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Background Keratins (KRTs) are intermediate filament proteins that interact with multiple regulatory proteins to initiate signaling cascades. Keratin 13 (KRT13) plays an important role in breast cancer progression and metastasis. The objective of this study is to elucidate the mechanism by which KRT13 promotes breast cancer growth and metastasis. Methods The function and mechanisms of KRT13 in breast cancer progression and metastasis were assessed by overexpression and knockdown followed by examination of altered behaviors in breast cancer cells and in xenograft tumor formation in mouse mammary fat pad. Human breast cancer specimens were examined by immunohistochemistry and multiplexed quantum dot labeling analysis to correlate KRT13 expression to breast cancer progression and metastasis. Results KRT13-overexpressing MCF7 cells displayed increased proliferation, invasion, migration and in vivo tumor growth and metastasis to bone and lung. Conversely, KRT13 knockdown inhibited the aggressive behaviors of HCC1954 cells. At the molecular level, KRT13 directly interacted with plakoglobin (PG, γ-catenin) to form complexes with desmoplakin (DSP). This complex interfered with PG expression and nuclear translocation and abrogated PG-mediated suppression of c-Myc expression, while the KRT13/PG/c-Myc signaling pathway increased epithelial to mesenchymal transition and stem cell-like phenotype. KRT13 expression in 58 human breast cancer tissues was up-regulated especially at the invasive front and in metastatic specimens (12/18) (p Conclusions This study reveals that KRT13 promotes breast cancer cell growth and metastasis via a plakoglobin/c-Myc pathway. Our findings reveal a potential novel pathway for therapeutic targeting of breast cancer progression and metastasis.

This article describes cell signaling network of metastatic prostate cancer (PCa) to bone and visceral organs in the context of tumor microenvironment and for the development of novel therapeutics. The article focuses on our recent progress in the understanding of: 1) The plasticity and dynamics of tumor–stroma interaction; 2) The significance of epigenetic reprogramming in conferring cancer growth, invasion and metastasis; 3) New insights on altered junctional communication affecting PCa bone and brain metastases; 4) Novel strategies to overcome therapeutic resistance to hormonal antagonists and chemotherapy; 5) Genetic-based therapy to co-target tumor and bone stroma; 6) PCa-bone-immune cell interaction and TBX2-WNTprotein signaling in bone metastasis; 7) The roles of monoamine oxidase and reactive oxygen species in PCa growth and bone metastasis; and 8) Characterization of imprinting cluster of microRNA, in tumor–stroma interaction. This article provides new approaches and insights of PCa metastases with emphasis on basic science and potential for clinical translation. This article referenced the details of the various approaches and discoveries described herein in peer-reviewed publications. We dedicate this article in our fond memory of Dr. Donald S. Coffey who taught us the spirit of sharing and the importance of focusing basic science discoveries toward translational medicine. Keywords: Prostate cancer, Castration resistance, Metastasis, Cancer-stromal interaction, Targeted therapy

Tumor metastasis depends on the dynamic regulation of cell adhesion through β1-integrin. The Cub-Domain Containing Protein-1, CDCP1, is a transmembrane glycoprotein which regulates cell adhesion. Overexpression and loss of CDCP1 have been observed in the same cancer types to promote metastatic progression. Here, we demonstrate reduced CDCP1 expression in high-grade, primary prostate cancers, circulating tumor cells and tumor metastases of patients with castrate-resistant prostate cancer. CDCP1 is expressed in epithelial and not mesenchymal cells, and its cell surface and mRNA expression declines upon stimulation with TGFβ1 and epithelial-to-mesenchymal transition. Silencing of CDCP1 in DU145 and PC3 cells resulted in 3.4-fold higher proliferation of non-adherent cells and 4.4-fold greater anchorage independent growth. CDCP1-silenced tumors grew in 100% of mice, compared to 30% growth of CDCP1-expressing tumors. After CDCP1 silencing, cell adhesion and migration diminished 2.1-fold, caused by loss of inside-out activation of β1-integrin. We determined that the loss of CDCP1 reduces CDK5 kinase activity due to the phosphorylation of its regulatory subunit, CDK5R1/p35, by c-SRC on Y234. This generates a binding site for the C2 domain of PKCδ, which in turn phosphorylates CDK5 on T77. The resulting dissociation of the CDK5R1/CDK5 complex abolishes the activity of CDK5. Mutations of CDK5-T77 and CDK5R1-Y234 phosphorylation sites re-establish the CDK5/CDKR1 complex and the inside-out activity of β1-integrin. Altogether, we discovered a new mechanism of regulation of CDK5 through loss of CDCP1, which dynamically regulates β1-integrin in non-adherent cells and which may promote vascular dissemination in patients with advanced prostate cancer.

Background Global disparities in prostate cancer (PCa) incidence highlight the urgent need to identify genomic abnormalities in prostate tumors in different ethnic populations including Asian men. Objective To systematically explore the genomic complexity and define disease-driven genetic alterations in PCa. Design, setting, and participants The study sequenced whole-genome and transcriptome of tumor-benign paired tissues from 65 treatment-naive Chinese PCa patients. Subsequent targeted deep sequencing of 293 PCa-relevant genes was performed in another cohort of 145 prostate tumors. Outcome measurements and statistical analysis The genomic alteration landscape in PCa was analyzed using an integrated computational pipeline. Relationships with PCa progression and survival were analyzed using nonparametric test, log-rank, and multivariable Cox regression analyses. Results and limitations We demonstrated an association of high frequency of CHD1 deletion with a low rate of TMPRSS2-ERG fusion and relatively high percentage of mutations in androgen receptor upstream activator genes in Chinese patients. We identified five putative clustered deleted tumor suppressor genes and provided experimental and clinical evidence that PCDH9, deleted/loss in approximately 23% of tumors, functions as a novel tumor suppressor gene with prognostic potential in PCa. Furthermore, axon guidance pathway genes were frequently deregulated, including gain/amplification of PLXNA1 gene in approximately 17% of tumors. Functional and clinical data analyses showed that increased expression of PLXNA1 promoted prostate tumor growth and independently predicted prostate tumor biochemical recurrence, metastasis, and poor survival in multi-institutional cohorts of patients with PCa. A limitation of this study is that other genetic alterations were not experimentally investigated. Conclusions There are shared and salient genetic characteristics of PCa in Chinese and Caucasian men. Novel genetic alterations in PCDH9 and PLXNA1 were associated with disease progression. Patient summary We reported the first large-scale and comprehensive genomic data of prostate cancer from Asian population. Identification of these genetic alterations may help advance prostate cancer diagnosis, prognosis, and treatment.

Treatment of prostate cancer (PC) by androgen suppression promotes the emergence of aggressive variants that are androgen receptor- (AR-) independent. Here we identify the transcription factor ONECUT2 (OC2) as a master regulator of AR networks in metastatic castration-resistant prostate cancer (mCRPC). OC2 acts as a survival factor in mCRPC models, suppresses the AR transcriptional program by direct regulation of AR target genes and the AR licensing factor FOXA1, and activates genes associated with neural differentiation and progression to lethal disease. OC2 appears active in a substantial subset of human prostate adenocarcinoma and neuroendocrine tumors. Inhibition of OC2 by a newly identified small molecule suppresses metastasis in mice. These findings suggest that OC2 displaces AR-dependent growth and survival mechanisms in many cases where AR remains expressed, but where its activity is bypassed. OC2 is also a potential drug target in the metastatic phase of aggressive PC.

Abnormalities in nuclear shape are a well-known feature of cancer, but their contribution to malignant progression remains poorly understood. Here, we show that depletion of the cytoskeletal regulator, Diaphanous-related formin 3 (DIAPH3), or the nuclear membrane–associated proteins, lamin A/C, in prostate and breast cancer cells, induces nuclear shape instability, with a corresponding gain in malignant properties, including secretion of extracellular vesicles that contain genomic material. This transformation is characterized by a reduction and/or mislocalization of the inner nuclear membrane protein, emerin. Consistent with this, depletion of emerin evokes nuclear shape instability and promotes metastasis. By visualizing emerin localization, evidence for nuclear shape instability was observed in cultured tumor cells, in experimental models of prostate cancer, in human prostate cancer tissues, and in circulating tumor cells from patients with metastatic disease. Quantitation of emerin mislocalization discriminated cancer from benign tissue and correlated with disease progression in a prostate cancer cohort. Taken together, these results identify emerin as a mediator of nuclear shape stability in cancer and show that destabilization of emerin can promote metastasis. Significance: This study identifies a novel mechanism integrating the control of nuclear structure with the metastatic phenotype, and our inclusion of two types of human specimens (cancer tissues and circulating tumor cells) demonstrates direct relevance to human cancer. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/21/6086/F1.large.jpg. Cancer Res; 78(21); 6086–97. ©2018 AACR.

Cancer metastasis is highly inefficient and complex. Common features of metastatic cancer cells have been observed using cancer cell lines and genetically reconstituted mouse and human tumor xenograft models. These include cancer cell interaction with the tumor microenvironment, and the ability of cancer cells to sense extracellular stimuli and adapt to adverse growth conditions. This review summarizes the coordinated response of cancer cells to soluble growth factors, such as RANKL, by a unique forward feedback mechanism employing coordinated upregulation of RANKL and c-Met with downregulation of androgen receptor. The RANK-mediated signal network was found to drive epithelial to mesenchymal transition in prostate cancer cells, promote osteomimicry and the ability of prostate cancer cells to assume stem cell and neuroendocrine phenotypes, and confer the ability of prostate cancer cells to home to bone. Prostate cancer cells with activated RANK-mediated signal network were observed to recruit and even transform the non-tumorigenic prostate cancer cells to participate in bone and soft tissue colonization. The coordinated regulation of cancer cell invasion and metastasis by the forward feedback mechanism involving RANKL, c-Met, transcription factors and VEGF-neuropilin could offer new therapeutic opportunities to target prostate cancer bone and soft tissue metastases.

Prostate cancer (PCa) metastasis to bone is lethal and there is no adequate animal model for studying the mechanisms underlying the metastatic process. Here, we report that receptor activator of NF-κB ligand (RANKL) expressed by PCa cells consistently induced colonization or metastasis to bone in animal models. RANK-mediated signaling established a premetastatic niche through a feed-forward loop, involving the induction of RANKL and c-Met, but repression of androgen receptor (AR) expression and AR signaling pathways. Site-directed mutagenesis and transcription factor (TF) deletion/interference assays identified common TF complexes, c-Myc/Max, and AP4 as critical regulatory nodes. RANKL–RANK signaling activated a number of master regulator TFs that control the epithelial-to-mesenchymal transition (Twist1, Slug, Zeb1, and Zeb2), stem cell properties (Sox2, Myc, Oct3/4, and Nanog), neuroendocrine differentiation (Sox9, HIF1α, and FoxA2), and osteomimicry (c-Myc/Max, Sox2, Sox9, HIF1α, and Runx2). Abrogating RANK or its downstream c-Myc/Max or c-Met signaling network minimized or abolished skeletal metastasis in mice. RANKL-expressing LNCaP cells recruited and induced neighboring non metastatic LNCaP cells to express RANKL, c-Met/activated c-Met, while downregulating AR expression. These initially non-metastatic cells, once retrieved from the tumors, acquired the potential to colonize and grow in bone. These findings identify a novel mechanism of tumor growth in bone that involves tumor cell reprogramming via RANK–RANKL signaling, as well as a form of signal amplification that mediates recruitment and stable transformation of non-metastatic bystander dormant cells.

Identification of factors that mediate visceral and bone metastatic spread and subsequent bone remodeling events is highly relevant to successful therapeutic intervention in advanced human prostate cancer. TBX2, a T-box family transcription factor that negatively regulates cell-cycle inhibitor p21, plays critical roles during embryonic development, and recent studies have highlighted its role in cancer. Here, we report that TBX2 is overexpressed in human prostate cancer specimens and bone metastases from xenograft mouse models of human prostate cancer. Blocking endogenous TBX2 expression in PC3 and ARCaPM prostate cancer cell models using a dominant-negative construct resulted in decreased tumor cell proliferation, colony formation, and invasion in vitro. Blocking endogenous TBX2 in human prostate cancer mouse xenografts decreased invasion and abrogation of bone and soft tissue metastasis. Furthermore, blocking endogenous TBX2 in prostate cancer cells dramatically reduced bone-colonizing capability through reduced tumor cell growth and bone remodeling in an intratibial mouse model. TBX2 acted in trans by promoting transcription of the canonical WNT (WNT3A) promoter. Genetically rescuing WNT3A levels in prostate cancer cells with endogenously blocked TBX2 partially restored the TBX2-induced prostate cancer metastatic capability in mice. Conversely, WNT3A-neutralizing antibodies or WNT antagonist SFRP-2 blocked TBX2-induced invasion. Our findings highlight TBX2 as a novel therapeutic target upstream of WNT3A, where WNT3A antagonists could be novel agents for the treatment of metastasis and for skeletal complications in prostate cancer patients. Cancer Res; 77(6); 1331–44. ©2017 AACR.

Background: Treatment of prostate cancer by hormone suppression leads to the appearance of aggressive variants of metastatic castration-resistant prostate cancer (mCRPC) with variable or no dependence on the androgen receptor (AR). Here we identify the neural transcription factor ONECUT2 as a negative regulator of the AR axis, that emerges in aggressive PC variants to control transcriptional networks linked to CRPC and neuroendocrine (NE) differentiation. We further demonstrate that ONECUT2 can be targeted with a small molecule that inhibits mCRPC metastasis in mice. Methods: ONECUT2 was confirmed as a mCRPC-relevant protein and to be targetable by computational modeling and bioinformatics, enforced expression, silencing, microarray, ChIP-Seq, immunohistochemistry, immunofluorescence, quantitative imaging, functional assays, in vivo experiments, and surface plasmon resonance. Results: We have performed a master regulator analysis using 260 mCRPC transcriptome profiles and developed a model transcription factor network for mCRPC that associates ONECUT2 for the first time with metastatic progression. Gene expression profiling of ONECUT2-engineered PC cell lines has allowed us to generate a ONECUT2 activity signature that reveals high positive correlation with pro-neural and aggressive PC signatures, and a negative correlation with AR activation pathways. We find that ONECUT2 is a negative regulator of AR expression and a repressor of its transcriptional program through direct binding to AR target genes. We also find that ONECUT2 is significantly increased in human NEPC and confers NE properties to CRPC through direct downregulation of the NEPC inhibitor FOXA1 and direct upregulation of the NEPC driver PEG10. Finally, we show that ONECUT2 is required for cell growth and survival and that it can be targeted with a small molecule that, by binding to the ONECUT2 C-terminal DNA binding domain, inhibits mCRPC metastasis in mice. Conclusions: OC2 is a master regulator of aggressive mCRPC variants that drives AR-dependent adenocarcinoma toward NEPC differentiation by blocking AR/FOXA1-activity and inducing PEG10. OC2 can be targeted with a drug-like small molecule that inhibits CRPC metastasis in mice. Patients with OC2 active tumors may benefit from OC2 inhibitor therapy. Citation Format: Mirja Rotinen, Sungyong You, Julie Yang, Simon Coetzee, Wen-Chin Huang, Fangjin Huang, Xinlei Pan, Alberto Yáñez, Dennis Hazelett, Chia-Yi Chu, Leland Chung, Stephen Freedland, Dolores Di Vizio, Isla Garraway, Ramachandran Murali, Beatrice Knudsen, Michael Freeman. ONECUT2 is a targetable master regulator of aggressive variants of castration-resistant prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A047.

Introduction and Objective: Keratins (KRTs) were reported to interact with multiple cellular proteins and initiate signaling cascades that promote cell proliferation, migration and metastasis in cancers. Keratin13 (KRT13), a member of the intermediate filament proteins, plays important role in breast cancer progression and metastasis. The objectives of this study are to determine the role and molecular mechanism of KRT13 in promoting breast cancer growth and metastatic progression. Methods and Results: We evaluated KRT13 protein expression in 58 primary breast cancer tissues by immunohistochesmistry and found that KRT13 is overexpressed in metastatic breast cancer specimens (12/18) (p Conclusion: KRT13 exerts a novel function in the regulation of breast cancer cell growth, progression and metastasis via alterations of nuclear translocation of plakoglobin that modulates downstream c-Myc-dependent signaling, inducing stem cell-like phenotype and metastasis of breast cancer. Our findings could reveal potential novel targeting of breast cancer progression and metastasis. Funded by an NCI P01 CA098912 and a Board of Governors Chair of Cancer Research fund from Cedars-Sinai Medical Center to LWK Chung. Citation Format: Lijuan Yin, Gina Chia-Yi Chu, Qinlong Li, Liyuan Yin, Michael Lewis, Mourad Tighiouart, Leland W. k. Chung, Haiyen E. Zhau. KRT13 promotes stemness and drives metastasis in breast cancer through direct interaction with plakoglobin-desmoplakin complexes regulating c-Myc signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5002.

Prostate cancer remains the most frequently diagnosed cancer in men in North America, and despite recent advances in treatment patients with metastatic disease continue to have poor five-year survival rates. Recent studies in prostate cancer have revealed the critical role of the tumor microenvironment in the initiation and progression to advanced disease. Experimental data have uncovered a reciprocal relationship between the cells in the microenvironment and malignant tumor cells in which early changes in normal tissue microenvironment can promote tumorigenesis and in turn tumor cells can promote further pro-tumor changes in the microenvironment. In the tumor microenvironment, the presence of persistent immune infiltrates contributes to the recruitment and reprogramming of other non-immune stromal cells including cancer-associated fibroblasts and a unique recently identified population of metastasis-initiating cells (MICs). These MICs, which can also be found as part of the circulating tumor cell (CTC) population in PC patients, promote cancer cell transformation, enhance metastatic potential and confer therapeutic resistance. MICs act can on other cells within the tumor microenvironment in part by secreting exosomes that reprogram adjacent stromal cells to create a more favorable tumor microenvironment to support continued cancer growth and progression. We review here the current data on the intricate relationship between inflammation, reactive stroma, tumor cells and disease progression in prostate cancer.

FYN is a SRC family kinase (SFK) that has been shown to be up-regulated in human prostate cancer (PCa) tissues and cell lines. In this study, we observed that FYN is strongly up-regulated in human neuroendocrine PCa (NEPC) tissues and xenografts, as well as cells derived from a NEPC transgenic mouse model. In silico analysis of FYN expression in prostate cancer cell line databases revealed an association with the expression of neuroendocrine (NE) markers such as CHGA, CD44, CD56, and SYP. The loss of FYN abrogated the invasion of PC3 and ARCaPM cells in response to MET receptor ligand HGF. FYN also contributed to the metastatic potential of NEPC cells in two mouse models of visceral metastasis with two different cell lines (PC3 and TRAMPC2-RANKL). The activation of MET appeared to regulate neuroendocrine (NE) features as evidenced by increased expression of NE markers in PC3 cells with HGF. Importantly, the overexpression of FYN protein in DU145 cells was directly correlated with the increase of CHGA. Thus, our data demonstrated that the neuroendocrine differentiation that occurs in PCa cells is, at least in part, regulated by FYN kinase. Understanding the role of FYN in the regulation of NE markers will provide further support for ongoing clinical trials of SFK and MET inhibitors in castration-resistant PCa patients.

Purpose: MicroRNAs in the delta-like 1 homolog–deiodinase, iodothyronine 3 (DLK1-DIO3) cluster have been shown to be critical for embryonic development and epithelial to mesenchymal transition (EMT). DLK1-DIO3 cluster miRNAs are elevated in the serum of patients with metastatic cancer. However, the biologic functions of these miRNAs in the EMT and metastasis of cancer cells are poorly understood. We previously demonstrated the oncogenic and metastatic role of miR-409-3p/5p, a member of this cluster, in prostate cancer. In this study, we defined the role of miR-154* and miR-379, two key members of this cluster, in prostate cancer progression and bone metastasis in both cell line models and clinical specimens. Experimental Design: Genetic manipulation of miR-154* and miR-379 was performed to determine their role in tumor growth, EMT, and bone metastasis in mouse models. We determined the expression of miR-154* in prostate cancer clinical samples and bone metastasis samples using in situ hybridization and quantum dot labeling. Results: Elevated expression of miR-154* and miR-379 was observed in bone metastatic prostate cancer cell lines and tissues, and miR-379 expression correlated with progression-free survival of patients with prostate cancer. Intracardiac inoculation (to mimic systemic dissemination) of miR-154* inhibitor-treated bone metastatic ARCaPM prostate cancer cells in mice led to decreased bone metastasis and increased survival. Conclusion: miR-154* and miR-379 play important roles in prostate cancer biology by facilitating tumor growth, EMT, and bone metastasis. This finding has particular translational importance because miRNAs in the DLK1-DIO3 cluster can be attractive biomarkers and possible therapeutic targets to treat bone metastatic prostate cancer. Clin Cancer Res; 20(24); 6559–69. ©2014 AACR.

Purpose: miR-409-3p/-5p is a miRNA expressed by embryonic stem cells, and its role in cancer biology and metastasis is unknown. Our pilot studies demonstrated elevated miR-409-3p/-5p expression in human prostate cancer bone metastatic cell lines; therefore, we defined the biologic impact of manipulation of miR-409-3p/-5p on prostate cancer progression and correlated the levels of its expression with clinical human prostate cancer bone metastatic specimens. Experimental Design: miRNA profiling of a prostate cancer bone metastatic epithelial-to-mesenchymal transition (EMT) cell line model was performed. A Gleason score human tissue array was probed for validation of specific miRNAs. In addition, genetic manipulation of miR-409-3p/-5p was performed to determine its role in tumor growth, EMT, and bone metastasis in mouse models. Results: Elevated expression of miR-409-3p/-5p was observed in bone metastatic prostate cancer cell lines and human prostate cancer tissues with higher Gleason scores. Elevated miR-409-3p expression levels correlated with progression-free survival of patients with prostate cancer. Orthotopic delivery of miR-409-3p/-5p in the murine prostate gland induced tumors where the tumors expressed EMT and stemness markers. Intracardiac inoculation (to mimic systemic dissemination) of miR-409-5p inhibitor–treated bone metastatic ARCaPM prostate cancer cells in mice led to decreased bone metastasis and increased survival compared with control vehicle–treated cells. Conclusion: miR-409-3p/-5p plays an important role in prostate cancer biology by facilitating tumor growth, EMT, and bone metastasis. This finding bears particular translational importance as miR-409-3p/-5p appears to be an attractive biomarker and/or possibly a therapeutic target to treat bone metastatic prostate cancer. Clin Cancer Res; 20(17); 4636–46. ©2014 AACR.

Tumor cells are inherently heterogeneous and often exhibit diminished adhesion, resulting in the shedding of tumor cells into the circulation to form circulating tumor cells (CTCs). A fraction of these are live CTCs with potential of metastatic colonization whereas others are at various stages of apoptosis making them likely to be less relevant to understanding the disease. Isolation and characterization of live CTCs may augment information yielded by standard enumeration to help physicians to more accurately establish diagnosis, choose therapy, monitor response, and provide prognosis. We previously reported on a group of near-infrared (NIR) heptamethine carbocyanine dyes that are specifically and actively transported into live cancer cells. In this study, this viable tumor cell-specific behavior was utilized to detect live CTCs in prostate cancer patients. Peripheral blood mononuclear cells (PBMCs) from 40 patients with localized prostate cancer together with 5 patients with metastatic disease were stained with IR-783, the prototype heptamethine cyanine dye. Stained cells were subjected to flow cytometric analysis to identify live (NIR(+)) CTCs from the pool of total CTCs, which were identified by EpCAM staining. In patients with localized tumor, live CTC counts corresponded with total CTC numbers. Higher live CTC counts were seen in patients with larger tumors and those with more aggressive pathologic features including positive margins and/or lymph node invasion. Even higher CTC numbers (live and total) were detected in patients with metastatic disease. Live CTC counts declined when patients were receiving effective treatments, and conversely the counts tended to rise at the time of disease progression. Our study demonstrates the feasibility of applying of this staining technique to identify live CTCs, creating an opportunity for further molecular interrogation of a more biologically relevant CTC population.

We have previously shown that human prostate cancer cells are capable of acquiring malignant attributes through interaction with stromal cells in the tumor microenvironment, while the interacting stromal cells can also become affected with both phenotypic and genotypic alterations. This study used a co-culture model to investigate the mechanism underlying the co-evolution of cancer and stromal cells. Red fluorescent androgen-dependent LNCaP prostate cancer cells were cultured with a matched pair of normal and cancer-associated prostate myofibroblast cells to simulate cancer-stromal interaction, and cellular changes in the co-culture were documented by tracking the red fluorescence. We found frequent spontaneous fusions between cancer and stromal cells throughout the co-culture. In colony formation assays assessing the fate of the hybrid cells, most of the cancer-stromal fusion hybrids remained growth-arrested and eventually perished. However, some of the hybrids survived to form colonies from the co-culture with cancer-associated stromal cells. These derivative clones showed genomic alterations together with androgen-independent phenotype. The results from this study reveal that prostate cancer cells are fusogenic, and cancer-stromal interaction can lead to spontaneous fusion between the two cell types. While a cancer-stromal fusion strategy may allow the stromal compartment to annihilate invading cancer cells, certain cancer-stromal hybrids with increased survival capability may escape annihilation to form a derivative cancer cell population with an altered genotype and increased malignancy. Cancer-stromal fusion thus lays a foundation for an incessant co-evolution between cancer and the cancer-associated stromal cells in the tumor microenvironment.

Introduction: FYN is a Src family kinase (SFK) member that has been shown to be upregulated in human prostate cancer tissues and cell lines (PCa). The majority of the studies on SRC kinases have focused on the classical c-SRC. We demonstrated previously that knockdown of FYN in PCa cell lines leads to reduced tumor growth in vitro and in vivo. We hypothesized that MET, an oncogenic tyrosine kinase receptor implicated in metastasis and therapeutic resistance of a variety of solid tumors is regulated by FYN expression in prostate cancer. Methods: We performed in vitro growth assays, migration and invasion assays and studied PCa cell response to HGF in wildtype, FYN-depleted and FYN-reconstituted cells. Additionally, we studied the role of FYN in promoting metastasis in vivo in xenograft mouse models. Summary: In this study, we demonstrate that knockdown of FYN leads to reduced migration, invasion and metastatic dissemination of PCa cells in vitro and in vivo. Conversely, ectopic expression of FYN promotes enhanced tumor growth and invasion. Hepatocyte growth factor (HGF) induced activation of MET is perturbed in FYN knockdown cells and overexpression of FYN leads to excessive MET activation suggesting a possible feedback loop between FYN and MET in prostate cancer. Consistent these findings, PC3 prostate cancer cells overexpressing FYN are resistant to treatment with a MET inhibitor upon ligand stimulation. More importantly, we show that FYN promotes epithelial to mesenchymal transition (EMT) of PCa cells and knockdown of FYN leads to reversal of EMT and alters radiation sensitivity of PCa cells. To further identify the molecular mechanism of FYN mediated regulation of tumor growth, we performed integrated RNA sequencing analysis of FYN knockdown cells and found MET and a network of molecular targets that appear to regulate FYN mediated tumor progression and metastasis. Conclusions: We demonstrate that FYN mediated MET activation promotes PCa invasion, EMT and metastasis. None of the SFK inhibitors currently in clinical trials are specific for SFKs alone. Therefore, it is imperative to develop and test novel inhibitors that selectively target FYN and other SFKs. Our findings have therapeutic implications since clinical trials are being performed for both SFK inhibitors and MET inhibitors in castration resistant PCa patients. Our studies reveal that FYN and MET interact in PCa cells and this interaction is clinically relevant given the success of XL-184 (a MET inhibitor) in phase III clinical trials in human prostate cancer. Clinical studies with translational endpoints are the need of the hour with agents that effectively inhibit FYN and MET in a variety of settings. Citation Format: Murali Gururajan, Margarit Sievert, Sheldon Mink, Chia-Yi (Gina) Chu, Matteo Morello, Jake Lichterman, Michael R. Freeman, Edwin M. Posadas. SRC family kinase FYN promotes MET tyrosine kinase activation, epithelial to mesenchymal transition and metastasis in human prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3459. doi:10.1158/1538-7445.AM2014-3459

BACKGROUND:Bone metastasis is the most lethal form of several cancers. The β2-microglobulin (β2-M)/hemochromatosis (HFE) complex plays an important role in cancer development and bone metastasis. We demonstrated previously that overexpression of β2-M in prostate, breast, lung and renal cancer leads to increased bone metastasis in mouse models. Therefore, we hypothesized that β2-M is a rational target to treat prostate cancer bone metastasis. RESULTS:In this study, we demonstrate the role of β2-M and its binding partner, HFE, in modulating radiation sensitivity and chemo-sensitivity of prostate cancer. By genetic deletion of β2-M or HFE or using an anti-β2-M antibody (Ab), we demonstrate that prostate cancer cells are sensitive to radiation in vitro and in vivo. Inhibition of β2-M or HFE sensitized prostate cancer cells to radiation by increasing iron and reactive oxygen species and decreasing DNA repair and stress response proteins. Using xenograft mouse model, we demonstrate that anti-β2-M Ab sensitizes prostate cancer cells to radiation treatment. Additionally, anti-β2-M Ab was able to prevent tumor growth in an immunocompetent spontaneous prostate cancer mouse model. Since bone metastasis is lethal, we used a bone xenograft model to test the ability of anti-β2-M Ab and radiation to block tumor growth in the bone. Combination treatment significantly prevented tumor growth in the bone xenograft model by inhibiting β2-M and inducing iron overload. In addition to radiation sensitive effects, inhibition of β2-M sensitized prostate cancer cells to chemotherapeutic agents. CONCLUSION:Since prostate cancer bone metastatic patients have high β2-M in the tumor tissue and in the secreted form, targeting β2-M with anti-β2-M Ab is a promising therapeutic agent. Additionally, inhibition of β2-M sensitizes cancer cells to clinically used therapies such as radiation by inducing iron overload and decreasing DNA repair enzymes.

Introduction and Objective: While prostate cancer (PCa) is curable at early diagnosis, metastatic PCa is terminal. Recent studies have shown that Receptor Activator of NF kappa-B Ligand, RANKL, markedly enhances bone metastasis (70-100%) by the LNCaP human PCa cell line in mice compared to neo controls (0%). Multivariable tumor and microenvironmental factors in vivo make it difficult to identify the specific cellular response induced by RANKL causing PCa cell homing and growth in bone. Current in vitro 2D cultures lack mechanical and biochemical properties mimicking the bone microenvironment. Our preliminary data showed integrin alpha2 is elevated in RANKL-over-expressing LNCaP cells. PCa interaction with the major bone matrix protein Col1 may contribute to the metastatic potential of LNCAP-RANKL cells. This study used 3D systems to explore the role of RANKL in PCa-bone matrix interactions. Methods: 3D cultures of RANKL-expressing (highly metastatic) or non-metastatic LNCaP cells were evaluated in 2D, suspension cultures, on type 1 collagen (Col1) coated polymeric meshes, embedded in Col1 gel or hydrogel mixed with Col1 for growth, morphology, motility, and gene expression. Time-dependent cell migration was assessed by a time-lapse imaging system. Results: LNCAP-RANKL cells and Neo-expressing controls grew as aggregates in all 3D cultures but not 2D cultures. Col1 promoted less compact and more widespread aggregation of LNCAP-RANKL cells in the 3D cultures, exhibiting more motile and directional migratory behavior. A molecular signature of 3D growth was generated and compared among different conditions. Consistent with 2D studies, 3D qRT-PCR data showed RANKL drove epithelial-to-mesenchymal transition (EMT), shown by increased expression of vimentin and N-cadherin but decreased E-cadherin. Decreased expression of androgen receptor and PSA was observed in RANKL-expressing PCa cells. Supporting the migration data, the presence of Col1 drove RANKL-expressing PCa cells to express higher levels of c-Met protooncogene, sensitizing PCa cell responses to the c-Met ligand, hepatocyte growth factor (HGF). Conclusions: 3D culture systems were established to study interactions between Col1 and metastatic or non-metastatic PCa cells. 3D culture systems supported PCa cell growth, while maintaining their classical markers, validating the reliability of our 3D systems. Bone metastatic PCa cells preferentially exhibited EMT, increased expression of α2 integrin, and c-Met protooncogene. These 3D systems will be used to assess liquid biopsy specimens harvested from human PCa patients to improve prognosis and therapeutic follow-up. Citation Format: Shabnam Ziaee, Shirly Sieh, Chia-Yi Chu, Ruoxiang Wang, Dietmar Hutmacher, Colleen Nelson, Chin-Lin Guo, Leland W.K. Chung. Using three-dimensional (3-D) culture systems to delineate the role of RANKL in metastatic prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B11.