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Metastatic prostate cancer (PCa) in bone induces bone-forming lesions that enhance PCa progression. How tumor-induced bone formation enhances PCa progression is not known. We have previously shown that PCa-induced bone originates from endothelial cells (ECs) that have undergone endothelial-to-osteoblast (EC-to-OSB) transition by tumor-secreted bone morphogenetic protein 4 (BMP4). Here, we show that EC-to-OSB transition leads to changes in the tumor microenvironment that increases the metastatic potential of PCa cells. We found that conditioned medium (CM) from EC-OSB hybrid cells increases the migration, invasion, and survival of PC3-mm2 and C4-2B4 PCa cells. Quantitative mass spectrometry (Isobaric Tags for Relative and Absolute Quantitation) identified Tenascin C (TNC) as one of the major proteins secreted from EC-OSB hybrid cells. TNC expression in tumor-induced OSBs was confirmed by immunohistochemistry of MDA PCa-118b xenograft and human bone metastasis specimens. Mechanistically, BMP4 increases TNC expression in EC-OSB cells through the Smad1-Notch/Hey1 pathway. How TNC promotes PCa metastasis was next interrogated by in vitro and in vivo studies. In vitro studies showed that a TNC-neutralizing antibody inhibits EC-OSB-CM-mediated PCa cell migration and survival. TNC knockdown decreased, while the addition of recombinant TNC or TNC overexpression increased migration and anchorage-independent growth of PC3 or C4-2b cells. When injected orthotopically, PC3-mm2-shTNC clones decreased metastasis to bone, while C4-2b-TNC-overexpressing cells increased metastasis to lymph nodes. TNC enhances PCa cell migration through α5β1 integrin-mediated YAP/TAZ inhibition. These studies elucidate that tumor-induced stromal reprogramming generates TNC that enhances PCa metastasis and suggest that TNC may be a target for PCa therapy.

Yes-associated protein 1 (YAP1), a key player in the Hippo pathway, has been shown to play a critical role in tumor progression. However, the role of YAP1 in prostate cancer cell invasion, migration, and metastasis is not well defined. Through functional, transcriptomic, epigenomic, and proteomic analyses, we showed that prolyl hydroxylation of YAP1 plays a critical role in the suppression of cell migration, invasion, and metastasis in prostate cancer. Knockdown (KD) or knockout (KO) of YAP1 led to an increase in cell migration, invasion, and metastasis in prostate cancer cells. Microarray analysis showed that the EMT pathway was activated in Yap1-KD cells. ChIP-seq analysis showed that YAP1 target genes are enriched in pathways regulating cell migration. Mass spectrometry analysis identified P4H prolyl hydroxylase in the YAP1 complex and YAP1 was hydroxylated at multiple proline residues. Proline-to-alanine mutations of YAP1 isoform 3 identified proline 174 as a critical residue, and its hydroxylation suppressed cell migration, invasion, and metastasis. KO of P4ha2 led to an increase in cell migration and invasion, which was reversed upon Yap1 KD. Our study identified a novel regulatory mechanism of YAP1 by which P4HA2-dependent prolyl hydroxylation of YAP1 determines its transcriptional activities and its function in prostate cancer metastasis.

Purpose: Radium-223 prolongs survival in a fraction of men with bone metastatic prostate cancer (PCa). However, there are no markers for monitoring response and resistance to Radium-223 treatment. Exosomes are mediators of intercellular communication and may reflect response of the bone microenvironment to Radium-223 treatment. We performed molecular profiling of exosomes and compared the molecular profile in patients with favorable and unfavorable overall survival. Experimental Design: We performed exosomal transcriptome analysis in plasma derived from our preclinical models (MDA-PCa 118b tumors, TRAMP-C2/BMP4 PCa) and from the plasma of 25 patients (paired baseline and end of treatment) treated with Radium-223. All samples were run in duplicate, and array data analyzed with fold changes +2 to −2 and P < 0.05. Results: We utilized the preclinical models to establish that genes derived from the tumor and the tumor-associated bone microenvironment (bTME) are differentially enriched in plasma exosomes upon Radium-223 treatment. The mouse transcriptome analysis revealed changes in bone-related and DNA damage repair–related pathways. Similar findings were observed in plasma-derived exosomes from patients treated with Radium-223 detected changes. In addition, exosomal transcripts detected immune-suppressors (e.g., PD-L1) that were associated with shorter survival to Radium-223. Treatment of the Myc-CaP mouse model with a combination of Radium-223 and immune checkpoint therapy (ICT) resulted in greater efficacy than monotherapy. Conclusions: These clinical and coclinical analyses showed that RNA profiling of plasma exosomes may be used for monitoring the bTME in response to treatment and that ICT may be used to increase the efficacy of Radium-223.

Renal cell carcinoma bone metastases (RCCBM) are typically osteolytic. We previously showed that BIGH3 (beta Ig-h3/TGFBI), secreted by 786-O renal cell carcinoma, plays a role in osteolytic bone lesion in RCCBM through inhibition of osteoblast (OSB) differentiation. To study this interaction, we employed three-dimensional (3D) hydrogels to coculture bone-derived 786-O (Bo-786) renal cell carcinoma cells with MC3T3-E1 pre-OSBs. Culturing pre-OSBs in the 3D hydrogels preserved their ability to differentiate into mature OSB; however, this process was decreased when pre-OSBs were cocultured with Bo-786 cells. Knockdown of BIGH3 in Bo-786 cells recovered OSB differentiation. Furthermore, treatment with bone morphogenetic protein 4, which stimulates OSB differentiation, or cabozantinib (CBZ), which inhibits VEGFR1 and MET tyrosine kinase activities, also increased OSB differentiation in the coculture. CBZ also inhibited pre-osteoclast RAW264.7 cell differentiation. Using RCCBM mouse models, we showed that CBZ inhibited Bo-786 tumor growth in bone. CBZ treatment also increased bone volume and OSB number, and decreased osteoclast number and blood vessel density. When tested in SN12PM6 renal cell carcinoma cells that have been transduced to overexpress BIGH3, CBZ also inhibited SN12PM6 tumor growth in bone. These observations suggest that enhancing OSB differentiation could be one of the therapeutic strategies for treating RCCBM that exhibit OSB inhibition characteristics, and that this 3D coculture system is an effective tool for screening osteoanabolic agents for further in vivo studies.

Purpose: Cabozantinib, an oral inhibitor of c-MET/VEGFR2 signaling, improved progression-free survival (mPFS) but not overall survival (OS) in metastatic castrate-resistant prostate cancer. We evaluated cabozantinib plus androgen deprivation therapy (ADT) in hormone-naïve metastatic prostate cancer (HNMPCa). Patients and Methods: Patients received ADT plus cabozantinib starting at 60 mg daily. The primary endpoint was castrate-resistant PFS by radiographic criteria, clinical progression, or receipt of additional therapy. Secondary endpoints included OS, safety, radiographic responses, and biomarker modulation. Results: Sixty-two patients received treatment. With a median follow-up of 31.2 months, the mPFS was 16.1 months (95% CI, 14.6–22.7 months), and mOS was not reached. Reductions in PSA ≥ 90%, bone-specific alkaline phosphatase ≥ 50%, and urine N-telopeptides ≥ 50% occurred in 83%, 87%, and 86% of evaluable patients, respectively. Responses in bone scan and measurable disease were observed in 81% of and 90% of evaluable patients, respectively. Most common grade 3 adverse events were hypertension (19%), diarrhea (6%), and thromboembolic events (6%), and dose reductions occurred in 85% of patients. Analysis of baseline cytokine and angiogenic factors (CAFs) revealed that higher plasma concentrations of Lumican, CXCL5, CD25, and CD30 were associated with shorter PFS as was high tumor expression of pFGFR1. Conclusions: Cabozantinib plus ADT has promising clinical activity in HNMPCa. CAF profiles and tissue markers suggest candidate prognostic and predictive markers of cabozantinib benefit and provide insights for rational therapy combinations.

Purpose: To determine the expression of glucocorticoid receptor (GR) and androgen receptor (AR) in salivary duct carcinoma (SDC) and to analyze the role of these proteins in the development and management of this disease entity. Experimental Design: We performed a phenotypic assessment of GR and AR localization and expression, and determined their association with clinicopathologic factors in 67 primary SDCs. In vitro functional and response analysis of SDC cell lines was also performed. Results: Of the 67 primary tumors, 12 (18%) overexpressed GR protein, 30 (45%) had constitutive expression, and 25 (37%) had complete loss of expression. Reciprocal GR and AR expression was found in 32 (48%) tumors, concurrent constitutive GR and AR expression in 23 (34%), and simultaneous loss of both receptors and high GR with AR expressions were found in 12 (18%). GR overexpression was significantly associated with worse clinical outcomes. In vitro ligand-independent AR activation was observed in both male- and female-derived cell lines. GR antagonist treatment resulted in decreased cell proliferation and survival in GR-overexpressing cells, irrespective of AR status. Reciprocal GR- and AR-knockdown experiments revealed an independent interaction. Conclusions: Our study, for the first time, demonstrates differential GR and AR expressions, autonomous GR and AR activation, and ligand-independent AR expression and activation in SDC cells. The findings provide critical information on the roles of GR and AR steroid receptors in SDC tumorigenesis and development of biomarkers to guide targeted steroid receptor therapy trials in patients with these tumors.

Disseminated tumor cells (DTCs) undergo a dormant state in the distant metastatic site(s) before becoming overt metastatic diseases. In prostate cancer (PCa), bone metastasis can occur years after prostatectomy, suggesting that bone may provide dormancy-inducing factors. To search for these factors, we prepared conditioned media (CM) from calvariae. Using live-cell imaging, we found that Calvarial-CM treatment increased cellular quiescence in C4-2B4 PCa cells. Mass spectrometry analysis of Calvarial-CM identified 132 secreted factors. Western blot and ELISA analyses confirmed the presence of several factors, including DKK3, BMP1, neogenin and vasorin in the Calvarial-CM. qRT-PCR analysis of total calvariae versus isolated osteoblasts showed that DKK3, BMP1, vasorin and neogenin are mainly expressed by osteoblasts, while MIA, LECT1, NGAL and PEDF are expressed by other calvarial cells. Recombinant human DKK3, BMP1, vasorin, neogenin, MIA and NGAL treatment increased cellular quiescence in both C4-2b and C4-2B4 PCa cells. Mechanistically, DKK3, vasorin and neogenin, but not BMP1, increased dormancy through activating the p38MAPK signaling pathway. Consistently, DKK3, vasorin and neogenin failed to induce dormancy in cells expressing dominant-negative p38αMAPK while BMP1 remained active, suggesting that BMP1 uses an alternative dormancy signaling pathway. Thus, bone secretes multiple dormancy-inducing factors that employ distinct signaling pathways to induce DTC dormancy in bone.

How disseminated tumour cells engage specific stromal components in distant organs for survival and outgrowth is a critical but poorly understood step of the metastatic cascade. Previous studies have demonstrated the importance of the epithelial–mesenchymal transition in promoting the cancer stem cell properties needed for metastasis initiation, whereas the reverse process of mesenchymal–epithelial transition is required for metastatic outgrowth. Here we report that this paradoxical requirement for the simultaneous induction of both mesenchymal–epithelial transition and cancer stem cell traits in disseminated tumour cells is provided by bone vascular niche E-selectin, whose direct binding to cancer cells promotes bone metastasis by inducing mesenchymal–epithelial transition and activating Wnt signalling. E-selectin binding activity mediated by the α1-3 fucosyltransferases Fut3/Fut6 and Glg1 are instrumental to the formation of bone metastasis. These findings provide unique insights into the functional role of E-selectin as a component of the vascular niche critical for metastatic colonization in bone.

Background Metastatic prostate cancer in bone is difficult to treat as the tumor cells are relatively resistant to hormonal or chemotherapies when compared to primary prostate cancer. Irreversible electroporation (IRE) is a minimally invasive ablation procedure that has potential applications in the management of prostate cancer in bone. However, a common limitation of IRE is tumor recurrence, which arises from incomplete ablation that allows remaining cancer cells to proliferate. In this study, we combined IRE with radium-223 (Ra-223), a bone-seeking radionuclide that emits short track length alpha particles and thus is associated with reduced damage to the bone marrow and evaluated the impact of the combination treatment on bone-forming prostate cancer tumors. Methods The antitumor activity of IRE and Ra-223 as single agents and in combination was tested in vitro against three bone morphogenetic protein 4 (BMP4)-expressing prostate cancer cell lines (C4-2B–BMP4, Myc-CaP–BMP4, and TRAMP-C2–BMP4). Similar evaluation was performed in vivo using a bone-forming C4-2B–BMP4 tumor model in nude mice. Results IRE and Ra-223 as monotherapy inhibited prostate cancer cell proliferation in vitro, and their combination resulted in significant reduction in cell viability compared to monotherapy. In vivo evaluation revealed that IRE with single-dose administration of Ra-233, compared to IRE alone, reduced the rate of tumor recurrence by 40% following initial apparent complete ablation and decreased the rate of proliferation of incompletely ablated tumor as quantified in Ki-67 staining (53.58 ± 16.0% for IRE vs. 20.12 ± 1.63%; for IRE plus Ra-223; p = 0.004). Histological analysis qualitatively showed the enhanced killing of tumor cells adjacent to bone by Ra-223 compared to those treated with IRE alone. Conclusion IRE in combination with Ra-223, which enhanced the destruction of cancer cells that are adjacent to bone, resulted in reduction of tumor recurrence through improved clearance of proliferative cells in the tumor region.

A fraction of patients undergoing androgen deprivation therapy (ADT) for advanced prostate cancer (PCa) will develop recurrent castrate-resistant PCa (CRPC) in bone. Strategies to prevent CRPC relapse in bone are lacking. Here we show that the cholesterol-lowering drugs statins decrease castration-induced bone marrow adiposity in the tumor microenvironment and reduce PCa progression in bone. Using primary bone marrow stromal cells (BMSC) and M2-10B4 cells, we showed that ADT increases bone marrow adiposity by enhancing BMSC-to-adipocyte transition in vitro. Knockdown of androgen receptor abrogated BMSC-to-adipocyte transition, suggesting an androgen receptor-dependent event. RNAseq analysis showed that androgens reduce the secretion of adipocyte hormones/cytokines including leptin during BMSC-to-adipocyte transition. Treatment of PCa C4-2b, C4-2B4, and PC3 cells with leptin led to an increase in cell cycle progression and nuclear Stat3. RNAseq analysis also showed that androgens inhibit cholesterol biosynthesis pathway, raising the possibility that inhibiting cholesterol biosynthesis may decrease BMSC-to-adipocyte transition. Indeed, statins decreased BMSC-to-adipocyte transition in vitro and castration-induced bone marrow adiposity in vivo. Statin pre-treatment reduced 22RV1 PCa progression in bone after ADT. Our findings with statin may provide one of the mechanisms to the clinical correlations that statin use in patients undergoing ADT seems to delay progression to "lethal" PCa.

Summary Cell type transition occurs during normal development and under pathological conditions. In prostate cancer bone metastasis, prostate cancer-secreted BMP4 induces endothelial cell-to-osteoblast (EC-to-OSB) transition. Such tumor-induced stromal reprogramming supports prostate cancer progression. We delineate signaling pathways mediating EC-to-OSB transition using EC lines 2H11 and SVR. We found that BMP4-activated pSmad1-Notch-Hey1 pathway inhibits EC migration and tube formation. BMP4-activated GSK3β-βcatenin-Slug pathway stimulates Osx expression. In addition, pSmad1-regulated Dlx2 converges with the Smad1 and β-catenin pathways to stimulate osteocalcin expression. By co-expressing Osx, Dlx2, Slug and Hey1, we were able to achieve EC-to-OSB transition, leading to bone matrix mineralization in the absence of BMP4. In human prostate cancer bone metastasis specimens and MDA-PCa-118b and C4-2b-BMP4 osteogenic xenografts, immunohistochemical analysis showed that β-catenin and pSmad1 are detected in activated osteoblasts rimming the tumor-induced bone. Our results elucidated the pathways and key molecules coordinating prostate cancer-induced stromal programming and provide potential targets for therapeutic intervention., Graphical abstract, Highlights • BMP4 upregulates several pathways essential for EC-to-OSB transition • BMP4 activates pSmad1-Notch-Hey1 and GSK3β-βcatenin-Slug-OSX pathways • pSmad1-regulated Dlx2 expression connects the pSmad1 and β-catenin pathways • Coexpression of OSX, Dlx2, Slug, and Hey1 is sufficient to induce EC-to-OSB transition, Molecular Biology ; Cell Biology ; Cancer

Background Patients with advanced primary and recurrent salivary duct carcinoma (SDC), a rare and lethal malignancy, have limited therapeutic options. Novel small-molecule agents aimed at targeting critical signaling associated with SDC tumorigenesis may lead to new therapeutic options for patients with these tumors. The human epidermal growth factor receptor 2 (HER2)/phosphoinositide 3-kinase (PI3K) axis, an important oncogenic pathway, has been targeted for therapy in several solid tumors. Currently, little is known about the role and clinical implications of alterations of the HER2/PI3K pathway in patients with SDC. Methods The authors investigated the clinicopathologic features, genetic alterations, and expression of key members of the HER2/PI3K pathway in 43 primary tumors and conducted in vitro functional and targeted drug-response analyses on cell lines derived from salivary epithelial carcinomas. Results In primary tumors, loss of phosphatase and tensin homolog (PTEN) expression was identified in 22 of 43 tumors (51%), overexpression of HER2 was observed in 12 of 43 tumors (28%), and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) mutations were identified in 12 of 43 tumors (28%). Phosphorylated protein kinase B (p-AKT) was highly expressed in most tumors. Most tumors (70%) displayed mutually exclusive alterations of PI3K members, whereas 8 tumors (19%) had 2 or more concurrent abnormalities. In vitro studies demonstrated a direct association between PTEN loss and PI3K pathway activation and evidence of response to combined PI3Kα and PI3Kβ and/or pan-PI3K inhibitors. Conclusions The current analyses reveal frequent PTEN loss and mutually exclusive alterations of key PI3K pathway members in SDC and demonstrate in vitro evidence of a response to pan-PI3K inhibitors. These results provide a framework for a biomarker-based substratification of patients with SDC in future targeted therapy. Cancer 2018;124:3523-32. © 2018 American Cancer Society.

Bone metastasis from prostate cancer can occur years after prostatectomy, due to reactivation of dormant disseminated tumor cells (DTC) in the bone, yet the mechanism by which DTCs are initially induced into a dormant state in the bone remains to be elucidated. We show here that the bone microenvironment confers dormancy to C4-2B4 prostate cancer cells, as they become dormant when injected into mouse femurs but not under the skin. Live-cell imaging of dormant cells at the single-cell level revealed that conditioned medium from differentiated, but not undifferentiated, osteoblasts induced C4-2B4 cellular quiescence, suggesting that differentiated osteoblasts present locally around the tumor cells in the bone conferred dormancy to prostate cancer cells. Gene array analyses identified GDF10 and TGFβ2 among osteoblast-secreted proteins that induced quiescence of C4-2B4, C4-2b, and PC3-mm2, but not 22RV1 or BPH-1 cells, indicating prostate cancer tumor cells differ in their dormancy response. TGFβ2 and GDF10 induced dormancy through TGFβRIII to activate phospho-p38MAPK, which phosphorylates retinoblastoma (RB) at the novel N-terminal S249/T252 sites to block prostate cancer cell proliferation. Consistently, expression of dominant-negative p38MAPK in C4-2b and C4-2B4 prostate cancer cell lines abolished tumor cell dormancy both in vitro and in vivo. Lower TGFβRIII expression in patients with prostate cancer correlated with increased metastatic potential and decreased survival rates. Together, our results identify a dormancy mechanism by which DTCs are induced into a dormant state through TGFβRIII–p38MAPK–pS249/pT252–RB signaling and offer a rationale for developing strategies to prevent prostate cancer recurrence in the bone. Significance: These findings provide mechanistic insights into the dormancy of metastatic prostate cancer in the bone and offer a rationale for developing strategies to prevent prostate cancer recurrence in the bone. Cancer Res; 78(11); 2911–24. ©2018 AACR.

Although emerging evidence suggests a potential role of calcium/calmodulin-dependent kinase II (CaMKII) in prostate cancer, its role in prostate cancer tumorigenesis is largely unknown. Here, we examine whether the acetyl CoA-CaMKII pathway, first described in frog oocytes, promotes prostate cancer tumorigenesis. In human prostate cancer specimens, metastatic prostate cancer expressed higher levels of active CaMKII compared with localized prostate cancer. Correspondingly, basal CaMKII activity was significantly higher in the more tumorigenic PC3 and PC3-mm2 cells relative to the less tumorigenic LNCaP and C4-2B4 cells. Deletion of CaMKII by CRISPR/Cas9 in PC3-mm2 cells abrogated cell survival under low-serum conditions, anchorage-independent growth and cell migration; overexpression of constitutively active CaMKII in C4-2B4 cells promoted these phenotypes. In an animal model of prostate cancer metastasis, genetic ablation of CaMKII reduced PC3-mm2 cell metastasis from the prostate to the lymph nodes. Knockdown of the acetyl-CoA transporter carnitine acetyltransferase abolished CaMKII activation, providing evidence that acetyl-CoA generated from organelles is a major activator of CaMKII. Genetic deletion of the β-oxidation rate-limiting enzyme ACOX family proteins decreased CaMKII activation, whereas overexpression of ACOXI increased CaMKII activation. Overall, our studies identify active CaMKII as a novel connection between organelle β-oxidation and acetyl-CoA transport with cell survival, migration, and prostate cancer metastasis. Significance: This study identifies a cell metabolic pathway that promotes prostate cancer metastasis and suggests prostate cancer may be susceptible to β-oxidation inhibitors. Cancer Res; 78(10); 2490–502. ©2018 AACR.

BACKGROUND : Bone metastasis is common in renal cell carcinoma (RCC), and the lesions are mainly osteolytic. The mechanism of bone destruction in RCC bone metastasis is unknown. METHODS : We used a direct intrafemur injection of mice with bone-derived 786-O RCC cells (Bo-786) as an in vivo model to study if inhibition of osteoblast differentiation is involved in osteolytic bone lesions in RCC bone metastasis. RESULTS : We showed that bone-derived Bo-786 cells induced osteolytic bone lesions in the femur of mice. We examined the effect of conditioned medium of Bo-786 cells (Bo-786 CM) on both primary mouse osteoblasts and MC3T3-E1 preosteoblasts and found that Bo-786 CM inhibited osteoblast differentiation. Secretome analysis of Bo-786 CM revealed that BIGH3 (Beta ig h3 protein), also known as TGFBI (transforming growth factor beta-induced protein), is highly expressed. We generated recombinant BIGH3 and found that BIGH3 inhibited osteoblast differentiation in vitro . In addition, CM from Bo-786 BIGH3 knockdown cells (786-BIGH3 KD) reduced the inhibition of osteoblast differentiation compared to CM from vector control. Intrafemural injection of mice with 786-BIGH3 KD cells showed a reduction in osteolytic bone lesions compared to vector control. Immunohistochemical staining of 18 bone metastasis specimens from human RCC showed strong BIGH3 expression in 11/18 (61%) and moderate BIGH3 expression in 7/18 (39%) of the specimens. CONCLUSIONS: These results suggest that suppression of osteoblast differentiation by BIGH3 is one of the mechanisms that enhance osteolytic lesions in RCC bone metastasis, and raise the possibilty that treatments that increase bone formation may improve therapy outcomes.

// Kai-Jie Yu 1, 5, 6, * , Jeffrey K. Li 1, * , Yu-Chen Lee 1, * , Guoyu Yu 1 , Song-Chang Lin 1 , Tianhong Pan 7 , Robert L. Satcher 7 , Mark A. Titus 2 , Li-Yuan Yu-Lee 4 , Wen Hui Weng 6 , Gary E. Gallick 2, 3 and Sue-Hwa Lin 1, 2, 3 1 Department of Translational Molecular Pathology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, USA 2 Department of Genitourinary Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, USA 3 The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, Texas, USA 4 Department of Medicine, Baylor College of Medicine, Houston, Texas, USA 5 Division of Urology, Department of Surgery, Chang Gung Memorial Hospital at Linkou, Chang Gung University College of Medicine, Taoyuan, Taiwan 6 Department of Chemical Engineering and Biotechnology and Graduate Institute of Biochemical and Biomedical Engineering, National Taipei University of Technology, Taipei, Taiwan 7 Department of Orthopedic Oncology, University of Texas MD Anderson Cancer Center, Houston, Texas, USA * Authors have contributed equally to this work Correspondence to: Sue-Hwa Lin, email: slin@mdanderson.org Keywords: cabozantinib, osteoblast, secretome, anchorage-independent growth, migration Abbreviations: CM: conditioned medium; PCa: prostate cancer; PMOs: primary mouse osteoblasts; real-time RT-PCR: reverse transcription followed with real-time polymerase chain reaction; ELISA: enzyme-linked immunosorbent assay Received: May 25, 2017 Accepted: July 14, 2017 Published: August 24, 2017 ABSTRACT Therapies that target cancer cells may have unexpected effects on the tumor microenvironment that affects therapy outcomes or render therapy resistance. Prostate cancer (PCa) bone metastasis is uniquely associated with osteoblastic bone lesions and treatment with cabozantinib, a VEGFR-2 and MET inhibitor, leads to a reduction in number and/or intensity of lesions on bone scans. However, resistance to cabozantinib therapy inevitably occurs. We examined the effect of cabozantinib on osteoblast differentiation and secretion in the context of therapy resistance. We showed that primary mouse osteoblasts express VEGFR2 and MET and cabozantinib treatment decreased osteoblast proliferation but enhanced their differentiation. A genome-wide analysis of transcriptional responses of osteoblasts to cabozantinib identified a set of genes accounting for inhibition of proliferation and stimulation of differentiation, and a spectrum of secreted proteins induced by cabozantinib, including pappalysin, IGFBP2, WNT 16, and DKK1. We determined that these proteins were upregulated in the conditioned medium of cabozantinib-treated osteoblasts (CBZ-CM) compared to control CM. Treatment of C4-2B4 or PC3-mm2 PCa cells with CBZ-CM increased the anchorage-independent growth and migration of these PCa cells compared to cells treated with control CM. These results suggest that the effect of cabozantinib on the tumor microenvironment may increase tumor cell survival and cause therapy resistance.

Osteoblasts communicate both with normal cells in the bone marrow, and with tumor cells that metastasized to bone. Here we show that osteoblasts release exosomes, we termed osteosomes, which may be a novel mechanism by which osteoblasts communicate with cells in their environment. We have isolated exosomes from undifferentiated/proliferating (D0 osteosomes) and differentiated/mineralizing (D24 osteosomes) primary mouse calvarial osteoblasts. The D0 and D24 osteosomes were found to be vesicles of 130–140 nm by dynamic light scattering analysis. Proteomics profiling using tandem mass spectrometry (LC-MS/MS) identified 206 proteins in D0 osteosomes and 336 in D24 osteosomes. The proteins in osteosomes are mainly derived from the cytoplasm (~47%) and plasma membrane (~31%). About 69% of proteins in osteosomes are also found in Vesiclepedia, and these canonical exosomal proteins include tetraspanins and Rab family proteins. We found that there are differences in both protein content and levels in exosomes isolated from undifferentiated and differentiated osteoblasts. Among the proteins that are unique to osteosomes, 169 proteins are present in both D0 and D24 osteosomes, 37 are unique to D0, and 167 are unique to D24. Among those 169 proteins present in both D0 and D24 osteosomes, 10 proteins are likely present at higher levels in D24 than D0 osteosomes, based on emPAI ratios of more than 5. These results suggest that osteosomes released from different cellular state of osteoblasts may mediate distinct functions. Using live-cell imaging, we measured the uptake of PKH26-labeled osteosomes into C4-2B4 and PC3-mm2 prostate cancer cells. In addition, we showed that cadherin-11, a cell adhesion molecule, plays a role in the uptake of osteosomes into PC3-mm2 cells as osteosome uptake was delayed by neutralizing antibody against cadherin-11. Together, our studies suggest that osteosomes could have a unique role in the bone microenvironment under both physiological and pathological conditions.

The overall goal of this study was to elucidate the role of FGFR1 induction in acquired resistance to MET and VEGFR2 inhibition by cabozantinib in prostate cancer (PCa) and leverage this understanding to improve therapy outcomes. The response to cabozantinib was examined in mice bearing patient-derived xenografts in which FGFR1 was overexpressed. Using a variety of cell models that reflect different PCa disease states, the mechanism underpinning FGFR1 signaling activation by cabozantinib was investigated. We performed parallel investigations in specimens from cabozantinib-treated patients to confirm our in vitro and in vivo data. FGFR1 overexpression was sufficient to confer resistance to cabozantinib. Our results demonstrate transcriptional activation of FGF/FGFR1 expression in cabozantinib-resistant models. Further analysis of molecular pathways identified a YAP/TBX5-driven mechanism of FGFR1 and FGF overexpression induced by MET inhibition. Importantly, knockdown of YAP and TBX5 led to decreased FGFR1 protein expression and decreased mRNA levels of FGFR1, FGF1, and FGF2. This association was confirmed in a cohort of hormone-naïve patients with PCa receiving androgen deprivation therapy and cabozantinib, further validating our findings. These findings reveal that the molecular basis of resistance to MET inhibition in PCa is FGFR1 activation through a YAP/TBX5-dependent mechanism. YAP and its downstream target TBX5 represent a crucial mediator in acquired resistance to MET inhibitors. Thus, our studies provide insight into the mechanism of acquired resistance and will guide future development of clinical trials with MET inhibitors.

Introduction: Metastatic prostate cancer (PCa) undergoes cycles of dormancy and reactivation, resulting in secondary metastasis, and leading to lethal disease. PCa preferentially metastasizes to bone where it can remain dormant for years after initial colonization. Previous work showed different growth and dormancy phenotypes in PCa tumors injected subcutaneously and intrafemorally, though the exact source of the dormancy-inducing factors remains undefined. We identified a novel receptor complex, the Perlecan-Semaphorin3A-PlexinA1-Neuropilin-1 (PSPN) complex, that is modulated by context-specific endothelial cell populations. The overall goal of this project is to identify the role of endothelial cells in stabilizing and silencing metastatic behavior via the PSPN complex, it's downstream pathways, and it's turnover by matrix metalloproteinase 7/Matrilysin (MMP-7). Methods: Live/dead assays coupled with immunolabeling and molecular approaches were used to determine the molecular impact of secreted endothelial cell factors on PCa signaling and behavior through the PSPN Complex in 2.5D and 3D microtumor cultures. Reverse phase protein arrays (RPPA) provided an unbiased means to assess the downstream molecular events in 3D co-cultures of PCa and microvascular cells. Results: Intact perlecan (P) induced clustering via engagement of PSPN complex. Destruction of complex components by MMP-7 activated growth and migration. FOXM1 was activated downstream of PSPN complex cleavage and resulting destabilization, consistent with escape from dormancy. Secreted factors from endothelial cells induced dormancy and reduced FOXM1 activation. Immunolocalization data showed internalization of plexin A1 with immunostaining both at the PCa cell surface and perinuclear, consistent with dynamic turnover associated with activation of signaling after loss of the PSPN complex. Conclusion: Intact PSPN complex at the PCa cell surface favors a dormant state that is reversed by MMP7 cleavage. In the cohesive state (PSPN complex intact) FOXM1 is “off”, inhibiting the transcription of growth and metastasis promoting factors. Context-specific endothelial cell populations secrete PSPN complex stabilizing proteins, silence downstream signaling, and result in a dormant phenotype. We propose that the PSPN complex at metastatic sites such as bone acts as a molecular toggle that can maintain dormancy until the switch is “flipped” by MMP-7 allowing reactivation, dyscohesion and favoring growth and metastasis. Citation Format: Tristen V. Tellman, Lindsey K. Sablatura, Lissette A. Cruz, Sue-Hwa Lin, Leland W. Chung, Mary C. Farach-Carson. The perlecan-semaphorin3A-plexinA1-neuropilin-1 Complex: Toggle control of prostate cancer cycles of dormancy and reactivation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3161.

// Hao Zeng 1 , Angelica Ortiz 2, 3, * , Peng-Fei Shen 1, * , Chien-Jui Cheng 4, 5 , Yu-Chen Lee 2 , Guoyu Yu 2 , Song-Chang Lin 2 , Chad J. Creighton 6 , Li-Yuan Yu-Lee 7 , Sue-Hwa Lin 2, 3, 8 1 Department of Urology, Institute of Urology, West China Hospital, Sichuan University, Chengdu, China 2 Department of Translational Molecular Pathology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, USA 3 The University of Texas Graduate School of Biomedical Sciences at Houston, Texas, USA 4 Department of Pathology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan 5 Department of Pathology, Taipei Medical University Hospital, Taipei, Taiwan 6 Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, Texas, USA 7 Department of Medicine, Baylor College of Medicine, Houston, Texas, USA 8 Department of Genitourinary Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, USA * These authors have contributed equally to this work Correspondence to: Hao Zeng, email: cdhx510@foxmail.com , kucaizeng@163.com Sue-Hwa Lin, email: slin@mdanderson.org Keywords: angiomotin, Hippo pathway, YAP, BMP4, proliferation Abbreviations: Angiomotin (AMOT), neurofibromatosis type 2 (NF2), prostate cancer (PCa) Received: October 11, 2016 Accepted: December 13, 2016 Published: December 29, 2016 ABSTRACT Angiomotin (AMOT) is a family of proteins found to be a component of the apical junctional complex of vertebrate epithelial cells and is recently found to play important roles in neurofibromatosis type 2 (NF-2). Whether AMOT plays a role in prostate cancer (PCa) is unknown. AMOT is expressed as two isoforms, AMOTp80 and AMOTp130, which has a 409 aa N-terminal domain that is absent in AMOTp80. Both AMOTp80 and AMOTp130 are expressed in LNCaP and C4-2B4, but at a low to undetectable level in PC3, DU145, and BPH1 cells. Further study showed that AMOTp130 and AMOTp80 have distinct functions in PCa cells. We found that AMOTp80, but not AMOT p130, functioned as a tumor promoter by enhancing PCa cell proliferation. Mechanistic studies showed that AMOTp80 signaled through the Hippo pathway by promoting nuclear translocation of YAP, resulting in an increased expression of YAP target protein BMP4. Moreover, inhibition of BMP receptor activity by LDN-193189 abrogates AMOTp80-mediated cell proliferation. Together, this study reveals a novel mechanism whereby the AMOTp80-Merlin-MST1-LATS-YAP-BMP4 pathway leads to AMOTp80-induced tumor cell proliferation.

// Mehmet Asim Bilen 1 , Kenneth R. Hess 2 , Matthew T. Campbell 3 , Jennifer Wang 3 , Russell R. Broaddus 4 , Jose A. Karam 5 , John F. Ward 5 , Christopher G. Wood 5 , Seungtaek L. Choi 6 , Priya Rao 4 , Miao Zhang 4 , Aung Naing 7 , Rosale General 3 , Diana H. Cauley 3 , Sue-Hwa Lin 8 , Christopher J. Logothetis 3 , Louis L. Pisters 5 , Shi-Ming Tu 3 1 Department of Hematology and Medical Oncology, Winship Cancer Institute of Emory University, Atlanta, GA, USA 2 Department of Biostatistics the University of Texas MD Anderson Cancer Center, Houston, Texas, USA 3 Department of Genitourinary Medical Oncology the University of Texas MD Anderson Cancer Center, Houston, Texas, USA 4 Department of Pathology the University of Texas MD Anderson Cancer Center, Houston, Texas, USA 5 Department of Urology the University of Texas MD Anderson Cancer Center, Houston, Texas, USA 6 Department of Radiation Oncology the University of Texas MD Anderson Cancer Center, Houston, Texas, USA 7 Department of Investigational Cancer Therapeutics the University of Texas MD Anderson Cancer Center, Houston, Texas, USA 8 Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA Correspondence to: Shi-Ming Tu, email: stu@mdanderson.org Keywords: testicular cancer, intratumoral heterogeneity, chemoresistance, nonseminomatous germ cell tumor, next-generation sequencing Received: August 15, 2016 Accepted: November 07, 2016 Published: November 16, 2016 ABSTRACT Background: Nonseminomatous germ cell tumor of the testis (NSGCT) is largely curable. However, a small group of patients develop refractory disease. We investigated the hypothesis that intratumoral heterogeneity contributes to the emergence of chemoresistance and the development of refractory tumor subtypes. Results: Our institution’s records for January 2000 through December 2010 included 275 patients whose primary tumor showed pure embryonal carcinoma (pure E); mixed embryonal carcinoma, yolk sac tumor, and teratoma (EYT); or mixed embryonal carcinoma, yolk sac tumor, seminoma, and teratoma (EYST). Patients with EYST had the highest cancer-specific mortality rate ( P = .001). They tended to undergo somatic transformation ( P = .0007). Two of 5 patients with clinical stage I EYST who had developed recurrence during active surveillance died of their disease. Materials and Methods: In this retrospective study, we evaluated consecutive patients who had been diagnosed with the three most common histological phenotypes of NSGCT. Chemoresistance was defined as the presence of teratoma, viable germ cell tumor, or somatic transformation in the residual tumor or the development of progressive or relapsed disease after chemotherapy. In a separate prospective study, we performed next-generation sequencing on tumor samples from 39 patients to identify any actionable genetic mutations. Conclusions: Our data suggest that patients with EYST in their primary tumor may harbor a potentially refractory NSGCT phenotype and are at increased risk of dying from disease. Despite intratumoral heterogeneity, improved patient selection and personalized care of distinct tumor subtypes may optimize the clinical outcome of patients with NSGCT.

BACKGROUND Intratumoral heterogeneity presents a major obstacle to the widespread implementation of precision medicine. The authors assessed the origin of intratumoral heterogeneity in nonseminomatous germ cell tumor of the testis (NSGCT) and identified distinct tumor subtypes and a potentially lethal phenotype. METHODS In this retrospective study, all consecutive patients who had been diagnosed with an NSGCT between January 2000 and December 2010 were evaluated. The histologic makeup of primary tumors and the clinical course of disease were determined for each patient. A Fine and Gray proportional hazards regression analysis was used to determine the prognostic risk factors, and the Gray test was used to detect differences in the cumulative incidence of cancer death. In a separate prospective study, next‐generation sequencing was performed on tumor samples from 9 patients to identify any actionable mutations. RESULTS Six hundred fifteen patients were included in this study. Multivariate analysis revealed that the presence of yolk sac tumor in the primary tumor (P = .0003) was associated with an unfavorable prognosis. NSGCT could be divided into 5 subgroups. Patients in the yolk sac‐seminoma subgroup had the poorest clinical outcome (P = .0015). These tumors tended to undergo somatic transformation (P, Whether intratumoral heterogeneity is derived from differentiation of aberrant progenitor cells or from mutation of driver genes is investigated in a relevant clinical model, and the results suggest that intratumoral heterogeneity is caused in part by differentiation of pluripotent progenitor cells. Integrated or multimodal therapy may be effective at addressing intratumoral heterogeneity and treating distinct tumor subtypes as well as potentially lethal tumor phenotypes; this finding has profound clinical implications regarding the origin of intratumoral heterogeneity and the implementation of precision medicine in testicular cancer and other solid tumors.

PURPOSE OF REVIEW: Prostate cancer bone metastasis is the lethal progression of the disease. The disease frequently presents with osteoblastic lesions in bone. The tumor-induced bone can cause complications that significantly hamper the quality of life of patients. A better understanding of how prostate cancer induces aberrant bone formation and how the aberrant bone affects the progression and treatment of the disease may improve the therapies for this disease. RECENT FINDINGS: Prostate cancer-induced bone was shown to enhance tumor growth and confer therapeutic resistance in bone metastasis. Clinically, Radium-223, an alpha emitter that selectively targets bone, was shown to improve overall survival in patients, supporting a role of tumor-induced bone in prostate cancer progression in bone. Recently, it was discovered that PCa-induced aberrant bone formation is due, in part, from tumor-associated endothelial cells that were converted into osteoblasts through endothelial-to-osteoblast (EC-to-OSB) conversion by tumor-secreted BMP4. SUMMARY: The unique bone-forming phenotype of prostate cancer bone metastasis plays a role in prostate cancer progression in bone and therapy resistance. Therapies that incorporate targeting the tumor-induced osteoblasts or EC-to-OSB conversion mechanism may reduce tumor-induced bone formation and improve therapy outcomes.

Purpose of review In the present review, we summarize the recent developments in the management of germ cell tumors (GCTs). Recent findings Treatment-related acute and late-onset toxicity remains a key challenge in the management of GCTs, with recent evidence showing that the adverse health outcomes of etoposide and cisplatin for four cycles in comparison to bleomycin, etoposide, and cisplatin for three cycles appear to be similar. Recent data showed that multidisciplinary clinic approach and management in experienced academic centers were associated with improved overall survival in GCT patients. There are currently multiple conventional-dose chemotherapy options for salvage therapy in patients with refractory or recurrent disease. In addition, more efficacious high-dose chemotherapy regimens continue to be developed. The role of salvage conventional-dose chemotherapy versus high-dose chemotherapy is currently being investigated prospectively. Recent reports suggested that brentuximab vedotin could be a potential salvage option for cluster of differentiation 30 positive refractory GCTs. On the other hand the results of the first phase II clinical trial investigating pembrolizumab in refractory GCTs were disappointing showing no clinical activity.Finally, deep exploration of the immune profile of GCTs using immunohistochemistry and gene expression profiling has identified that advanced GCT stage was associated with decreased T-cell and Natural killer-cell signatures, whereas T regulatory, neutrophil, mast cell, and macrophage signatures increased with advanced stage. Even though these results indicated that activated T-cell infiltration correlated with seminoma histology and good prognosis, and could be used in the future as a biomarker, this approach needs to be validated in a large cohort. Summary Remaining challenges to be addressed include minimizing therapeutic toxicity, and improving outcomes in patients with refractory/recurrent GCTs.

Resistance to currently available targeted therapies significantly hampers the survival of patients with prostate cancer with bone metastasis. Here we demonstrate an important resistance mechanism initiated from tumor-induced bone. Studies using an osteogenic patient-derived xenograft, MDA-PCa-118b, revealed that tumor cells resistant to cabozantinib, a Met and VEGFR-2 inhibitor, reside in a “resistance niche” adjacent to prostate cancer-induced bone. We performed secretome analysis of the conditioned medium from tumor-induced bone to identify proteins (termed “osteocrines”) found within this resistance niche. In accordance with previous reports demonstrating that activation of integrin signaling pathways confers therapeutic resistance, 27 of the 90 osteocrines identified were integrin ligands. We found that following cabozantinib treatment, only tumor cells positioned adjacent to the newly formed woven bone remained viable and expressed high levels of pFAK-Y397 and pTalin-S425, mediators of integrin signaling. Accordingly, treatment of C4-2B4 cells with integrin ligands resulted in increased pFAK-Y397 expression and cell survival, whereas targeting integrins with FAK inhibitors PF-562271 or defactinib inhibited FAK phosphorylation and reduced the survival of PC3-mm2 cells. Moreover, treatment of MDA-PCa-118b tumors with PF-562271 led to decreased tumor growth, irrespective of initial tumor size. Finally, we show that upon treatment cessation, the combination of PF-562271 and cabozantinib delayed tumor recurrence in contrast to cabozantinib treatment alone. Our studies suggest that identifying paracrine de novo resistance mechanisms may significantly contribute to the generation of a broader set of potent therapeutic tools that act combinatorially to inhibit metastatic prostate cancer. Cancer Res; 75(22); 4949–59. ©2015 AACR.

Bone metastases from renal cell carcinoma (RCC) are typically lytic, destructive, and resistant to treatment regimens. Current in vitro models for studying metastasis introduce artifacts that limit their usefulness. Many features of tumors growing in bone are lost when human RCC cells are cultured in two-dimensional (2D) plastic substrata. In this study, we established that RCC spheroids, consisting of aggregates of cells, can be grown in a three-dimensional (3D) hyaluronate hydrogel-based culture system. The bone-derived human 786-O RCC subline proliferated and survived long term in these hydrogels. Additionally, RCC spheroids in 3D hydrogels demonstrated lower proliferation rates than their counterparts grown in 2D. Overall, gene expression patterns of RCC spheroids in 3D more closely mimicked those observed in vivo than did those of cells grown in 2D. Of particular importance, selected adhesion molecules, angiogenesis factors, and osteolytic factors that have been shown to be involved in RCC bone metastasis were found to be expressed at higher levels in 3D than in 2D cultures. We propose that the 3D culture system provides an improved platform for RCC bone metastasis studies compared with 2D systems.

Sex associated differences in bone metastasis formation from breast, lung and prostate cancer exist in clinical studies, but have not been systematically reviewed. Differences in the bone marrow niche can be attributed to sexual dimorphism, to genetic variations that affect sex hormone levels, or to the direct effects of sex hormones, natural or exogenously delivered. This review describes the present understanding of sex associated and sex hormone level differences in the marrow niche and in formation of bone metastasis during the transition of these three cancers from treatable disease to an often untreatable, lethal metastatic one. Our purpose is to provide insight into some underlying molecular mechanisms for hormonal influence in bone metastasis formation, and to the potential influence of sexual dimorphism, genetic differences affecting sex assignment, and sex hormone level differences on the bone niche and its favorability for metastasis formation. We reviewed publications in PubMed and EMBASE, including full length manuscripts, case reports and clinical studies of relevance to our topic. We focused on bone metastasis formation in breast, lung, and prostate cancer because all three commonly present with bone metastases. Several clear observations emerged. For breast cancer bone metastasis formation, estrogen receptor signaling pathways indicate a role for estrogen receptor beta (ERβ). Estrogen influences the bone microenvironment, creating and conditioning a favorable niche for colonization and breast cancer progression. For lung cancer, studies support the hypothesis that females have a more favorable bone microenvironment for metastasis formation. For prostate cancer, a decrease in the relative androgen to estrogen balance or a “feminization” of bone marrow favors bone metastasis formation, with a potentially important role for ERβ that may be similar to that in breast cancer. Long term estrogen administration or androgen blockade in males may feminize the bone marrow niche to one more favorable for bone metastases in prostate cancer. Administration of androgens in females, especially combined with mastectomy, may reduce risk of developing bone metastatic breast cancer. We conclude that it should be considered that females, those with female-leaning genetic variations, or hormonal states that feminize the bone marrow, may offer favorable sites for bone metastases.

Prostate cancer (PCa) bone metastasis is frequently associated with bone-forming lesions, but the source of the osteoblastic lesions remains unclear. We show that the tumor-induced bone derives partly from tumor-associated endothelial cells that have undergone endothelial-to-osteoblast (EC-to-OSB) conversion. The tumor-associated osteoblasts in PCa bone metastasis specimens and patient-derived xenografts (PDXs) were found to co-express endothelial marker Tie-2. BMP4, identified in PDX-conditioned medium, promoted EC-to-OSB conversion of 2H11 endothelial cells. BMP4 overexpression in non-osteogenic C4-2b PCa cells led to ectopic bone formation under subcutaneous implantation. Tumor-induced bone was reduced in trigenic mice (Tie2cre/Osxf/f/SCID) with endothelial-specific deletion of osteoblast cell-fate determinant OSX compared with bigenic mice (Osxf/f/SCID). Thus, tumor-induced EC-to-OSB conversion is one mechanism that leads to osteoblastic bone metastasis of PCa.

We evaluated the patterns of progression and determined clinical predictors of survival in patients with castration-resistant prostate cancer (CRPCa) who received sipuleucel-T. We retrospectively analyzed 56 consecutive patients with asymptomatic or minimally symptomatic CRPCa treated with sipuleucel-T. Age, number of bone metastases, history of prior systemic treatment, and alkaline phosphatase level (ALP) were tested as predictors of survival in a multivariate Cox proportional hazards regression model. The Kaplan–Meier method was used to estimate event-free probabilities. The 56 patients were a median age of 67 years (range 51–84 years). After sipuleucel-T treatment, 25 patients developed bone progression after a median of 22 months of follow-up (54% of patients were event free at 2 years) and 10% (6/56 patients) developed rapid progression. Eleven deaths were observed after a median of 28 months of follow-up. Forty-eight patients were included in the multivariate analysis for overall survival. The analysis showed that age >70 years (p = 0.012), number of bone metastases >20 (p = 0.018), prior systemic treatment (p = 0.018), and ALP level >90 IU/L (p = 0.010) significantly predicted worse overall survival. Two-year overall survival was 36% among the 16 patients with two or more of these factors and was 93% among the 32 patients with one or none of these factors (p = 0.0004). CRPCa patients with age (>70 years), increased tumor burden in bone (>20 metastases and/or elevated ALP level), and/or prior systemic treatment are more likely to experience rapid deterioration after sipuleucel-T. These results need to be prospectively validated.

Talins are adaptor proteins that regulate focal adhesion signaling by conjugating integrins to the cytoskeleton. Talins directly bind integrins and are essential for integrin activation. We previously showed that β1 integrins are activated in metastatic prostate cancer (PCa) cells, increasing PCa metastasis to lymph nodes and bone. However, how β1 integrins are activated in PCa cells is unknown. In this study, we identified a novel mechanism of β1 integrin activation. Using knockdown experiments, we first demonstrated that talin1, but not talin2, is important in β1 integrin activation. We next showed that talin1 S425 phosphorylation, but not total talin1 expression, correlates with metastatic potential of PCa cells. Expressing a non-phosphorylatable mutant, talin1(S425A), in talin1-silenced PC3-MM2 and C4-2B4 PCa cells, decreased activation of β1 integrins, integrin-mediated adhesion, motility and increased the sensitivity of the cells to anoikis. In contrast, reexpression of the phosphorylation-mimicking mutant talin1(S425D) led to increased β1 integrin activation and generated biologic effects opposite to talin1(S425A) expression. In the highly metastatic PC3-MM2 cells, expression of a non-phosphorylatable mutant, talin1(S425A), in talin1-silenced PC3-MM2 cells, abolished their ability to colonize in the bone following intracardiac injection, while reexpression of phosphorylation-mimicking mutant talin1(S425D) restored their ability to metastasize to bone. Immunohistochemical staining demonstrated that talin S425 phosphorylation is significantly increased in human bone metastases when compared with normal tissues, primary tumors or lymph node metastases. We further showed that p35 expression, an activator of Cdk5, and Cdk5 activity were increased in metastatic tumor cells, and that Cdk5 kinase activity is responsible for talin1 phosphorylation and subsequent β1 integrin activation. Together, our study reveals Cdk5-mediated phosphorylation of talin1 leading to β1 integrin activation is a novel mechanism that increases metastatic potential of PCa cells.

Cadherin-11 (CDH11) is a member of the cadherin superfamily mainly expressed in osteoblasts but not in epithelial cells. However, prostate cancer cells with a propensity for bone metastasis express high levels of cadherin-11 and reduced levels of E-cadherin. Downregulation of cadherin-11 inhibits interaction of prostate cancer cells with osteoblasts in vitro and homing of prostate cancer cells to bone in an animal model of metastasis. These findings indicate that targeting cadherin-11 may prevent prostate cancer bone metastasis. To explore this possibility, a panel of 21 monoclonal antibodies (mAb) was generated against the extracellular (EC) domain of cadherin-11. Two antibodies, mAbs 2C7 and 1A5, inhibited cadherin-11–mediated cell–cell aggregation in vitro using L-cells transfected with cadherin-11. Both antibodies demonstrated specificity to cadherin-11, and neither antibody recognized E-cadherin or N-cadherin on C4-2B or PC3 cells, respectively. Furthermore, mAb 2C7 inhibited cadherin-11–mediated aggregation between the highly metastatic PC3-mm2 cells and MC3T3-E1 osteoblasts. Mechanistically, a series of deletion mutants revealed a unique motif, aa 343-348, in the cadherin-11 EC3 domain that is recognized by mAb 2C7 and that this motif coordinated cell–cell adhesion. Importantly, administration of mAb 2C7 in a prophylactic setting effectively prevented metastasis of PC3-mm2 cells to bone in an in vivo mouse model. These results show that targeting the extracellular domain of cadherin-11 can limit cellular adhesion and metastatic dissemination of prostate cancer cells. Implications: Monotherapy using a cadherin-11 antibody is a suitable option for the prevention of bone metastases. Mol Cancer Res; 11(11); 1401–11. ©2013 AACR.

Recently, many therapeutic agents for prostate cancer have been approved that target the androgen receptor and/or the prostate tumor microenvironment. Each of these therapies has modestly increased patient survival. A better understanding of when in the course of prostate cancer progression specific therapies should be applied, and of what biomarkers would indicate when resistance arises, would almost certainly improve survival due to these therapies. Thus, applying the armamentarium of therapeutic agents in the right sequences in the right combination at the right time is a major goal in prostate cancer treatment. For this to occur, an understanding of prostate cancer evolution during progression is required. In this review, we discuss the current understanding of prostate cancer progression, but challenge the prevailing view by proposing a new model of prostate cancer progression, with the goal of improving biologic classification and treatment strategies. We use this model to discuss how integrating clinical and basic understanding of prostate cancer will lead to better implementation of molecularly targeted therapeutics and improve patient survival. Significance: Rapid development of drugs with efficacy against prostate cancer now makes it possible to consider applying these agents with curative intent in men with currently incurable cancers. However, when to apply these new drugs, as well as those under development, to obtain the best outcomes is a challenge that must be addressed. To meet this challenge, better classification of the disease based on the underlying molecular mechanisms of progression will facilitate the implementation of current and emerging therapies. Cancer Discov; 3(8); 849–61. ©2013 AACR.

Disseminated prostate cancer cells must survive in circulation for metastasis to occur. Mechanisms by which these cells survive are not well understood. By immunohistochemistry of human tissues, we found that levels of β1 integrins and integrin-induced autophosphorylation of FAK (pFAK-Y397) are increased in prostate cancer cells in primary prostate cancer and lymph node metastases, suggesting that β1 integrin activation occurs in metastatic progression of prostate cancer. A conformation-sensitive antibody, 9EG7, was used to examine β1 integrin activation. We found that β1 integrins are constitutively activated in highly metastatic PC3 and PC3-mm2 cells, with less activation in low metastatic LNCaP and C4-2B4 cells. Increased β1 integrin activation as well as the anoikis resistance in prostate cancer cells correlated with metastatic potential in vivo. Knockdown of β1 integrin abrogated anoikis resistance in PC3-mm2 cells. In agreement with β1 integrin activation, PC3-mm2 cells strongly adhered to type I collagen and fibronectin, a process inhibited by the β1 integrin-neutralizing antibody mAb 33B6. mAb 33B6 also inhibited the phosphorylation of β1 integrin downstream effectors, focal adhesion kinase (FAK) and AKT, leading to a 3-fold increase in PC3-mm2 apoptosis. Systemic delivery of mAb 33B6 suppressed spontaneous metastasis of PC3-mm2 from the prostate to distant lymph nodes following intraprostatic injection and suppressed metastasis of PC3-mm2 to multiple organs following intracardiac injection. Thus, constitutively activated β1 integrins play a role in survival of PC3-mm2 cells in circulation and represent a potential target for metastasis prevention. Visual Overview: http://mcr.aacrjournals.org/content/11/4/405/F1.large.jpg. Mol Cancer Res; 11(4); 405–17. ©2013 AACR.

Introduction: This study reviewed patients’ demographic, clinical and treatment characteristics to identify prognostic factors associated with survival of prostate cancer after developing bone metastases. We explored the racial disparities in these factors and how they relate with survival. Methods: We conducted a retrospective study on 79 men diagnosed with bone metastasis secondary to prostate cancer who underwent surgery at a single institution from November 1977 to June 2011. Descriptive statistics were used to summarize patients’ characteristics. The Kaplan-Meier method was used to estimate characteristics of the survival distribution using two origination points—diagnosis and surgery. Cox hazard regression explored the relationship between prognostic factors and overall survival. Results: The majority of men were White (n = 63; 80%) followed by Black (n = 7; 9%), Hispanic (n = 7; 9%), and Asian (n = 2; 2%). Multivariate factors associated with poorer survival after bone metastasis surgery included race (Black), Gleason score > 8, and radiation treatment. Patients not receiving radiation had a longer survival experience relative to patients who received radiation before or after surgery (10.3 vs 6.5 months; P = 0.030). There was an association of PSA level at the time of bone metastasis diagnosis with survival following diagnosis but prior to surgery. The median time interval (Tm in months) between prostate cancer diagnosis and bone metastasis diagnosis was 39.1 (White), 31.2 (Hispanic), 15 (Blacks) and 43 (Asians). Patients with Tm m > 35 months (HR = 3.22; P Conclusion: The median survival and time interval from prostate cancer diagnosis to bone metastasis diagnosis was shorter in Blacks with respect to other races. The more aggressive nature of the disease in Blacks is likely due to the biology of the disease rather than access to treatment.

Bone metastases (BMs) occur commonly in patients with metastatic renal cell carcinoma (mRCC). Tyrosine kinase inhibitors (TKIs) have improved the outcomes for patients with mRCC. However, data on the outcomes of mRCC patients with BMs treated with TKIs are limited. We describe the outcomes of patients with BMs treated with TKI therapy and compare them with the outcomes from a pre-TKI group.Using an institutional tumor registry, a retrospective review of patients with mRCC from 2002 to 2003 and 2006 to 2007 was performed. The baseline characteristics were analyzed, and overall survival (OS) was estimated using the Kaplan-Meier method. The predictors of OS were analyzed using Cox regression analysis.The data from 375 patients were reviewed. Of these patients, 188 (50%) started treatment with TKIs and 187 (50%) had started treatment in the pre-TKI era. The distribution of patient characteristics was similar. The sites of organ metastases were equally distributed, including BMs in 48% of the patients in each cohort. The median OS for the patients treated in the TKI era was 22 months (95% confidence interval [CI], 17-25 months) compared with 14 months (95% CI, 10-19 months; P .01) for the historical controls. A subset analysis of patients with BM in the TKI era demonstrated a median OS of 24 months (95% CI, 17-28 months) compared with 18 months (95% CI, 10-21 months; P .01) in pre-TKI era. The predictors of shorter OS were a higher Memorial Sloan Kettering Cancer Center score; liver, lung, and brain metastases; and multiple sites of BMs (hazard ratio, 1.38; 95% CI, 1.02-1.91; P = .04). The rate of new BM development was the same in the pre- and post-TKI era.The rate of BM development was the same in the pre- and post-TKI era. The management of BMs in patients with mRCC remains challenging.

Mutational processes and signatures that drive early tumorigenesis are centrally important for early cancer prevention. Yet, to date, biomarkers and risk factors for polyps (adenomas) that inordinately and rapidly develop into colon cancer remain poorly defined. Here, we describe surprisingly high mutational profiles through whole-genome sequence (WGS) analysis in 2 of 4 pairs of benign colorectal adenoma tissue samples. Unsupervised hierarchical clustered transcriptomic analysis of a further 7 pairs of adenomas reveals distinct mutational signatures regardless of adenoma size. Transitional single nucleotide substitutions of C:G>T:A predominate in the adenoma mutational spectrum. Strikingly, we observe mutations in the TGF-β pathway and CEA-associated genes in 4 out of 11 adenomas, overlapping with the Wnt pathway. Immunohistochemical labeling reveals a nearly 5-fold increase in CEA levels in 23% of adenoma samples with a concomitant loss of TGF-β signaling. We also define a functional role by which the CEA B3 domain interacts with TGFBR1, potentially inactivating the tumor suppressor function of TGF-β signaling. Our study uncovers diverse mutational processes underlying the transition from early adenoma to cancer. This has broad implications for biomarker-driven targeting of CEA/TGF-β in high-risk adenomas and may lead to early detection of aggressive adenoma to CRC progression.

Stem cells have long been implicated in prostate gland formation. The prostate undergoes regression after androgen deprivation and regeneration after testosterone replacement. Regenerative studies suggest that these cells are found in the proximal ducts and basal layer of the prostate. Many characteristics of prostate cancer indicate that it originates from stem cells. For example, the putative androgen receptor-negative (AR(-)) status of prostate stem cells renders them inherently insensitive to androgen blockade therapy. The androgen-regulated gene fusion TMPRSS2-ERG could be used to clarify both the cells of origin and the evolution of prostate cancer cells. In this review, we show that the hypothesis that distinct subtypes of cancer result from abnormalities within specific cell types-the stem cell theory of cancer-may instigate a major paradigm shift in cancer research and therapy. Ultimately, the stem cell theory of cancers will affect how we practice clinical oncology: our diagnosis, monitoring, and therapy of prostate and other cancers.

The microenvironment is critical to the growth of prostate cancer (PCa) in the bone. Thus, for clinical efficacy, therapies must target tumor–microenvironment interactions. Several protein tyrosine kinases have been implicated in the development and growth of PCa bone metastasis. In this review, specific protein tyrosine kinases that regulate these complex interactions, including PDGFR, the EGFR family, c-Src, VEGFR, IGF-1R, FGFR and c-Met will be discussed, with an emphasis on why these kinases are promising therapeutic targets for metastatic PCa treatment. For each of these kinases, small-molecule inhibitors have reached clinical trials. Current results of these trials and future prospects for the use of tyrosine kinase inhibitors for the treatment of PCa bone metastases are also discussed.

Induction of new bone formation is frequently seen in the bone lesions from prostate cancer. However, whether osteogenesis is necessary for prostate tumor growth in bone is unknown. Recently, 2 xenografts, MDA-PCa-118b and MDA-PCa-133, were generated from prostate cancer bone metastases. When implanted subcutaneously in severe combined immunodeficient (SCID) mice, MDA-PCa-118b induced strong ectopic bone formation while MDA-PCa-133 did not. To identify the factors that are involved in bone formation, we compared the expression of secreted factors (secretome) from MDA-PCa-118b and MDA-PCa-133 by cytokine array. We found that the osteogenic MDA-PCa-118b xenograft expressed higher levels of bone morphogenetic protein BMP4 and several cytokines including interleukin-8, growth-related protein (GRO), and CCL2. We showed that BMP4 secreted from MDA-PCa-118b contributed to about a third of the osteogenic differentiation seen in MDA-PCa-118b tumors. The conditioned media from MDA-PCa-118b induced a higher level of osteoblast differentiation, which was significantly reduced by treatment with BMP4 neutralizing antibody or the small molecule BMP receptor 1 inhibitor LDN-193189. BMP4 did not elicit an autocrine effect on MDA-PCa-118b, which expressed low to undetectable levels of BMP receptors. Treatment of SCID mice bearing MDA-PCa-118b tumors with LDN-193189 significantly reduced tumor growth. Thus, these studies support a role of BMP4-mediated osteogenesis in the progression of prostate cancer in bone. Cancer Res; 71(15); 5194–203. ©2011 AACR.

Men with castration-resistant prostate cancer (PCa) frequently develop metastasis in bone. The reason for this association is unclear. We have previously shown that cadherin-11 (also known as OB-cadherin), a homophilic cell adhesion molecule that mediates osteoblast adhesion, plays a role in the metastasis of PCa to bone. Here, we report that androgen deprivation therapy upregulates cadherin-11 expression in PCa. In human PCa specimens, immunohistochemical staining showed that 22 of 26 (85%) primary PCa tumors from men with castration-resistant PCa expressed cadherin-11. In contrast, only 7 of 50 (14%) androgen-dependent PCa tumors expressed cadherin-11. In the MDA-PCa-2b xenograft animal model, cadherin-11 was expressed in the recurrent tumors following castration. In the PCa cell lines, there is an inverse correlation between expression of cadherin-11 and androgen receptor (AR), and cadherin-11 is expressed in very low levels or not expressed in AR positive cell lines, including LNCaP, C4-2B4, and VCaP cells. We showed that AR likely regulates cadherin-11 expression in PCa through an indirect mechanism. Although re-expression of AR in the AR-negative PC3 cells led to the inhibition of cadherin-11 expression, depletion of androgen from the culture medium or down regulation of AR by RNA interference in the C4-2B4 cells or VCaP cells only produce a modest increase of cadherin-11 expression. Promoter analysis indicated that cadherin-11 promoter does not contain a typical AR binding element, and AR elicits a modest inhibition of cadherin-11 promoter activity, suggesting that AR does not regulate cadherin-11 expression directly. Together, these results suggest that androgen deprivation upregulates cadherin-11 expression in prostate cancer and this may contribute to the metastasis of PCa to bone. Our study suggests that therapeutic strategies that block cadherin-11 expression or function may be considered when applying androgen ablation therapy.

After prostate cancer cells (PCa) arrive in bone, interactions with cells that include long bone osteoblasts (LBOB) and bone marrow stromal cells (BMSC) lead to metastasis formation. The effect of heterotypic cell-cell contact between PCa cells and BMSC or LBOB on PCa cell gene expression is poorly understood. To establish the role of heterotypic contact in bone metastasis formation, we mixed and co-cultured PC3 cells with rat BMSC, LBOB, or human prostate stromal cells (PS15). PC3 cells were then re-isolated for gene array analysis, and imaged using in situ hybridization to confirm that heterotypic contact regulates gene expression. The gene expression was examined using focused gene arrays containing 96 each of tumor metastasis genes or osteogenesis genes. A total of 18 out of 192 genes in PC3 cells were found to be under or over expressed subsequent to heterotypic contact with BMSC when analyzed. A total of 15 genes out of 192 were regulated in co-culture with LBOB, and 19 genes with PS15. Only two genes, uPA and Collagen III, were regulated by contact with BMSC or LBOB (both are bone derived cells), but not by contact with PS15. The relationship between cell-cell contact and uPA expression was further explored by varying cell ratios in co-culture. uPA over-expression in PC3 was related to the BMSC:PC3 ratio, and was maximum at a 10:1 ratio, where most PC3 cells would be in contact with BMSC, as predicted by a theoretical model of heterotypic contact. In situ staining of micropatterned PC3 and BMSC cells showed that uPA over-expression localizes to regions of heterotypic cell-cell contact. Taken together, our results suggest that heterotypic cell-to-cell contact between PC3 and BMSC proportionally enhances gene expression for uPA, providing a mechanism for localized control of invasiveness.

In a placebo-controlled randomised study of the platelet-derived growth factor receptor (PDGFR) inhibitor imatinib mesylate and docetaxel in metastatic prostate cancer with bone metastases (n=116), no significant differences in progression-free and overall survival were observed. To evaluate pharmacodynamic correlates of outcomes, we assessed the association of plasma platelet-derived growth factor (PDGF) isoform kinetics and PDGFR inhibition with progression-free and overall survival by individual treatment arm. We found that in the docetaxel–placebo arm alone, the probability of decrease in PDGFR phosphorylation (Pr-Decr-pPDGFR) above 0.5 (vs ⩽0.5) was associated with a sharp increase in all measured plasma PDGF isoforms (P=0.006 for AA, 0.002 for BB, 0.045 for AB); a decreased median progression-free survival of 3.3 months vs 6.8 months (hazard ratio (HR) 2.5; P=0.006 in log-rank test) and an inferior median overall survival of 20 months vs >30 months (HR 3.1; P=0.04 in log-rank test). By contrast, in the docetaxel plus imatinib arm, the association of Pr-Decr-pPDGFR >0.5 with a rise in plasma PDGF isoform concentrations and inferior survival was not observed. The data suggest that dynamic changes in PDGFR phosphorylation in peripheral blood leukocytes predict docetaxel efficacy. Rising plasma PDGF concentrations may explain and/or mark docetaxel resistance. Validation and mechanistic studies addressing these unexpected findings should anticipate a confounding influence of concurrent PDGFR inhibitor therapy.

Bone is the most common site of metastases from prostate cancer. The mechanism by which prostate cancer cells metastasize to bone is not fully understood, but interactions between prostate cancer cells and bone cells are thought to initiate the colonization of metastatic cells at that site. Here, we show that cadherin-11 (also known as osteoblast-cadherin) was highly expressed in prostate cancer cell line derived from bone metastases and had strong homophilic binding to recombinant cadherin-11 in vitro. Down-regulation of cadherin-11 in bone metastasis–derived PC3 cells with cadherin-11–specific short hairpin RNA (PC3-shCad-11) significantly decreased the adhesion of those cells to cadherin-11 in vitro. In a mouse model of metastasis, intracardiac injection of PC3 cells led to metastasis of those cells to bone. However, the incidence of PC3 metastasis to bone in this model was reduced greatly when the expression of cadherin-11 by those cells was silenced. The clinical relevance of cadherin-11 in prostate cancer metastases was further studied by examining the expression of cadherin-11 in human prostate cancer specimens. Cadherin-11 was not expressed by normal prostate epithelial cells but was detected in prostate cancer, with its expression increasing from primary to metastatic disease in lymph nodes and especially bone. Cadherin-11 expression was not detected in metastatic lesions that occur in other organs. Collectively, these findings suggest that cadherin-11 is involved in the metastasis of prostate cancer cells to bone. (Mol Cancer Res 2008;6(8):1259–67)

The tendency of prostate cancer to produce osteoblastic bone metastases suggests that cancer cells and osteoblasts interact in ways that contribute to cancer progression. To identify factors that mediate these interactions, we compared gene expression patterns between two bone-derived prostate cancer cell lines that produce osteoblastic (MDA PCa 2b) or osteolytic lesions (PC-3). Both cell lines expressed Wnt ligands, including WNT7b, a canonical Wnt implicated in osteogenesis. PC-3 cells expressed 50 times more Dickkopf-1 (DKK1), an inhibitor of Wnt pathways, than did MDA PCa 2b cells. Evaluation of the functional role of these factors (in cocultures of prostate cancer cells with primary mouse osteoblasts (PMOs) or in bone organ cultures) showed that MDA PCa 2b cells activated Wnt canonical signaling in PMOs and that DKK1 blocked osteoblast proliferation and new bone formation induced by MDA PCa 2b cells. MDA PCa 2b cells did not induce bone formation in calvaria from mice lacking the Wnt co-receptor Lrp5. In human specimens, WNT7b was not expressed in normal prostate but was expressed in areas of high-grade prostate intraepithelial neoplasia, in three of nine primary prostate tumor specimens and in 16 of 38 samples of bone metastases from prostate cancer. DKK1 was not expressed in normal or cancerous tissue but was expressed in two of three specimens of osteolytic bone metastases (P=0.0119). We conclude that MDA PCa 2b induces new bone formation through Wnt canonical signaling, that LRP5 mediates this effect, and that DKK1 is involved in the balance between bone formation and resorption that determines lesion phenotype. Fil: Li, Z. G.. University of Texas; Estados Unidos Fil: Yang, J.. University of Texas; Estados Unidos Fil: Vazquez, Elba Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Rose, D.. University of Texas; Estados Unidos Fil: Vakar Lopez, F.. University of Texas; Estados Unidos Fil: Mathew, P.. University of Texas; Estados Unidos Fil: Lopez, A.. University of Texas; Estados Unidos Fil: Logothetis, C. J.. University of Texas; Estados Unidos Fil: Lin, S. H.. University of Texas; Estados Unidos Fil: Navone, N. M.. University of Texas; Estados Unidos

ErbB-3, an ErbB receptor tyrosine kinase, has been implicated in the pathogenesis of several malignancies, including prostate cancer. We found that ErbB-3 expression was up-regulated in prostate cancer cells within lymph node and bone metastases. Despite being a plasma membrane protein, ErbB-3 was also detected in the nuclei of the prostate cancer cells in the metastatic specimens. Because most metastatic specimens were from men who had undergone androgen ablation, we examined the primary tumors from patients who have undergone hormone deprivation therapy and found that a significant fraction of these specimens showed nuclear localization of ErbB3. We thus assessed the effect of androgens and the bone microenvironment on the nuclear translocation of ErbB-3 by using xenograft tumor models generated from bone-derived prostate cancer cell lines, MDA PCa 2b, and PC-3. In subcutaneous tumors, ErbB-3 was predominantly in the membrane/cytoplasm; however, it was present in the nuclei of the tumor cells in the femur. Castration of mice bearing subcutaneous MDA PCa 2b tumors induced a transient nuclear translocation of ErbB-3, with relocalization to the membrane/cytoplasm upon tumor recurrence. These findings suggest that the bone microenvironment and androgen status influence the subcellular localization of ErbB-3 in prostate cancer cells. We speculate that nuclear localization of ErbB-3 may aid prostate cancer cell survival during androgen ablation and progression of prostate cancer in bone. (Mol Cancer Res 2007;5(7):675–84)

We performed combinatorial peptide library screening in vivo on a novel human prostate cancer xenograft that is androgen-independent and induces a robust osteoblastic reaction in bonelike matrix and soft tissue. We found two peptides, PKRGFQD and SNTRVAP, which were enriched in the tumors, targeted the cell surface of androgen-independent prostate cancer cells in vitro, and homed to androgen receptor-null prostate cancer in vivo. Purification of tumor homogenates by affinity chromatography on these peptides and subsequent mass spectrometry revealed a receptor for the peptide PKRGFQD, α-2-macroglobulin, and for SNTRVAP, 78-kDa glucose-regulated protein (GRP78). These results indicate that GRP78 and α-2-macroglobulin are highly active in osteoblastic, androgen-independent prostate cancer in vivo. These previously unidentified ligand–receptor systems should be considered for targeted drug development against human metastatic androgen-independent prostate cancer.

Purpose Clinicians may have reservations about using strontium-89 for the treatment of bone metastases because of concerns that it may limit future use of chemotherapy. We assessed the rate of bone marrow failure in patients with prostate cancer who had received a dose of strontium-89. Patients and Methods This subgroup analysis involved 34 patients with androgen-independent prostate cancer who had been given a dose of strontium-89 and six weekly doses of doxorubicin after response to induction chemotherapy. We assessed subsequent hematotoxicity in terms of bone marrow failure and the ability to tolerate additional treatments during a median of 25 months (range, 7 to 76 months) after the strontium-89 was administered. Results No patients developed bone marrow failure within 6 months of receiving strontium-89. Five (15%) of 34 patients developed bone marrow failure at a median 23 months (range, 6 to 53 months) after the strontium-89 treatment. Bone marrow biopsy performed in two of these five patients showed complete replacement of the marrow by tumor. Thirty-one patients (91%) received subsequent cytotoxic treatments at a median 11 months (range, 1 to 33 months) after the strontium-89 treatment. Conclusion This analysis demonstrated that a single dose of strontium-89 combined with chemotherapy did not affect the delivery of subsequent courses of chemotherapy in a select group of patients. However, a majority of these therapies were given off protocol and were administered at a dose schedule that might be considered inappropriate or inadequate. The clinical role and safety profile of radiopharmaceuticals combined with chemotherapy in prostate cancer therapy deserve further exploration.

Prostate cancer is the most common cancer in men in America. Currently, steroid receptor coactivators have been proposed to mediate the development and progression of prostate cancer, at times in a steroid-independent manner. Steroid receptor coactivator-3 (SRC-3, p/CIP, AIB1, ACTR, RAC3, and TRAM-1) is a member of the p160 family of coactivators for nuclear hormone receptors including the androgen receptor. SRC-3 is frequently amplified or overexpressed in a number of cancers. However, the role of SRC-3 in cancer cell proliferation and survival is still poorly understood. In this study, we show that SRC-3 is overexpressed in prostate cancer patients and its overexpression correlates with prostate cancer proliferation and is inversely correlated with apoptosis. Consistent with patient data, we have observed that reduction of SRC-3 expression by small interfering RNA decreases proliferation, delays the G1-S transition, and increases cell apoptosis of different prostate cancer cell lines. Furthermore, with decreased SRC-3 expression, proliferating cell nuclear antigen and Bcl-2 expression, as well as bromodeoxyuridine incorporation in prostate cancer cells are reduced. Finally, knockdown of SRC-3 with inducible short hairpin RNA expression in prostate cancer cells decreased tumor growth in nude mice. Taken together, these findings indicate that SRC-3 is an important regulator of prostate cancer proliferation and survival.

Metastasis to bone is common in lung, kidney, breast and prostate cancers. However, prostate cancer is unique in that bone is often the only clinically detectable site of metastasis, and the resulting tumours tend to be osteoblastic (bone forming) rather than osteolytic (bone lysing). The interaction between host cells and metastatic cancer cells is an important component of organ-specific cancer progression. How can this knowledge lead to the development of more effective therapies?

Prostate cancer (PCa) has a propensity to metastasize to bone. The identification of molecules that mediate the biological interactions between PCa cells and the bone environment is crucial to the understanding of PCa bone metastasis. The present study has used protein purification to identify bone metastasis-related factors present in bone marrow aspirates from PCa patients with bone metastasis. MDA-BF-1 was the first bone metastasis factor to be identified and is a secreted isoform of the ErbB3 growth factor receptor. To determine which cell types in PCa bone metastases express MDA-BF-1, MDA-BF-1 expression was studied in both primary and metastatic PCa cells, using an antibody to the extracellular domain (Ab10) and another to the cytoplasmic domain (RTJ.2) of ErbB3 to distinguish MDA-BF-1 from p180-ErbB3. It was found that epithelial cells in primary PCa did not express MDA-BF-1. In contrast, epithelial cells in 41 of 45 PCa metastases (18 of 19 lymph node metastases and 23 of 26 bone metastases), and activated osteoblasts in bone metastases, expressed MDA-BF-1. In addition, newly formed bone matrices adjacent to activated osteoblasts were also immunopositive for MDA-BF-1, suggesting that activated osteoblasts secrete MDA-BF-1. These observations indicate that MDA-BF-1 is up-regulated in metastatic PCa and raise the interesting possibility that MDA-BF-1 may play a role in the metastasis and progression of PCa, particularly in bone.