unaltered by rapamycin, except in PC3 cells in which Smad3 was induced and Smad5 was decreased. Rapamycin did not seem to change the levels of mTOR, PTEN or Akt1 in those cells. The magnitude of phospho-Smad activation by 200 nmol/L rapamycin appeared to correlate with that of growth suppression by rapamycin.
We examined the ability of rapamycin to activate Smads 1, 5 and/or 8 in a dose- and time-dependent manner in LNCaP cells. Cells were treated with (0-1000 nmol/L) rapamycin for 24 h or from 0 to 72 h with 200 nmol/L rapamycin and were then subjected to Western blot analysis using antibodies against phospho-Smads, cyclin D1 and cyclin D2 (Fig. 3.2a (top), 3.2b (top)). Phosphorylation of Smad1/5/8 at 24 h occurred at 12.5 nmol/L rapamycin and was maximal between 100 and 200 nmol/L, and inversely correlated with changes in the cyclin Ds. Rapamycin induced levels of phospho-Smad1/5/8 by 12 h, with maximal induction by 72 h. We confirmed that rapamycin activated Smads 1/5/8 rather than just inducing their phosphorylation under
these conditions, by transfecting cells in a parallel experiment with an Id1 promoter-
luciferase construct, which contains a number of BMP response elements [81], and
measuring luciferase activity various times later (Fig 3.2a (below), 3.2b (middle)). As
anticipated, Id1 promoter was activated with 12.5 nmol/L rapamycin at 24 h; 200 nmol/L
rapamycin activated this promoter by 5-fold after 24 h to 45-fold by 72 h (Fig. 3.2b
(middle)). Furthermore, rapamycin induced Id1 mRNA levels by 24 h (Fig. 2b (bottom).
Similar results were obtained in PC3 and in the non-tumorigenic rat cell line NRP-152 138
(Fig. 3.3). Rapamycin significantly enhanced the ability of BMP4 to induce cell death
(by Hoechst 33258/Flow cytometry/ cell number assays) and activate the Id1 promoter
(Fig. 3.2c, Fig. 3.3a-d, 3.4, and 3.5).
Rapamycin-mediated cell death requires Smad1 and Smad5 but not Smad8.
We used a lentiviral delivery system for inducible expression of specific small hairpin
(sh) RNAs [193], to efficiently and stably silence expression of Smads 1, 5, 1&5, and 8 by >95% in LNCaP and PC3 cells (Fig. 3.6a). Using those cell lines we assessed which of these Smads were activated by rapamycin (200 nmol/L at 24 h) (Fig. 3.6b (top), Fig.
3.7a&b). Our analysis revealed that silencing Smads 1 and 5 but not Smad8 repressed the levels of p-Smad1/5/8 activated by rapamycin, suggesting that rapamycin activates both
Smad1 and Smad5, but not Smad8. Silencing Smads 1 or 5 also repressed activation of
Smad3 by rapamycin in PC3 cells, suggesting that Smads 1 and 5 may be essential to the activation of Smad3 by rapamycin. Intriguingly, blocking Smad8 enhanced rapamycin- induced Smad activation (including Smad3 in PC3 cells), indicating that Smad8 functions as a negative regulator of the activation of Smad1 and 5 by rapamycin. Similarly, we observed that shSmad1 and shSmad5 each suppressed rapamycin-induced Id1 promoter activity, whereas shSmad8 enhanced such activity (Fig. 3.6b (lower). Moreover, Smad1- and Smad5-silenced cells were less responsive than the LacZ control silenced cells to rapamycin, as measured by changes in cell viability (trypan blue dye exclusion), total adherent cells (Coulter counter), and induction of Id1 promoter activity (Fig. 3.6 b-d, Fig.
3.8). In contrast, Smad8 silenced cells showed increased sensitivity to the above 139
responses by rapamycin. A similar pattern of Smad dependence on cell death was observed with BMP4; however, silencing either Smad1 or Smad5 alone completely
reversed cell death (Fig. 3.6d). In contrast, silencing of Smads 1, 5 or 8 each alone did
not reverse BMP4-induced Id-1 promoter activity (Fig. 3.8).
Overexpression of Smad1 or Smad5 enhances Rapamycin-induced Id1 promoter
activation and cell death in LNCaP cells.
Based on the above results, we tested whether overexpression of Smad1, Smad5,
Smad1+5, or Smad8 would enhance rapamycin’s ability to induce Id1 luciferase promoter and cell death. We efficiently over-expressed Smad1, Smad5, and Smad8 using the pLPCX retrovirus vector in LNCaP and PC3 cells (Fig. 3.9a, data not shown). The stable cell lines were treated with rapamycin (200 nmol/L, overnight) and analyzed by
Western blot , Id1 promoter activity, or cell death (Fig. 3.9b-d, Supplementary Fig 3.10a- c). Overexpression of Smad1 and Smad5 enhanced rapamycin-induced activation of
Smads, Id1 promoter activity and cell death, whereas overexpression of pLPCX-Smad8 diminished such responses in LNCaP cells (Fig 3.9). Similar results were observed in
PC3 cells over-expressing Smad1, Smad5, and Smad8 (Fig. 3.10). Taken together, these
data supports that both Smad1 and Smad5 play an important role in mediating
rapamycin-induced cell death, whereas Smad8 functions as a negative regulator of this
response.
BMPRI kinase activity is involved in cellular responses to rapamycin.
140
Since rapamycin promotes the activation Smads 1 and 5, which are activated by BMP, we examined whether inhibition of BMPRI would abrogate rapamycin’s ability to induce phospho-Smad activation, Id1 luciferase promoter activity or cell death. We examined the above activities in prostate cancer cell lines pre-treatment with a selective kinase inhibitor (LDN-191389) of BMP type I receptors (ALK2, ALK3, and ALK6) [194] or a selective kinase inhibitor (HTS-466284) of TGF-β type I receptors (ALK4, ALK5,
ALK7) [195]. PC3 cells were treated with LDN-193189 (500 nmol/L) or HTS-466284 (2
μmol/L) 2 h prior to 20 h treatment with rapamycin (200 nmol/L) and then subjected to
Western blot analysis (Fig. 3.11a (top). As in Fig.1a, rapamycin (200 nmol/L) induced expression of both phospho-Smad1/5/8 (top band) and phospho-Smad3 (lower band).
LDN-193189 repressed activation of Smad1/5/8 (top band), and also repressed phospho-
Smad3 levels, whereas HTS-466284 blocked phospho-Smad3, and the combined treatment with both inhibitors abrogated all phospho-Smads. The concentrations of inhibitors were chosen based their minimal concentrations that selectively and efficiently inhibited Smad activation by BMP4 or TGF-β. That 500 nmol/L LDN-193189 weakly blocks activation of Smad3 by TGF-β1 [194] suggest that activation of Smad3 by rapamycin was dependent on the kinase activity of a BMP type I receptor. Similarly, we treated the same panel of cell lines with variable concentrations of LDN-193189 (0-500 nmol/L) 2 h prior to a 70 h treatment with 200 nmol/L rapamycin, and then measured the total adherent cells using a Coulter counter (Fig. 3.11a (below), Fig. 3.12 a&b).
Inhibition of BMPRI reversed rapamycin-induced cell death in all cases.
141
We next examined whether LDN-193189 could block rapamycin’s ability to induce
Id1 mRNA or Id1 transcriptional regulation. Semi-quantitative RT-PCR (Fig. 3.11b
(top)) and real-time quantitative PCR (Fig. 3.11b (bottom)) was used to assess the ability of BMPRI inhibitor to suppress rapamycin-induced levels of Id1 mRNA and cell death in
LNCaP cells (Fig. 3.11c). Rapamycin-induced expression of Id1 mRNA by 6-fold as determined by real-time PCR (RTqPCR), and LDN-193189 was able to fully suppress rapamycin-induced Id1 mRNA. Similarly BMP4 was able to enhance Id1 mRNA levels by 20-fold, and when combined with rapamycin this activity was heightened to 42-fold, whereas inclusion of LDN-193189 abrogated such induction.
Next, we examined the role of Id1 induction in growth suppression by rapamycin in
LNCaP and DU145 cells by silencing Id1with shRNA as previously described [188].
Cells were infected with either vehicle control pLKO.1-shGFP or pLKO.1-shId1#1 for 48 h prior to a 3 day treatment with rapamycin (200 nmol/L) and/or BMP4 (5 ng/ml), and subjected to Western blot, cell number and morphological analysis (Fig. 3.11d, Fig. 3.12c
& Fig. 3.13). We found that silencing Id1 alone suppressed cell growth, but did not
reverse rapamycin-induced cell death in both cell lines. Taken together, these data
support that Id1 induction does not mediate growth suppression by rapamycin.
Silencing mTOR, raptor or rictor enhances rapamycin’s ability to activate the Id1
promoter.
We examined the role of mTOR and its binding partners, raptor and rictor, in the
ability of rapamycin or BMP4 to induce the Id1 promoter. For this we utilized NRP-152
cells that were stably silenced for mTOR, raptor, and rictor by an shRNA lentiviral 142
delivery system [188]. The stably silenced cell lines were transfected with Id1 luciferase
promoter and then treated with ±LDN-19189 (500 nmol/L) followed 2 h later by
treatment with ±rapamycin (200 nmol/L) 20 h prior to a 4 h treatment with BMP4 (5
ng/ml) (Fig. 3.14a-b). Silencing mTOR, raptor, or rictor alone each activated the Id1
promoter and enhanced both rapamycin- and BMP4-induced activation of the Id1
promoter. Taken together, these results suggest that both mTORC1 and mTORC2 are
involved in the ability of rapamycin to activate Smad1/5 signaling, and that both mTOR
complexes are endogenous inhibitors of BMP4- or rapamycin-activated Smads.
Rapalogs enhance levels of phopho-Smad1/5/8 in human prostate cancer tissues,
correlating with loss of both mTOR activity and survivin expression.
Complementing our cell culture studies, we next addressed whether rapamycin would
affect Smad activation in vivo. Here we implanted PC3 cells (3x106 cells) s.c. in athymic nude male mice and once tumors were evident (~100 mm3), animals were administered
daily with either vehicle or rapamycin (5.0 mg kg-1) by i.p. for either 48 h or 6 days.
Tumors were then fixed in formalin and processed for IHC analysis of phospho-
Smad1/5/8, phospho-S6 and survivin expression (Fig. 3.15a). Administration of
rapamycin for 2 days clearly enhanced staining for phospho-Smad1/5/8 expression and
suppressed that for phospho-S6 and survivin, with greater effects by 6 days. H-score
analysis (% positive stained cells x intensity of staining (0-3)) provided statistically
significant and quantifiable changes in the pattern of expression (Fig. 3.15b).
To show the relevance of these findings in humans, we utilized radical prostectomy
tumor tissue sections from a phase II clinical trial in which patients with newly diagnosed 143
high-risk prostate cancer were administered everolimus (5 or 10 mg/daily orally)
continuously for 8 weeks before undergoing radical prostatectomy. At the time of
conducting these experiments, prostate cancer tissue sections from 6 patients were
available. Tumor tissue specimens were evaluated for phospho-Smad1/5/8 and phospho-
S6 via IHC (Fig 3.15c, Fig. 3.16). We compared these results side-by-side with those
from a larger (22 biopsy cores) and a comparable (localized prostate adenocarcinoma
stages II-III) cohort of non-treated human prostate tumor tissues in a microarray (PR8011
series) obtained from US BioMax, Inc. H-score analysis was used to determine the
relative expression of phospho-Smad1/5/8 and phospho-S6 in the non-treated control
group (n=22) against the everolimus treatment group (n=6) (Fig 3.15d). Although the
sample size (n=6) of this ongoing clinical trial is currently small, statistically significant
differences in relative levels of phospho-S6 and phospho-Smad1/5/8 between the
everolimus-treated and non-treated groups were generated, with reduced phospho-S6 levels and increased phospho-Smad1/5/8 levels in the everolimus group compared to the
untreated group. Our data suggest that 4/6 (67%) of patients responded to everolimus
treatment by loss of phospho-S6 and also showed enhanced phospho-Smad1/5/8
expression, supporting that suppression of mTOR by everolimus enhanced the activation
of Smad1/5/8. Once this trial is completed, all specimens will be re-analyzed in similar
fashion. Further, we also compared p-Smad 1, 5, 8 compared to p-S6 expression in
variable stages of human prostate cancer progression in vivo (Fig. 3.17). As prostate
cancer progresses, p-Smad staining decrease, whereas p-S6 expression increases.
144
Discussion
The discovery of rapamycin as an anticancer agent provided enormous impetus to
identify its target, mTOR, which was later shown to be a key regulator of protein
synthesis, cell metabolism and cell growth. mTOR is activated by mitogenic signals
through the receptor tyrosine kinase/PI3K/Akt pathway [83, 196]. Importantly, Akt and
mTOR are hyperactivated in many cancers including prostate cancer, principally through
loss of PTEN and constitutive activation of PI3K [107-109, 177, 197]. However, the
underlying molecular mechanism(s) by which mTOR promotes the pathogenesis of prostate cancer remains incompletely explored. Despite strong evidence for the importance of the hyperactivation of mTOR in promoting tumor cell growth, most
cancers show limited growth suppression by rapalogs [185], attributed largely to reversal
of the negative feedback of mTORC1 on IRS-1, and enhanced oncogenic activation of
Akt by mTORC2 [114, 186, 187, 198]. Moreover, recent studies demonstrate that
mTORC2 rather than mTORC1 is critical to the development of prostate tumors in PTEN
knockout mice [199], suggesting that selective targeting of mTORC2 over mTORC1 may
hold more therapeutic promise.
We suggest that components of the BMP and TGF-β signaling pathways,
particularly the expression of Smads 1, 5 and 3 may be critical to the anti-tumor activity
of rapalogs. Our recent report that IGF-I abrogates BMP4 signaling through activating
mTORC1 and mTORC2 [188], provided the first functional connection between
activation of mTOR and subsequent loss of the tumor suppressor function of BMP4 in
prostate cancer cells, and suggested that suppressing mTOR signaling may restore tumor 145
suppression by BMP4. Our current study here extends those findings and provides the first evidence that rapalogs induce the activation of Smads 1 and/or 5 in human prostate cancer cells in culture, in tumor xenografts and in human prostate cancer tissues. Our cell culture studies support that such Smad activation is associated with suppression of mTOR’s function, and requires Smad1, Smad5 and the kinase activity of a BMP type I receptor (ALKs 2, 3 and/or 6); however, Smad8 represses such activation. Moreover, we show that Smad1, Smad5 and the BMP type I receptor play critical roles in the ability of rapamycin to suppress growth or induce apoptosis, whereas Smad8 reverses rapamycin- induced growth suppression.
A recent pharmacodynamic study of rapamycin in patients with intermediate- to high-risk prostate cancer demonstrated that administration of daily doses of 3 mg rapamycin for 14 days suppressed tumoral levels of p-S6 and increased nuclear expression of p27, with no significant differences in the expression of key proliferative and apoptotic markers (pAktSer473, Ki-67, cleaved caspase-3)[142]. That pAkt levels did
not change suggested growth suppression by rapamycin was not opposed by the
activation of an IRS-1 feedback loop in those tumors. It is possible that the course of
treatment was too short for detecting significant changes in the above markers. The
preliminary result of our neoadjuvant everolimus trial, in which the duration of therapy
was 4 times longer, indicates that this rapalog robustly activates the tumor suppressors
Smad1/5, but it is unclear whether this suppresses tumor burden. Further work will be
necessary to assess if everolimus can suppresses growth of clinically localized prostate
cancer or whether it can improve surgical success by inhibiting inflammation and/or micro-metastases. 146
In spite of the importance of mTOR in preclinical models, the above clinical
studies and others suggest that rapalogs activate a number of potential compensatory
mechanisms in prostate tumors. Our findings suggest that BMP Smads also activate
oncogenic signals such Id1 [188] that may counteract the therapeutic efficacy of rapalogs.
Another likely compensatory mechanism well known to be activated by rapamycin is
autophagy [200, 201], which is a cell survival mechanism activated in response to
metabolic stress [202]. Although autophagy initially promotes cell survival through
inhibiting apoptosis, sustained autophagy by rapamycin and/or in combination with other
cellular stresses may favor the induction of apoptosis [203, 204]. This may provide at least part of the mechanistic basis for enhanced therapeutic efficacy of rapalogs when combined with autophagy inducers such as PI3K/Akt inhibitors [205], radiation [206,
207] or anti-androgens [208-210].
Given substantial evidence for the oncogenic function of BMP and TGF-β
signaling in a number of late-stage cancers including prostate and breast cancer [211],
activation of BMP Smads may contribute to reduced therapeutic efficacy of rapalogs in
late-stage cancers. If so, combined therapeutics of rapalogs with a BMP receptor kinase
inhibitor (i.e., LDN-189193) may prove efficacious for such late-stage cancers. Further
research is thus warranted to more fully explore the roles of BMRII, Smad1/5 and Smad8 as prognostic markers and therapeutic targets of rapalog-based neoadjuvant modalities.
Acknowledgements. We thank Tracy Krebs for technical assistant and Dr. Cristina
Magi Galluzzi for help with pathological assessment and critiquing this manuscript. This
work was supported by NIH grants R01CA092102, R01CA102074 and R01 CA134878
147
(D. Danielpour), a pre-doctoral fellowship (R. Wahdan-Alaswad) from Case
Comprehensive Cancer Center’s Research Oncology Training Grant 5T32CA059366-15
(2009) and National Research Service Award Individual Fellowship Application
1F31CA142311-01 (2010), and the Case Comprehensive Cancer Center P30 CA-43703
(for Cytometry core) (P30 CA43703).
148
FIGURES
Figure 3.1. Rapamycin-mediated cell death and Smad activation in prostate cancer cell lines.
Effect of various doses of rapamycin for 72 h on the viability LNCaP, C4-2, C4-2B, PC3,
DU145 cells was assessed by a microtiter MTT assay. B, Effect of rapamycin (200 nmol/L) on changes in cell growth was enumerated using a Coulter counter. C, Effect of rapamycin treatment (200 nmol/L , 24 h) on activation of Smads was assessed by
Western blot analysis using three different phospho-specific antibodies to the c-terminal
phospho-serines of Smads: p-Smad2, p-Smad1/3/5/8, and pSmad1/5/8, the latter two of
which allowed for identification of p-Smad3 (lower band) from p-Smad1/5/8 (upper
band). The blot was reprobed for expression of total each of the total Smads, Akt and
mTOR. Values represent averages of triplicate determinations ± S.E.
149
(Figure 3.1)
150
Figure 3.2. Characterization of rapamycin induced phosphorylation and activation of Smad1/5/8.
A, Dose-dependent effect of rapamycin (for 24 h) on phosphorylation of Smad1/5/8,
repression of cyclin Ds (top, Western blot) and activation of the Id1 promoter (bottom,
luciferase reporter assay) was assessed in LNCaP cells. B, Time-dependent effects 200
nmol/L rapamycin on the phosphorylation of Smad1/5/8 (top, Western blot), activation of
the Id1 promoter (middle, luciferse reporter assay), and induction of Id1 mRNA (bottom,
by RT-PCR) were assessed in LNCaP cells. C, Combined effects of rapamycin (200
nmol/L) and BMP4 (5 ng/ml) on Id1 promoter activity (28 h treatment, left panel) and apoptosis (72 h treatment, right panel) were assessed in LNCaP cells. Apoptotic cells
were enumerated under fluorescence and phase contrast microscopy at 200X following
staining them Hoechst dye. In the case of the promoter assay, BMP4 was added 4 h prior
to harvesting cells. Columns, average of triplicate determinations; bar, ±S.E. Data is
representative of three independent experiments.
151
(Figure 3.2)
A. LNCaP B. LNCaP
Rapamycin (nM) 0 12.5 25 50 100 200 500 1000 Rapamycin (200 nM)(h) 00.5 1 3 6 12 24 48 72 P-Smad1,5,8 P-Smad1,5,8 Smad 1,5,8 Smad 1,5,8 CyclinD1 β-actin
50 CyclinD2
β-actin 40
7 30 6 20 5 4 10 (Id-1 promoter/cmv-renilla) (Id-1 Relative Luciferase Activity 3 0 2 Rapamycin ( 200 nM) (h) 00.51 3 612244872 1 (Id-1 promoter/cmv-renilla) Relative Luciferase Activity Luciferase Relative Rapamycin (200 nM)(D) -1236- 0 Rapamycin (nM) 0 12.5 25 50 100 200 500 1000 BMP4 (5 ng/ mL) - ---- + Id-1 C. β-actin LNCaP LNCaP 20 Vehicle 75 Vehicle Rapamycin Rapamycin 15 50 10
25
5 % Apoptosis (Id-1 promoter/cmv-renilla) Relative Luciferase Activity Luciferase Relative 0 0 Rapamycin (200 nM) -+-+ -+-+ BMP4 (5 ng/ml) --++ --++
152
Figure 3.3. Rapamycin-mediated phospho-Smad1/5/8 and Id1 luciferase activity is time and dose-dependent in mediating cell death of human and rat prostate epithelial cell lines (PC3 and NRP-152).
A, PC3 cells were treated with 200 nmol/L rapamycin or vehicle control for 0-72 h prior
to examining phospho-Smad1/5/8 expression by western blot analysis. B, NRP-152 cells
were treated as described above prior to examining phospho-Smad1/5/8 expression by
western blot analysis (left), or treated ±rapamycin (200 nmol/L) for a period of 5 days, and adherent cells were enumerated using a Coulter counter (right). C, NRP-152 cells
were co-transfected with Id1 promoter as described previously for 24 h prior to
±rapamycin in dose response (0-1000nmol/L) for 24 h (left) or a in time-course
±rapamycin (200 nmol/L) for 0-72 h (right), and luciferase activity was measured
asindicated. Columns, average of triplicate determinations; bar, ±S.E. Data is
representative of three independent experiments.
153
(Figure 3.3)
154
Figure 3.4. Rapamycin-mediated cell death is enhanced in the presence of BMP4 as examined in human and rat prostate epithelial cell lines.
A, PC3 cells were treated with ±rapamycin (200nmol/L) or vehicle control 2 h prior to treating the cells with BMP4 (5 ng/ml) for an additional 5 days followed by examining cell number by Coulter counter. B, NRP-152 cells were co-transfected with Id1- luciferase as described above for 24 h then treated with rapamycin (200 nmol/L) or vehicle 24 h to ±BMP4 (5 ng/ml) for an additional 4 h, and luciferase activity was then measured. C. LNCaP, NRP-152, and PC3 cells were treated with ±rapamycin
(200nmol/L) or vehicle control 2 h prior to treating the cells with BMP4 (5 ng/ml) for 72 h prior to harvesting the cells and total adherent cells were enumerated with a Coulter counter. D, NRP-152 and PC3 cells were treated with rapamycin (200 nmol/L) or vehicle
2 h prior to ±BMP4 (5 ng/ml) for 72 h, stained with Hoechst dye, and examined under fluorescence and phase contrast microscopy at 200X. Columns, average of triplicate determinations; bar, ±S.E. Data is representative of three independent experiments.
155
(Figure 3.4)
A. PC3 B. NRP-152 ) 100 4 Vehicle BMP4 Rapamycin 75 BMP4 + Rapamycin
50
25 Adhernt cells (x10 0 0 1 2 3 4 5 Days
C. LNCaP NRP-152 PC3 8 Vehicle 5 Vehicle ) ) 75 Vehicle 4 ) 7 BMP4 4 4 BMP-4 BMP4 6 4
5 50 3 4
3 2 25 2 1 Adherent Cells (x10 Adherent Cells
Adherent Cells (x10 Adherent 1 Adherent Cells (x10 Adherent Cells 0 0 0 BMP4 (5 ng/ml) - + -+ - + -+ - + -+ Rapamycin (200 nM) - - ++ - - ++ - - ++ D. NRP-152 PC3 75 75 Vehicle Vehicle Rapamycin Rapamycin
50 50
25 25 % Apoptosis % Apoptosis
0 0 Rapamycin (200 nM) -+-+ -+ -+ BMP4 (5 ng/ml) --++ --++
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Figure 3.5. mTOR inhibition in the presence of BMP4 enhance apoptosis in PC3 cells.
A, PC3 cells were treated ±rapamycin (200nmol/L) 2 h prior to treating the cells with
BMP4 (5 ng/ml) for an period of 0-72 h prior to harvesting cells and examining cell fractions in either Sub G1 (apoptotic fraction), G1, or G2/M transition as examined by
flow cytometry.
157
(Figure 3.5)
15 24 h PC3 48 h 72 h 10
5 %Cells in Sub G1
0 Control Rapamycin BMP4 BMP4+Rapamycin
100
75
50
% Cells inG1 25
0 Control Rapamycin BMP4 BMP4+Rapamycin
20
10 % Cells in G2/M
0 Control Rapamycin BMP4 BMP4+Rapamycin
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Figure 3.6. Silencing Smad1 and Smad5 repress rapamycin-induced Smad activation, Id1 promoter activity and cell death.
A, Smad1, Smad5, and Smad8 were effectively silenced in LNCaP cells by transduction with shRNA lentiviruses. B, Impact of silencing endogenous Smads 1, 5, 8 on rapamycin
(200 nmol/L, 24 h)-induced activation of phospho-Smads (top, Western blot) activation of Id1 promoter (bottom). C,D LNCaP cells stably expressing shSmad1, shSmad5,
shSmad1/5, and shSmad8 were treated with rapamycin (200 nmol/L) for 72 h and
examined for apoptosis by Hoechst-33258 dye staining (C) or total adherence cells
(enumerated by a coulter counter) (D); cells were treated with BMP4 alone or in
combination with rapamycin (D) for comparative analysis. Columns, average of triplicate
determinants; bar, ±S.E and experiments were run in triplicates.
159
(Figure 3.6)
160
Figure 3.7. Silencing Smad1, Smad5, and/or Smad1/5 cells enhance rapamycin- mediated cell death.
A & B, Smads1, Smad5, Smad1/5 and Smad8 were stably silenced in both PC3 (A) and
LNCaP cells (B) and treated with rapamycin (200 nmol/L) for 24 h prior to examining phospho-Smad1/5/8 and/or total Smads expression was analyzed using western blot.
161
(Figure 3.7)
162
Figure 3.8. Silencing Smad1, Smad5, and/or Smad1/5 cells enhance rapamycin- mediated Id1 promoter activation.
LNCaP-shLacZ, LNCaP-shSmad1, LNCaP-shSmad5, and LNCaP-shSmad8 co-
transfected with Id1 promoter 24 h prior to rapamycin (200 nmol/L) treatment and
BMP4 (5 ng/ml) was added 4 h before measuring luciferase activity. Columns, average of triplicate determinants; bar, ±S.E and experiments were run in triplicates.
163
(Figure 3.8)
450 LNCaP-sh-LacZ 400 LNCaP-sh-Smad1 350 LNCaP-sh-Smad5 300 LNCaP-sh-Smad8 250 200 150 100
6
4 (Id-1 promoter/cmv-renilla) (Id-1 Relative Luciferase Activity Luciferase Relative 2
0 Rapamycin (200 nM) -+-+ -+-+ -+-+ -+-+ BMP4 (5 ng/ml) --++ --++ --++ --++
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Figure 3.9. Overexpression of Smad1 and Smad5 enhance rapamycin-induced Smad activation, Id1 promoter activity and cell death in LNCaP cells.
A, Smad1, Smad5, and Smad8 were overexpressed using pLPCX retrovirus as shown by
Western blot analysis in LNCaP cells. B, LNCaP-pLPCX-control, -Smads 1, 5 or 8 cells were treated with rapamycin (200 nmol/L) for 24 h and examined by Western blot analysis. C, Stably overexpressing LNCaP-Smads 1, 5, and/or 8 were subjected to Id1 promoter activity following treatment of cells with rapamycin (200 nmol/L) for 24 h prior to BMP4 (5 ng/ml) for 4 h. D, LNCaP-pLPCX-control or -Smad1,-Smad5, and/or -
Smad8 stably overexpressing cells were treated with rapamycin (200 nmol/L) for 72 h prior to examining % viable adherent cells. Columns, average of triplicate determinants; bar, ±S.E and experiments were run in triplicates.
165
(Figure 3.9)
166
Figure 3.10. Overexpression of Smad1 and Smad5 enhance rapamycin-mediated Smad activation and Id1 promoter activity and cell death in PC3 cells.
A, PC3-pLPCX-control, -Smad-1,-5 or-8 cells were treated with rapamycin (200 nmol/L) for 24 h and examined by western blot analysis. B, Stably overexpressing PC3-Smad1, -
Smad5, and/or -Smad8 were subjected to Id1 promoter activity following treatment of cells with rapamycin (200 nmol/L) for 24 h. C, LNCaP-pLPCX-control or Smad1,-
Smad5, and/or -Smad8 stably overexpressing cells were treated rapamycin (200 nmol/L) for 24 h prior to examining luciferase activity. Columns, average of triplicate determinants; bar, ±S.E and experiments were run in triplicates.
167
(Figure 3.10)
A. PC3
-+ -+ -+ -+ 200nM Rapamycin P-Smad158
P-Smad158
Survivin
β-actin
B. PC3 C. LNCaP
LNCaP-pLPCX-Control 70 Rapamycin 200nM LNCaP-pLPCX-Smad1 * 10.0 60 *:P <0.001 LNCaP-pLPCX-Smad5 LNCaP-pLPCX-Smad8
50 7.5 40 * 5.0 30 *
% Viable Cells 20 2.5 (Id-1 promoter/cmv-renilla) (Id-1 Relative Luciferase Activity Luciferase Relative 10
0.0 0 Rapamycin (200 nM) -++ - -++ - . .
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Figure 3.11. Rapamycin-induced Smad1/5/8 activation, Id1 promoter activity, and cell death requires BMPRI.
A, PC3 cells were treated with either BMPRI inhibitor (LDN-193189 (500 nmol/L) or
TβRI inhibitor (HTS466284 (2 μmol/L ) 2 h prior to rapamycin (200 nmol/L, 20h) treatment; BMP4 (5 ng/ml) was added 4 h prior to harvesting. Cell lysates were subjected to Western blot analysis of phospho-Smads (top). Effect of various doses of
LDN-193189 reversing the ability of rapamycin (200 nmol/L, 3 days) to decrease the number of adherent cells as assessed in PC3 (bottom). B, Effects LDN-193189 (500 nmol/L, added 2 h before rapamycin addition) on the ability of rapamycin (200 nmol/L,
44 h) or BMP4 (5 ng/ml, 24 h) to induce Id1 mRNA expression in LNCaP cells, as measured by RT-PCR (top) or real-time quantitative PCR (RT-q-PCR) (below). C,
LNCaP cells were co-transfected with Id1 promoter prior to treating the cells as described in A and luciferase activity (top) or total adherent cell number (below) were monitored.
D, Id1 efficiently silenced in LNCaP cells (LNCaP-shGFP v.s. LNCaP-shId1#1) and in
DU-145 cells (DU145-shGFP v.s. DU145-shId1#1) were treated with rapamycin (200 nmol/L) 2 h prior to BMP4 (5 ng/ml) stimulation for 70 h and the total adherent cells were enumerated with a Coulter counter (bottom). Columns, average of triplicate
determinants; bar, ±S.E and data are representative of three independent experiments.
169
(Figure 3.11)
170
Figure 3.12. Rapamycin-induced Id1 promoter activity and expression requires BMPRI.
A & B, LNCaP and NRP-152 cells were treated with various concentrations of LDN-
193189 (nmol/L) 2 h prior to treatment with rapamycin (200 nmol/L, 3 days) to reverse rapamycin-mediated cell death. C, Stably silenced LNCaP-shGFP or LNCaP-shId1 were treated with rapamycin (200 nmol/L) 2 h prior to BMP4 (5 ng/ml) stimulation for 24 h and the Id1 expression was analyzed by western blot. Data are representative of three independent experiments.
171
(Figure 3.12)
LNCaP A. Vehicle )
4 Rapamycin (200 nM) 20
10 Adherent Cell (x10
0 0 31 62 125 250 500
NRP-152 B. 20 Vehicle )
4 Rapamycin (200 nM)
10 Adherent Cell (x10 Cell Adherent
0 LDN-193189 (nM) 0 31 62 125 250 500
C. LNCaP-ShRNA pLKO.1 shGFP pLKO.1 shID-1#1 BMP4 (5 ng/ml) -+-+-+-+ Rapamycin (200 nM) --++--++
Id-1
β-actin
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Figure 3.13. Silencing Id1 enhanced rapamycin-mediated cell death in LNCaP cells.
A, LNCaP-shGFP or LNCaP-shId1#1 cells were treated with rapamycin (200 nmol/L) 2 h prior to BMP4 (5 ng/ml) stimulation for an additional 70 h where morphological alterations of cells were examined by phase contrast microscope at 200x.
173
(Figure 3.13)
Rapamycin Vehicle BMP4 Rapamycin +BMP PLKO.1-Sh-GFP PLKO.1-Sh-ID-1#1
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Figure 3.14. Rictor, Raptor and mTOR block rapamycin-mediated Id1 promoter expression in NRP-152 prostate epithelial cells requires BMPRI.
A, NRP-152-tTR-sh-LacZ, NRP-152-tTR-sh-Rictor, NRP-152 tTR-sh-Raptor, or NRP-
152-tTR-sh-mTOR cells were co-transfected with Id1 promoter, and then treated with rapamycin (200 nmol/L) for 24 h before measuring luciferase activity. B, Inducible silenced cells from A were co-transfected with Id1 luciferase promoter for 24 h prior to rapamycin (200 nmol/L) treatment alone, which was 2 h prior to BMP4 (5 ng/ml) addition and luciferase activity was measured 24 h later. Columns, average of triplicate determinants; bar, ±S.E.
175
(Figure 3.14)
30 A. sh-LacZ-I sh-Rictor shmTOR sh-LacZ-A 20 sh-Raptor
10 (Id-1 promoter/cmv-renilla) (Id-1 Relative Luciferase Activity Luciferase Relative 0 Rapamycin (200 nM) -+ -+ -+ -+ -+
70 sh-LacZ(I) B. sh-Rictor 60 sh-mTOR sh-Raptor 50
40
30
20
(Id-1 promoter/cmv-renilla) (Id-1 10 Relative Luciferase Activity Luciferase Relative
0 Rapamycin (200 nM) -+-+ -+-+ -+-+ -+-+ BMP4 (5 ng/ml) --++ --++ --++ --++
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Figure 3.15. Rapamycin and everolimus (RAD001) enhances phospho-Smad1/5/8 expression in vivo in PC3 Xenografts in tumors of patients with newly diagnosed localized prostate cancer.
A, Expression of p-Smad1/5/8, p-S6 and survivin were assessed by IHC of PC3 tumor xenografts implanted in (6-7 week old ) Ncr:NU athymic male mice that received either
vehicle control (n=5), or rapamycin treatment for 48 h (n=5) or 6 day (n=5) as described
in methods (left); staining results were quantified by measuring H-score (% positive
stained cells x staining intensity (0-3)) of matched sections (right). B, Expression of p-
Smad1/5/8 and p-S6 (by IHC) in prostate tumor sections from patients on a phase II
clinical trial who were treated with everolimus (5 mg or 10 mg/day for 8 weeks) as neo-
adjuvant therapy in patients with high-risk prostate cancer (n=6), and compared to non-
treated control patients with prostate adenocarcinoma stage II-III (n=22) (left); H-score
(% positive stained cells x staining intensity (0-3)) of non-treated control matched cores
were compared to everolimus (RAD001) clinical trial matched sections (right).
177
(Figure 3.15)
178
Figure 3.16. Smad1/5/8 expression is enhanced in RAD001 treatment compared to non treated controls in vivo.
A, Immunostaining of normal prostate gland and prostate adenocarcinoma (stage III) stained with H&E, phospho-Smad1/5/8, PTEN, or phospho-S6. B, Randomized phase II study of two different does of RAD001 (Everolimus) (5mg or 10 mg) as neo-adjuvant
therapy in patients with localized prostate cancer radical prostatectomy matched prostate
sections (n=3) were stained with phospho-Smad1/5/8 or phospho-S6 and analyzed at
200x.
179
(Figure 3.16)
180
Figure 3.17. Smad1/5/8 and Phospho-S6 expression in variable stages of human prostate cancer progression in vivo.
A, Immunohistochemistry of normal prostate hyperplasia, Stage II, Stage III, or Stave IV prostate cancer section from PR8011 microtissue array. Relative H-score for each stage is plotted and examined by three independent investigators.
181
(Figure 3.17)
182
Chapter 4 Summary, Discussion and Future Directions
4.1 Summary Bone Morphogenetic Proteins (BMPs) are pleiotropic cytokines that play integral roles in
embryogenesis, osteogenesis, organogenesis. BMPs belong to the transforming growth
factor-β (TGF-β) superfamily and are reported to function as a tumor suppressor in early
preneoplastic lesions of the prostate and a tumor promoter in late stage prostate
adenocarcinoma. Moreover, BMPs have been recently shown to be pivotal in controlling
prostate tumorigenesis, and loss of BMP receptor function has been correlated to a higher
Gleason grade in prostate cancer patients. During advanced prostate cancer, reports have
shown that the IGF-I/PI3K/Akt/mTOR pathway is hyperactive in 50% of patients
examined. In this light, we provide evidence that support that the IGF-I signaling axis
inhibits BMP4-induced apoptosis, Smad-mediated gene expression, and BMP specific
downstream targets. Suppression of the BMP4 signaling by IGF-I was reversed by direct
genetic manipulation using enforced expression of wt-PTEN or DN-PI3K, use of
chemical inhibitors against PI3K/Akt/mTOR, or small hairpin RNA-mediated silencing
of mTORC1/2 subunits Raptor or Rictor. Our results provided evidence that IGF-I
blocked BMP-induced transcription of Id1, Id2, and Id3 all downstream targets of BMP
through a PI3K/Akt/mTOR dependant mechanism. Using various rat and human prostate
epithelial cell lines as well as human prostate pathological specimens, we provide the first
evidence that mTOR mediates inhibition of BMP-induced Smad1/5/8 activation.
Deregulation of mTOR-mediated inhibition of BMP signaling pathway may be crucial to
halting progression of prostate cancer formation. Furthermore, we report that direct
inhibition of mTOR by rapamycin or rapalogs are able to enhance BMP-mediated Smad1
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and Smad5 activation in human prostate cancer cell lines and tissue. Utilization of
lentivirus-based silencing and retroviral-based overexpression enabled us to show that
Smad1 and Smad5 mediate rapamycin-induced cell death and activation of BMP target
gene Id1. On the other hand, we showed that Smad8 represses rapamycin’s action in
human prostate cell lines. All in all, the studies described here provide novel
implications in BMP and mTOR signaling axis in prostate cancer, and may possibly
provide new potential targets for the therapeutic intervention of this malignancy.
Chapter 2: IGF-I suppresses BMP4-induced cell death through a PI3K/Akt/mTOR dependent mechanism. In chapter 2 of this dissertation, I was able to successfully generate constructs
aimed at understanding BMP-mediated pathway in prostate epithelial cell lines. My project focused on the investigation BMPs and their function in prostate cancer
development. I began addressing this question by designing Flag-tagged-Smad1, -
Smad5, -Smad8, -BMPRII, -ACTRII, -MISRII constructs that provided me with the tools
to address my hypothesis (Fig. 4-1, Fig. 4-2). Further, I examined the literature and
identified a useful luciferase-promoter that contains a number of BMP-response elements
(Id luciferase promoter) that would aid our understanding of BMP-mediated
transcriptional regulation of specific target genes [81]. With the aid of fellow graduate
student (Dorjee Shola) and my advisor (Dr. Danielpour) we designed an Id1-luciferase promoter and tested the response in a number of prostate epithelial cells lines. In conjunction with generating these tools, I also helped build our Immunohistochemistry
(IHC) platform that we would use to analyze our in vivo mouse xenographs or human tissue sections that were embedded in paraffin. Last, we used NRP-152 cells as an
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integral model system to understand BMP-mediated signaling in “normal” prostate
epithelium. NRP-152 cells were derived from the preneoplastic region of the prostate of
Lobund-Wistar rat, which was developed and characterized[146] for their unique sensitivity to TGF-β-mediated cell death (Fig. 2.1). Use of NRP-152 cells along with alternate prostate epithelial cell lines aided in completing the first aim of this dissertation.
In chapter 2, we reported that IGF-I intercepts BMP responses by suppressing the
C-terminal phosphorylation of Smad1/5/8, Id1 transcriptional regulation, and apoptosis in prostate epithelial cell lines (Fig. 2.3, 2.7, 2.9). Further, using chemical inhibitors and genetic modulation of the PI3K/Akt/mTOR pathway, or shRNA-mediated silencing of mTORC1 or mTORC2 complex (mTOR, rictor, and raptor), suppression of the BMP pathway by IGF-I was relieved (Fig. 2.10 – 2.13). Last, immunohistochemical analysis of normal human prostate or tumors from stage II- stage IV prostate cancer provided the first in vivo evidence that IGF-I signaling pathway blocks BMP-mediated Smad1/5/8 expression (Fig. 2.14). Completion of aim provided the first in vitro and in vivo evidence that IGF-I signaling suppresses BMP-mediated tumor suppressive responses, and repression of IGF-I signaling pathway with chemical inhibitors against PI3K/Akt/mTOR will help us restore BMP-mediated tumor suppression and aid in the therapeutic intervention of prostate cancer.
Chapter 3: Rapamycin (an mTOR inhibitor) reverses mTOR-mediated inhibition of BMP signaling, thus leading to enhanced BMP-mediated activation of Smad(s) 1/5/8, Id1 transcriptional regulation, and cell death in vitro and in vivo In chapter 3, I show direct inhibition of mTOR by rapamycin and rapamycin analogs enhanced activation of Smad1 and Smad5 in human prostate cancer cells and
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human prostate tissue sections. Our previous findings in chapter 2 provided the first
evidence that IGF-I suppressed BMP-mediated Smad1/5/8 activation. Due to antibody
specificity, we could not determine which Smad was involved in this inhibition. Using
lentivirus based silencing strategy, I successfully silenced endogenous Smad1, Smad5,
Smad1/5, and Smad8 with an efficiency of >95% and generated stable cell lines using
PC3 and LNCaP human prostate epithelial cells (Fig. 3.6). Further, a fellow graduate
student (Dorjee Shola) and I also designed and characterized retroviral-based
overexpression of Smad1, Smad5, and Smad8 to demonstrate that rapamycin enhanced
cell death in Smad1 and Smad5 stable cell lines, whereas Smad8 blocked rapamycin-
induced apoptosis and Id1 transcriptional activation (Fig. 3.9). We were the first to
demonstrate that Smad1 and Smad5 mediate rapamycin responses in prostate epithelial
cell lines. Moreover, blocking BMP-type I receptor kinase inhibitor (LDN-193189)
abrogated rapamycin-mediated actions. In addition, I showed rapamycin-mediated activation of Smad1 and Smad5 was enhanced by silencing mTOR, rictor, or raptor, whereas Smad8 was blocked (Fig. 3.14). Interestingly, IHC analysis was performed on human tissue sections from radical prostectomy biopsy sections from a phase II clinical trial in which patients with newly diagnosed clinically localized prostate cancer were administered the rapalog, RAD001 (5 or 10 mg/daily by p.o.), continuously for 8 weeks before undergoing radical prostatectomy. Human tissue sections were evaluated for phospho-Smad1/5/8 and phospho-S6 via IHC. Tissue samples from patients treated with
RAD001 suppressed phospho-S6 expression and showed enhanced phospho-Smad1/5/8 expression (Fig. 3.15, 3.16). This data was further supported by our PC3 human xenograft study, in which mice were administered rapamycin (i.p., 5 mg/kg/day for 2 to 6
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days) versus vehicle control. The treated group showed enhanced phospho-Smad1/5/8 staining relative to reduced phospho-S6 and survivin expression (Fig. 3.15). All in all, our data directly implicates the pivotal roles Smad1, Smad5 as prognostic markers and therapeutic target for rapamycin-mediated neoadjuvant based therapy of prostate cancer.
4.2 Discussion and Future Directions for Chapter 2
4.2.1 BMPRI, BMPRII, Smad1, Smad5, and Smad8-mediated interactions with mTOR, Rictor and Raptor in LNCaP cells. BMPs and their receptors play a pivotal role in bone formation, osteogenesis, morphogenesis, and development, and have been directly implicated in prostate cancer, specifically advanced stage bone metastasis [68, 73, 165, 212-214]. Much of our understanding of BMP has been centered on BMP4; however, we have not examined alternate BMP ligands such as BMP2, BMP6, BMP7, and BMP9 that also have been implicated in prostate cancer. Collectively, BMPs and their receptors play important role during the development of prostate cancer, but we have not defined the exact mechanisms of IGF-I mediated inhibition of BMP signaling. In order to clearly define the molecular interaction between the IGF-I signaling pathway and BMP mediated signaling, we could examine the physical interaction of the mTORC1 and mTORC2 complexes with
BMPRIA/B, BMPRII, Smad1, Smad5, and/or Smad8. To test this, we would perform coimmunoprecipitation (Co-IP) of mTORC1/2 complex with BMPRI, BMPRII, and
Smad1/5/or 8 proteins. In our lab, under the aid of Dr. Kyung Song, we are testing this hypothesis, and studies are ongoing. Once we determine whether components of mTORC1/2 interact with BMPRI, BMPRII, or Smads1/5/8, then this information would further enhance our understanding of the direct mechanism of IGF-I mediated inhibition
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of the BMP pathway. One possibility based on data from Fig. 3.6-3.8 is that mTOR will
specifically bind to Smad1, Smad5 but not Smad8, thus suppressing BMP-mediated
phosphorylation of Smads and blocking downstream Id1 transcriptional regulation and
apoptosis. In order to examine this hypothesis, we would use Smad1, Smad5 and Smad8
truncated proteins that were generated by a graduate student in our lab (Dorjee Shola) to
define the exact region where mTOR binds. Last, I designed a BMPRII (Δ1-172aa)
truncated receptor (LNCaP-pLPCX-BMPRII(Δ1-172aa)), where we were able to inhibit
BMP mediated Smad 1/5/8 activation, Id1 transcription and BMP-induced apoptosis (Fig.
4.3a-c). We can use the cell line that stably over-expresses BMPRII truncated receptor to define whether direct inhibition of BMPRII further enhances mTOR-mediated signaling
(phospho-S6), phospho-rictor, or phospho-raptor level of expression as analyzed by
Western blot analysis.
4.2.2 BMP4-mediated apoptosis and survivin in prostate epithelial cell lines. We reported that BMP4 induce apoptosis in a time and dose dependent manner in
NRP-152, DP-153 (rat prostate epithelial cells) and PC3, LNCaP, and DU-145 (human prostate epithelial cells [189]. Although the direct mechanism of BMP-mediated apoptosis is still poorly understood, we have preliminary evidence that support BMP4 inhibits survivin expression, a member of the inhibitor of apoptosis protein family.
Based on our data from chapter 3, we further postulate that Smad1 or Smad5 may repress survivin activity. By using our shRNA stably silenced LNCaP-shLacZ, LNCaP-shSmad1 or LNCaP-shSmad5 cell lines we can examine whether blocking Smad1 or Smad5 reverse the ability of BMP4 to suppress survivin expression by Western blot analysis.
Our lab previously reported that TGF-β downregulates survivin expression in prostate
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epithelial cells through Rb/E2F4-mediated mechanism involving Smad2 and Smad3
[162]. Using the methods described in this paper, we can also determine whether BMP4
down-regulates survivin expression by treating LNCaP or PC3 cells with BMP4 (5
ng/mL) for 0-48h time course and examine p-survivin expression by Western blot
analysis. In addition, we can also examine whether BMP-suppresses survivin mRNA expression using RT-PCR and RTq-PCR. Using a survivin (Suv-829) or empty vector control, we can transfect these constructs into LNCaP cells to test whether BMP4-inhibits
survivin transcriptional activation. Last, we can also use survivin promoter deletion constructs (generated by a graduate student in the lab Jaiyi Yang), to directly map which region of the survivin promoter is involved in BMP-mediated repression. An alternate method would be to overexpress survivin in LNCaP cells to test whether overexpression of survivin would block phospho-Smad activity. I predict that overexpression of
Survivin should abrogate Smad 1/5/8 activation. All in all, these experiments will aid our understanding of BMP-mediated cell death and how pro-survival pathways may alter or inhibit BMP-mediated tumor suppression in prostate epithelial cells.
4.2.3 IGF-I and BMP-induced apoptosis in prostate epithelial cells. Due to the limited data on BMP-mediated apoptosis in prostate cancer
development, further clarification on this topic would aid our efforts in restoring BMPs
tumor suppressive function in prostate epithelial cells. Our microarray analysis from
Chapter 2 provided the first evidence that BMP-induced GADD45 expression was
repressed in the presence of IGF-I (Fig. 2.14A). GADD45 proteins are well known for
their roles as a pro-apoptotic protein, stress signaling sensor, cell cycle arrest, and DNA
repair [215-219]. Reports have shown Smad1/Runx2 activation upregulates the
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expression of GADD45β, which is found in nuclei of chondrocytes [215, 216]. By
blocking GADD45 using a lentiviral based silencing strategy, would BMP-mediated
apoptosis be suppressed? Successful repression of GADD45 expression may possibly
suppress BMP-mediated apoptosis, thereby implicating GADD45 as a positive modulator
of BMP-induced apoptosis in prostate epithelial cells. Similarly, by designing GADD45
overexpression construct, can we accelerate BMP-mediated apoptosis in prostate
epithelial cells? All in all, this data will provide us with clear direction of BMP-mediate
apoptosis in prostate epithelial cells.
4.3 Future Directions for Chapter 3
4.3.1 FKBP12-mediated inhibition of BMP signaling. Rapamycin, an mTOR inhibitor, exerts its effects by binding to FK506-binding
protein 12 (FKBP12) directly upstream from mTOR kinase domain. Rapamycin has
been documented to promote cytostatic growth arrest in prostate cancer cell lines (Fig.
3.1), and enhance phospho-Smad1/5/8 activation in human prostate cancer tissue sections
(Fig. 3.2). Although the underlying mechanism of such action is still unclear, I speculate
FKBP12 may play a role in blocking Smad1 and Smad5 activation in prostate epithelial
cell lines and human tissue sections. Previous reports show FKBP12 to be negative regulators of human type I TGF-β receptor (TβRI) as shown in the crystal structure [220].
Using the resolved crystal structure as a template, alternate groups identified 68% sequence identity with BMPRI-FKBP12 model, and Q249 residue on the GS domain and
L45 loop (C-terminus) of BMPRIB makes contact with the FKPB12 [54]. Further, in some cancers, the Q249 residue contains a mutation (Q249R) which is speculated to enhance interaction between BMPRIB and FKBP12, leading to a stronger inhibition of
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receptor activity. Although not much data has been generated to show BMPRI and
FKBP12 interactions, BMPRIA receptor was able to bind to FKBP12, and this binding
was suppressed by rapamycin [221]. To date, we have never confirmed whether
overexpressed BMPRIA, BMPRIB or BMPRII are able to bind to FKBP12 via
coimmunoprecipitation experiments in HEK293 cell. Much of our understanding of
mTORC1-mediated inhibition of BMP-signaling pathway has been based on our study in
chapter 2 where we silenced mTOR, rictor and raptor which relieved mTOR-mediated
inhibition of BMP-Smad1/5/8 expression (Western blot) (Fig. 2.12b&c) and Id1 promoter
activity (luciferase assay) (Fig. 2.12D). As proposed in 4.2.1, we would want to overexpress mTOR, rictor, raptor and test whether components of mTORC1/2 complex directly bind to BMPRI, BMPRII, or Smads 1, 5, 8. Our data shows that components of mTORC1/2 can block BMP signaling, and rapamycin can attenuate this inhibition. We speculate that both mTORC1/2 components may directly inhibit Smad1, Smad5, and
Smad8 phosphorylation, and FKBP12-mediates inhibition of BMPRIA and BMPRIB. In
co-IP experiments we will explore whether FKBP12-BMPRIA/B or FKBP12-BMPRII
protein interactions are altered with rapamycin. This information will provide us with
two possible modes of inhibition of BMP pathway: one through mTORC1/2 complex
which inhibits Smad1/5/8 interactions, and the other is through FKBP12-mediated
inhibition of the BMPRI or BMPRII.
4.3.2 Androgen Receptor and BMP-mediated Smad1/5/8 activation and Id1 promoter activation. Prostate cancer progresses from a state of androgen-dependence toward that of hormone-independent state [222]. Although the direct mechanism of this transition is not
191
well understood, a number of models suggest that androgen receptor (AR) is activated in the absence of ligand in androgen refractory prostate cancer [223, 224]. Further, Erk has been shown to modify Smad1 through phosphorylation of the middle linker region, which then allows the modified Smad1 to bind to AR [89]. Once Smad1 is bound to androgen- activated androgen receptor, BMP represses growth and gene transcription of androgen- sensitive prostate cancer cells. Recent reports show AR enhances mTOR activity in prostate epithelial cells exposed to dihydrotestosterone (DHT) [225]. Similarly, direct inhibition of mTOR resulted in enhanced AR activity [173, 174]. Based on these findings, we speculate that using anti-androgen therapy (casodex) in conjunction with mTOR inhibitors (rapalogs) would enhance Smad1 and Smad5 activation in prostate epithelial cells and restore BMP-mediated inhibition of prostate cancer growth, as shown in chapter 3. Using shRNA lentiviral based silencing strategy or chemical inhibitors against mTOR and AR could possibly reverse suppression of BMP-mediated Smad1/5/8 activation.
Previous data from our lab shows that AR can directly bind to GST-Smad1 and
GST-Smad3 and not GST-control fusion proteins [91]. Although little is known regarding whether Smad5 and/or Smad8 can directly bind to AR, further studies will be performed to address this question. In addition, we want to examine whether androgen- bound AR can inhibit transcriptional activation of BMP response elements Id1. To test this we would transfect LNCaP cells with Id1 luciferase promoter as previously described with Smad1, Smad5, Smad8, or empty vector control then treat the transfected cells ±
DHT in a dose dependent manner. We predict that Smads 1, 5, and 8 would activate the
Id1 promoter, whereas DHT would inhibit Id1 transcriptional activation in a dose- 192
dependent manner. Smad8 may or may not respond to AR-mediated inhibition (based on
our data from chapter 3). Further, blocking both AR and mTOR mediated inhibition of
BMP signaling pathway will help us restore BMP tumor suppressive effects which will
halt growth of prostate epithelial cells.
4.3.3 Smad1, Smad5, and/or Smad8 as prognostic markers for prostate cancer. During the past 20 years, limited strides have been made in the development of
new detection methods to diagnose prostate cancer. Other than prostate-specific antigen
(PSA) screening, there has been significant interest in developing new molecular-based
prognostic factors that can be used identify benign prostate hyperplasia (BPH) from advanced hormone-refractory malignant prostate cancer cases. Current preclinical procedures have adapted new surrogate markers in their staging of prostate cancer malignancy, potential molecular markers include P70S6K, Akt, cyclinD1, p27, KI67,
PTEN and caspase-3 [139, 142, 226-229]. In addition to these prognostic markers, we propose that Smad1, Smad5, Smad8 and survivin to be used as potential surrogate makers based on our data that show high levels of Smad1 and Smad5 are responsive to rapamycin-based therapy, where expression of Smad8 would not be responsive to treatment modality. To date, we have not fully screened staged prostate cancer tissue to evaluate the expression of Smad1, Smad5, Smad8, and survivin in human prostate tissue sections by IHC. I would perform IHC on staged biopsy section for expression of both total and phospho-Smad1, Smad5, Smad8 and Survivin to determine whether there is a correlation between aggressiveness of tumor stage against expression of these potential surrogate markers. Strategies aimed at detecting expression levels of phospho-Smad1 or phospho-Smad5 has been difficult to monitor in vivo due to lack of specific antibodies
193
that would aid our therapeutic strategy in using mTOR inhibitors. Before proceeding, we would need to generate highly-specific antibody for phospho-Smad1, -Smad5, and –
Smad8. In addition to the proposed surrogate markers above, additional markers could be used such as IRS-1, raptor, rictor, PRAS40, 4EBP-1, TROC2, Akt activity, measurements of proliferation and apoptosis would aid our efforts in determining favorable effects of mTOR inhibitors [116, 199, 230, 231].
194
FIGURES
Figure 4.1. Smad1, Smad5, and Smad8-Flag tagged constructs in pDC516 vector plasmid.
Smad1, Smad5, and Smad8 were inserted into a Flag-taged-pDC516 vector plasmid.
195
(Figure 4.1)
196
Figure 4.2. BMPRIIA (iso1 and iso2), ActRIIB, MISRII-Flag tagged constructs.
BMPRII, ActRIIB, and MISRII were inserted into Flag-tagged pCDNA3 vector.
197
(Figure 4.2)
198
Figure 4.3. Overexpression of BMPRII (Δ1-172aa) blocks BMP-induced Id1 promoter activation and apoptosis in NRP-152 cells.
A, NRP-152-pLPCX empty vector control or NRP-152-pLPCX-BMPRII(Δ1-172aa) stably overexpressing cells were transfected with Id1-luciferase promoter 24 h prior to treating cells with 200 nM rapamycin 2 h prior to ±BMP4 (5 ng/mL) and examining Id1
luciferase activity 24 h later. B, NRP-152-pLPCX empty vector control or NRP-152-
pLPCX-BMPRII(Δ1-172aa) stably overexpressing cells were treated with 200 nM
rapamycin 2 h prior to ±BMP4 (5 ng/mL) and examining total adherent cells 72 hr later
by Coulter counter. C, NRP-152-pLPCX empty vector control or NRP-152-pLPCX-
BMPRII(Δ1-172aa) stably overexpressing cells were treated as described in B and
apoptosis was measured 72 h later by Hoechst staining.
199
(Figure 4.3)
A. B. NRP-152-pLPCX-EV Control 150 7.5 Vehicle
NRP-152-pLPCx-BMPRII(Δ1-172aa) ) 4 100 Rapamycin
50 5.0
20
2.5 10 Total Adherent Cells (x10 (Id-1 promoter/cmv-renilla) (Id-1 Relative Luciferase Activity Luciferase Relative 0.0 0 Rapamycin (200 nM) -+-+ -+-+ Rapamycin (200 nM) - + - + -+- + BMP4 (5 ng/ml) --++ --++ BMP4 (5 ng/ml) - - ++- + - + NRP-152-pLPCX-Control ++++ - - -- NRP-152-pLPCX-BMPRII ----++++ (Δ1-172aa)
100 C. Vehicle Rapamycin 75
50 %Apoptosis 25
0 Rapamycin (200 nM) - + - + -+- + BMP4 (5 ng/ml) - - ++- + - + NRP-152-pLPCX-Control ++++ - - -- NRP-152-pLPCX-BMPRII ----++++ (Δ1-172aa)
200
Figure 4.4. BMP-inhibits survivin in PC3 cells.
PC3 cells were treated ±200 nM rapamcyin 2 h prior to BMP4 (5 ng/ml) treatment for an additional 46 h, where expression of survivin, p-Smad1/3/5/8 and total Smads, BMPRII is examined by Western blot.
201
(Figure 4.4)
202
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