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Clinical studies have shown that subsets of patients with cancer achieve a significant benefit from Aurora kinase inhibitors, suggesting an urgent need to identify biomarkers for predicting drug response. Chromodomain helicase DNA binding protein 1 (CHD1) is involved in chromatin remodeling, DNA repair, and transcriptional plasticity. Prior studies have demonstrated that CHD1 has distinct expression patterns in cancers with different molecular features, but its impact on drug responsiveness remains understudied. Here, we show that CHD1 promotes the susceptibility of prostate cancer cells to inhibitors targeting Aurora kinases, while depletion of CHD1 impairs their efficacy in vitro and in vivo. Pan-cancer drug sensitivity analyses revealed that high expression of CHD1 was associated with increased sensitivity to Aurora kinase A (AURKA) inhibitors. Mechanistically, KPNA2 served as a direct target of CHD1 and suppressed the interaction of AURKA with the coactivator TPX2, thereby rendering cancer cells more vulnerable to AURKA inhibitors. Consistent with previous research reporting that loss of PTEN elevates CHD1 levels, studies in a genetically engineered mouse model, patient-derived organoids, and patient samples showed that PTEN defects are associated with a better response to AURKA inhibition in advanced prostate cancer. These observations demonstrate that CHD1 plays an important role in modulating Aurora kinases and drug sensitivities, providing new insights into biomarker-driven therapies targeting Aurora kinases for future clinical studies. Significance: CHD1 plays a critical role in controlling AURKA activation and promoting Aurora kinase inhibitor sensitivity, providing a potential clinical biomarker to guide cancer treatment.

Prostate cancer (PCa) typically spreads to the bone, and this distribution is attributed to the central role of the microenvironment in progression. However, metastasis to the adrenal glands, while not as common, does occur. The biology that accounts for adrenal metastases may be attributed to the unique local steroid metabolome and co-clinical characterization may elucidate the role steroid biosynthesis plays in PCa progression.Three patients with metastatic PCa who had archived tumor tissue from an adrenalectomy were retrospectively identified, and one adrenal metastasis was developed into a xenograft (MDA-PCa-250). The adrenal metastases were characterized by performing somatic DNA whole exome sequencing (WES), RNA-Seq, immunohistochemistry (IHC), and steroid metabolite quantitation. The influence of steroid metabolites on adrenal metastasis cells and tumor growth was tested in vitro and in vivo.Clinically, adrenalectomy was performed during castration-resistant oligometastatic disease, and two men experienced resensitization to leuprolide. Somatic DNA WES revealed heterogeneous alterations in tumor suppressor and DNA damage repair pathway genes. Adrenal metastases had active androgen receptor (AR) signaling by IHC, and RNA-Seq supported a potential role for adrenal androgen precursor metabolism in activating the AR. Steroid quantitation suggested the adrenal androgen precursors were converted into testosterone in these metastases, and stable isotope tracing of an organoid from MDA-PCa-250 confirmed the capability of adrenal metastases to biosynthesize testosterone from adrenal precursors. In vitro testing of a cell line derived from MDA-PCa-250 showed that testosterone and cortisol stimulated tumor cell growth. In vivo experiments demonstrated that MDA-PCa-250 grew in intact mice with circulating testosterone, but not in castrated mice.PCa adrenal metastases depend upon AR signaling driven by androgen precursors, androstenedione and dehydroepiandrosterone, available in the microenvironment, despite the presence of heterogeneous somatic DNA alterations. Moreover, MDA-PCa-250 provides a preclinical model that can recapitulate the unique androgen-dependence of adrenal metastases.This study does not report the clinical results of a clinical trial, but it does use samples from a completed clinical trial that is registered with clinicaltrials.gov (NCT01254864).

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.

Metastatic prostate cancer in the bone induces bone-forming lesions that contribute to progression and therapy resistance. Prostate cancer–induced bone formation originates from endothelial cells (EC) that have undergone endothelial-to-osteoblast (EC-to-OSB) transition in response to tumor-secreted BMP4. Current strategies targeting prostate cancer–induced bone formation are lacking. Here, we show that activation of retinoic acid receptor (RAR) inhibits EC-to-OSB transition and reduces prostate cancer–induced bone formation. Treatment with palovarotene, an RARγ agonist being tested for heterotopic ossification in fibrodysplasia ossificans progressiva, inhibited EC-to-OSB transition and osteoblast mineralization in vitro and decreased tumor-induced bone formation and tumor growth in several osteogenic prostate cancer models, and similar effects were observed with the pan-RAR agonist all-trans-retinoic acid (ATRA). Knockdown of RARα, β, or γ isoforms in ECs blocked BMP4-induced EC-to-OSB transition and osteoblast mineralization, indicating a role for all three isoforms in prostate cancer–induced bone formation. Furthermore, treatment with palovarotene or ATRA reduced plasma Tenascin C, a factor secreted from EC-OSB cells, which may be used to monitor treatment response. Mechanistically, BMP4-activated pSmad1 formed a complex with RAR in the nucleus of ECs to activate EC-to-OSB transition. RAR activation by palovarotene or ATRA caused pSmad1 degradation by recruiting the E3-ubiquitin ligase Smad ubiquitination regulatory factor1 (Smurf1) to the nuclear pSmad1/RARγ complex, thus blocking EC-to-OSB transition. Collectively, these findings suggest that palovarotene can be repurposed to target prostate cancer–induced bone formation to improve clinical outcomes for patients with bone metastasis. Significance: This study provides mechanistic insights into how RAR agonists suppress prostate cancer–induced bone formation and offers a rationale for developing RAR agonists for prostate cancer bone metastasis therapy. See related commentary by Bhowmick and Bhowmick, p. 2975

Genetic inactivation of PTEN is common in prostate cancer and correlates with poorer prognosis. We previously identified CHD1 as an essential gene in PTEN-deficient cancer cells. Here, we sought definitive in vivo genetic evidence for, and mechanistic understanding of, the essential role of CHD1 in PTEN-deficient prostate cancer. In Pten and Pten/Smad4 genetically engineered mouse models, prostate-specific deletion of Chd1 resulted in markedly delayed tumor progression and prolonged survival. Chd1 deletion was associated with profound tumor microenvironment (TME) remodeling characterized by reduced myeloid-derived suppressor cells (MDSC) and increased CD8+ T cells. Further analysis identified IL6 as a key transcriptional target of CHD1, which plays a major role in recruitment of immunosuppressive MDSCs. Given the prominent role of MDSCs in suppressing responsiveness to immune checkpoint inhibitors (ICI), our genetic and tumor biological findings support combined testing of anti-IL6 and ICI therapies, specifically in PTEN-deficient prostate cancer. Significance: We demonstrate a critical role of CHD1 in MDSC recruitment and discover CHD1/IL6 as a major regulator of the immunosuppressive TME of PTEN-deficient prostate cancer. Pharmacologic inhibition of IL6 in combination with immune checkpoint blockade elicits robust antitumor responses in prostate cancer. This article is highlighted in the In This Issue feature, p. 1241

Background: The methylthioadenosine phosphorylase (MTAP) gene encodes an essential enzyme for the salvage pathway of adenosine synthesis and is frequently lost in different types of cancer including urothelial bladder carcinoma. Therefore, MTAP-deficient tumors are theoretically very sensitive to anti-folate agents such as pemetrexed that can effectively block the de novo pathway of adenosine synthesis and as a result, create a state of synthetic lethality. We thus hypothesize that tumor MTAP gene deficiency is associated with response to pemetrexed therapy in bladder cancer. Methods: In this study, we investigated MTAP gene deficiency rates in the TCGA database and confirmed MTAP protein loss by immunohistochemistry using a tumor tissue microarray containing bladder tumor tissues from 151 patients. We then performed in vitro and in vivo studies using MTAP-proficient and MTAP-deficient human bladder cancer cell lines. Functional loss of MTAP was verified with mass spectrometry, which detects its substrate methylthioadenosine (MTA) levels. We also correlated these pre-clinical studies with clinical response data on patients with metastatic bladder cancer treated with pemetrexed. Results: We identified that 27.8% bladder cancer patients have MTAP protein deficiency, which is consistent with exome sequencing data from the TCGA database. In vitro data showed MTAP-deficient human bladder cancer cell lines were significantly more sensitive to pemetrexed, with IC50 at least 40 times lower than MTAP-proficient cell lines. Subsequent knockdown of the MTAP gene in MTAP-proficient cell lines increased sensitivities to pemetrexed treatment. Consistent with the in vitro data, pemetrexed significantly inhibited the growth of MTAP-deficient or knockdown xenograft tumors but not MTAP-proficient tumors. Furthermore, 4 of 4 (100%) patients with MTAP-deficient metastatic bladder cancer responded to pemetrexed treatment, whereas only 1 of 11 (9%) patients with MTAP-proficient metastatic bladder cancer responded to pemetrexed. Conclusion: Our data demonstrate that MTAP gene loss in urothelial bladder cancer leads to a metabolic state of synthetic lethality with pemetrexed therapy. Therefore, bladder tumor MTAP loss should be further investigated as a potential biomarker for selection of patients for anti-folate therapy. Citation Format: Jianfeng Chen, Omar Alhalabi, Guangchun Han, Wei-Lien Wang, Xin-Qiao Zhang, Jian H. Song, Lidia P. Lopez, Sumankalai Ramachandran, Anh G. Hoang, Tyrone Garnett, Matthew Campbell, Amishi Y. Shah, Jennifer Wang, Arlene O. Siefker-Radtke, Shi-Ming Tu, Mark Titus, Charles C. Guo, Gary E. Gallick, Eleni Efstathiou, William F. Benedict, Christopher J. Logothetis, Thai H. Ho, Linghua Wang, Jianjun Gao. MTAP gene deficiency creates vulnerability to anti-folate therapy in urothelial bladder carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 385.

4521 Background: The MTAP gene encodes an essential enzyme for the salvage pathway of nucleotide synthesis and is frequently deleted in UBC. Anti-folate agents such as pemetrexed can effectively inhibit the de novo pathway of nucleotide synthesis and as a result, create a synthetic lethality in MTAP deficient UBC. We hypothesize that MTAP gene loss correlates with enhanced response to pemetrexed in UBC. Methods: We investigated MTAP gene deletion rates in the TCGA database and determined MTAP protein loss rates by immunohistochemistry (IHC) using a UBC tissue microarray (TMA) from 151 patients (pts). We then performed in vitro and in vivo studies using MTAP proficient and MTAP deficient bladder cancer cell lines. At the clinical level, we performed a retrospective analysis based on MTAP status of pts treated with pemetrexed as 2nd line at our institution between 2014 and 2018. We are now enrolling pts in a single-arm, open-label, phase II clinical trial (NCT02693717) with pemetrexed in pts with MTAP deficient UBC. Results: Per our TCGA and TMA IHC analyses, MTAP deficiency rate was 25.9% and 27.8%, respectively. MTAP deficient UBC cell lines were at least 40 times more sensitive to pemetrexed than MTAP proficient lines. Knockdown of the MTAP gene increased apoptosis rate by pemetrexed from approximately 20% to 60%. Additionally, pemetrexed significantly inhibited the growth of MTAP deficient or knockdown xenograft tumors but not MTAP proficient tumors. Retrospective analysis of 12 pts using RECIST criteria indicated that all 4 MTAP deficient UBC pts responded to pemetrexed whereas only 1 of 8 (12.5%) MTAP proficient UBC pts responded. Of the 6 pts enrolled on the clinical trial, 3 (50%) had complete or partial response, 1 had stable disease, 1 was not evaluable and 1 had disease progression. Combined analysis of the entire experience demonstrates a higher response rate in MTAP deficient UBC (70%) as compared to MTAP proficient UBC (12.5%). Conclusions: Our preclinical and clinical data demonstrate that MTAP loss in UBC leads to a state of synthetic lethality when treated with pemetrexed and should be further investigated as a novel biomarker to predict response to anti-folate agents.

Purpose: To investigate the antitumor effects of targeting Src and tubulin in mucinous ovarian carcinoma. Experimental Design: The in vitro and in vivo effects and molecular mechanisms of KX-01, which inhibits Src pathway and tubulin polymerization, were examined in mucinous ovarian cancer models. Results: In vitro studies using RMUG-S and RMUG-L cell lines showed that KX-01 inhibited cell proliferation, induced apoptosis, arrested the cell cycle at the G2–M phase, and enhanced the cytotoxicity of oxaliplatin in the KX-01–sensitive cell line, RMUG-S. In vivo studies showed that KX-01 significantly decreased tumor burden in RMUG-S and RMUG-L mouse models relative to untreated controls, and the effects were greater when KX-01 was combined with oxaliplatin. KX-01 alone and in combination with oxaliplatin significantly inhibited tumor growth by reducing cell proliferation and inducing apoptosis in vivo. PTEN knock-in experiments in RMUG-L cells showed improved response to KX-01. Reverse phase protein array analysis showed that in addition to blocking downstream molecules of Src family kinases, KX-01 also activated acute stress-inducing molecules. Conclusion: Our results showed that targeting both the Src pathway and tubulin with KX-01 significantly inhibited tumor growth in preclinical mucinous ovarian cancer models, suggesting that this may be a promising therapeutic approach for patients with mucinous ovarian carcinoma. Clin Cancer Res; 19(23); 6532–43. ©2013 AACR.

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.

The receptor tyrosine kinase, MET, has been implicated in tumorigenesis and metastasis of many solid tumors, by multiple mechanisms, including cross talk with epidermal growth factor receptor. In this study, we examined the role of insulin-like growth factor receptor-1 (IGF-1R) signaling in MET activation, focusing on prostate cancer cells. Stimulation of the prostate cancer cell line PC3 with IGF-1 induces a delayed phosphorylation of MET at multiple sites (indicative of full activation), reaching a maximum 18 hr after IGF-1 addition. MET activation does not require the sole MET ligand hepatocyte growth factor (HGF), but does require transcription to occur. Furthermore, direct injection of IGF-1 is sufficient to induce MET activation in vivo, in a PC3 xenograft model. Pharmacologic or genetic inhibition of the tyrosine kinase, Src, abolishes MET phosphorylation, and expression of activated Src is sufficient to induce Met phosphorylation in the absence of IGF-1 stimulation. Activated MET is essential for IGF-1-mediated increased migration of PC3 cells, demonstrating an important biologic effect of IGF-1-mediated MET activation. Finally, we demonstrate that IGF-1-induced delayed MET activation occurs in multiple cell lines which express both the receptors, suggesting that IGF-1R-mediated MET activation may contribute to tumorigenic properties of multiple cancer types when both growth factor receptors are expressed. The results further suggest that MET may be activated by multiple receptor tyrosine kinase receptors, and dual targeting of these receptors may be important therapeutically.

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.

BACKGROUND Dasatinib, an inhibitor of Src-family kinases, combined with docetaxel in men with castrate-resistant prostate cancer (CRPC), affects bone turnover markers in a phase I/II clinical trial in metastatic CRPC. Only a subset of men benefit from this therapy, and predictive markers are lacking. We hypothesized a role for insulin-like growth factor-1 (IGF-1) as a predictive marker, since IGF-1 is important in both prostate cancer progression and bone development. Hence, we determined the association of IGF-1 expression to treatment response, and whether this expression resulted from tumor cells, the microenvironment, or their interactions. METHODS We measured serum IGF-1 levels in men with CRPC treated with dasatinib plus docetaxel. To investigate the source of IGF-1, we utilized two different mouse models harboring human prostate cancer cells, and used species-specific IGF-1 ELISA kits (mouse vs. human). RESULTS In men with CRPC, an increase in IGF-1 levels after one cycle of treatment with dasatinib and docetaxel is associated with a higher response rate and longer duration of treatment. Xenograft experiments with subcutaneous and intratibial injection of prostate cancer cells suggest that direct interaction of prostate cancer cells with bone microenvironment is necessary for IGF-1 induction, is entirely host-derived, and occurs only in mice that respond to dasatinib-based therapy. CONCLUSION Our results support a role for serum IGF-1 as a potential biomarker for benefit from dasatinib-based combination treatments in CRPC. Prostate 73: 979–985, 2013. © 2013 Wiley Periodicals, Inc.

MicroRNA (miRNA) expression profile can be used as prognostic marker for human cancers. We aim to explore the significance of miRNAs in colorectal cancer (CRC) metastasis.We performed miRNA microarrays using primary CRC tissues from patients with and without metastasis, and validated selected candidates in 85 CRC samples by quantitative real-time PCR (qRT-PCR). We tested metastatic activity of selected miRNAs and identified miRNA targets by prediction algorithms, qRT-PCR, western blot and luciferase assays. Clinical outcomes were analysed in six sets of CRC cases (n=449), including The Cancer Genome Atlas (TCGA) consortium and correlated with miR-224 status. We used the Kaplan-Meier method and log-rank test to assess the difference in survival between patients with low or high levels of miR-224 expression.MiR-224 expression increases consistently with tumour burden and microsatellite stable status, and miR-224 enhances CRC metastasis in vitro and in vivo. We identified SMAD4 as a miR-224 target and observed negative correlation (Spearman Rs=-0.44, p0.0001) between SMAD4 and miR-224 expression in clinical samples. Patients with high miR-224 levels display shorter overall survival in multiple CRC cohorts (p=0.0259, 0.0137, 0.0207, 0.0181, 0.0331 and 0.0037, respectively), and shorter metastasis-free survival (HR 6.51, 95% CI 1.97 to 21.51, p=0.0008). In the TCGA set, combined analysis of miR-224 with SMAD4 expression enhanced correlation with survival (HR 4.12, 95% CI 1.1 to 15.41, p=0.0175).MiR-224 promotes CRC metastasis, at least in part, through the regulation of SMAD4. MiR-224 expression in primary CRC, alone or combined with its targets, may have prognostic value for survival of patients with CRC.

While several new therapies are FDA-approved for bone-metastatic prostate cancer (PCa), patient survival has only improved marginally. Here, we report that chitosan nanoparticle-mediated delivery of miR-34a, a tumor suppressive microRNA that downregulates multiple gene products involved in PCa progression and metastasis, inhibited prostate tumor growth and preserved bone integrity in a xenograft model representative of established PCa bone metastasis. Expression of miR-34a induced apoptosis in PCa cells, and, in accord with downregulation of targets associated with PCa growth, including MET and Axl and c-Myc, also induced a form of non-canonical autophagy that is independent of Beclin-1, ATG4, ATG5 and ATG7. MiR-34a-induced autophagy is anti-proliferative in prostate cancer cells, as blocking apoptosis still resulted in growth inhibition of tumor cells. Thus, combined effects of autophagy and apoptosis are responsible for miR-34a-mediated prostate tumor growth inhibition, and have translational impact, as this non-canonical form of autophagy is tumor inhibitory. Together, these results provide a new understanding of the biological effects of miR-34a and highlight the clinical potential for miR-34a delivery as a treatment for bone metastatic prostate cancer.

Contains fulltext : 154473.pdf (Publisher’s version ) (Open Access) To study the role of FAK signaling complexes in promoting metastatic properties of prostate cancer (PCa) cells, we selected stable, highly migratory variants, termed PC3 Mig-3 and DU145 Mig-3, from two well-characterized PCa cell lines, PC3 and DU145. These variants were not only increased migration and invasion in vitro, but were also more metastatic to lymph nodes following intraprostatic injection into nude mice. Both PC3 Mig-3 and DU145 Mig-3 were specifically increased in phosphorylation of FAK Y861. We therefore examined potential alterations in Src family kinases responsible for FAK phosphorylation and determined only Yes expression was increased. Overexpression of Yes in PC3 parental cells and src-/-fyn-/-yes-/- fibroblasts selectively increased FAK Y861 phosphorylation, and increased migration. Knockdown of Yes in PC3 Mig-3 cells decreased migration and decreased lymph node metastasis following orthotopic implantation of into nude mice. In human specimens, Yes expression was increased in lymph node metastases relative to paired primary tumors from the same patient, and increased pFAK Y861 expression in lymph node metastases correlated with poor prognosis. These results demonstrate a unique role for Yes in phosphorylation of FAK and in promoting PCa metastasis. Therefore, phosphorylated FAK Y861 and increased Yes expression may be predictive markers for PCa metastasis.

Prostate cancer has a proclivity to metastasize to bone. The mechanism by which prostate cancer cells are able to survive and progress in the bone microenvironment is not clear. Identification of molecules that play critical roles in the progression of prostate cancer in bone will provide essential targets for therapy. Ribosomal S6 protein kinases (RSK) have been shown to mediate many cellular functions critical for cancer progression. Whether RSK plays a role in the progression of prostate cancer in bone is unknown. IHC analysis of human prostate cancer specimens showed increased phosphorylation of RSK in the nucleus of prostate cancer cells in a significant fraction of human prostate cancer bone metastasis specimens, compared with the primary site or lymph node metastasis. Expression of constitutively active myristylated RSK in C4-2B4 cells (C4-2B4/RSK) increased their survival and anchorage-independent growth compared with C4-2B4/vector cells. Using an orthotopic bone injection model, it was determined that injecting C4-2B4/RSK cells into mouse femurs enhanced their progression in bone compared with control cells. In PC3-mm2 cells, knockdown of RSK1 (RPS6KA1), the predominant RSK isoform, but not RSK2 (RPS6KA2) alone, decreased anchorage-independent growth in vitro and reduced tumor progression in bone and tumor-induced bone remodeling in vivo. Mechanistic studies showed that RSK regulates anchorage-independent growth through transcriptional regulation of factors that modulate cell survival, including ING3, CKAP2, and PTK6. Together, these data provide strong evidence that RSK is an important driver in prostate cancer progression in bone. Implications: RSK, an important driver in prostate cancer progression in bone, has promising potential as a therapeutic target for prostate cancer bone metastasis. Mol Cancer Res; 13(2); 348–57. ©2014 AACR.

Background: Gastric cancer is the primary cause of cancer-specific mortality worldwide. PODXL (podocalyxin-like protein, or podocalyxin) is a cell-surface glycoprotein that belongs to CD34 family of sialomucins. PODXL-v1 is widely expressed on vascular endothelium, mesothelial cells, and platelets; in contrast, PODXL-v2, a truncated short form of PODXL is specifically expressed or up-regulated in numerous cancer cell types as well as in small blood vessels in tumor tissues. There remains considerable interest in generating monoclonal antibodies (mAbs) directed against specific tumor targets for antigen (Ag) discovery, diagnosis and therapy. Methods: Mixed live cells from four human GC cell lines were used as the immunogen in A/J mice. MS17-38 mAb was generated by hybridoma technology and from high throughput screening (HTS), with GC cell lines versus normal human PBMC live cells. Purified MS17-38 mAb was used to confirm the target Ag through a combination of western blot (WB), immunoprecipitation (IP) and mass spectrometry (MS). Peptide microarrays of the identified Ag were layered on chips and double screening was performed to achieve epitope mapping of binding of the mAb its target. Dose-dependent treatment of GC cells with MS17-38 mAb and target siRNA-was followed by staining with CCK-8 and CyQuant GR dyes and read at 450nm and 480/520nm for cell proliferation and transwell migration assays. GC MKN-45 cells were injected subcutaneously (s.c.) into the flank of nu/nu mice treated with MS17-38 mAb or control mAb. In addition, tumor tissues from 42 human GC patients were stained by immunohistochemistry (IHC) and evaluated for PODXL expression using MS17-38, and the survival of the GC patients compared to target antigen expression levels. Results: MS17-38 mAb was generated by live cells HTS and was demonstrated to bind to a specific conformational epitope of PODXL-v2 on GC cells. PODXL-v2 expression inhibition by PODXL-v2 siRNA or the PODXL-v2-neutralizing antibody MS17-38 resulted in inhibition of GC cell growth and prevention of GC cell migration in vitro. Furthermore, in vivo studies demonstrated that MS17-38 mAb can potently inhibit tumor growth and prevent MKN-45 metastasis to the lungs in nu/nu mouse models. Finally, high expression of PODXL-v2 was associated with advanced GC stage and short survival time (P Conclusions: Our findings indicate that PODXL-v2 is specifically expressed in the extracellular matrix of GC, and MS17-38 mAb directed against a conformational epitope of PODXL-v2 identified through HTS can functionally inhibit GC cancer cell growth and migration/metastasis both in vitro and in vivo. MS17-38 is a promising functional antibody directed against GC that could potentially be developed into a therapeutic mAb for clinical application. Citation Format: Runhua Feng, Ming Li, Yuling Wang, Dongqing Zhang, Xiaolian Gao, Xuehua Chen, Joe X. Zhou, Victor G. Prieto, Jian H. Song, Shenying Fang, Binya Liu, Zhenggang Zhu, Xiangcang Ye, Lee M. Ellis, Mason Lu, Jeffery E. Lee. MS17-38 mAb targeting of PODXL-v2 inhibits gastric cancer growth and metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 599.

Metastatic Prostate Cancer (PCa) is the second leading cause of cancer related-deaths in men in the United States. Understanding the molecular events critical to PCa metastasis should lead to novel therapies for treatment of metastatic disease. To assess molecular alterations required for increased PCa cell migration, a critical step in metastasis, multiple rounds of selection of PC3 and DU145 PCa cells were performed using a modified Boyden chamber assay. Highly migratory cells (termed PC3-Mig-3 and DU145-Mig-3) were obtained, a phenotype that has remained stable. PC3-Mig-3 and DU145-Mig-3 cells were also increased in invasiveness, decreased in adhesion and proliferation; thus acquiring additional properties of metastatic cells. Following orthotopic injection into nude mice, migratory variants were significantly increased in number of lymph node metastases (P Citation Format: Tanushree Chatterji, Jian H. Song, Nila U. Parikh, Chien-Jui Cheng, Sue-Hwa Lin, Gary E. Gallick. Differential phosphorylation of focal adhesion kinase and activation of Yes kinase are associated with increased metastatic potential of 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 4056. doi:10.1158/1538-7445.AM2014-4056

166 Background: Dasatinib, an inhibitor of Src-family kinases, combined with docetaxel in men with castrate-resistant prostate cancer (CRPC), affects bone turnover markers. Only a subset of men benefit from this therapy, and predictive markers are lacking. We hypothesized a role for insulin-like growth factor-1 (IGF-1) as a predictive marker, since IGF-1 is important in both prostate cancer (PCa) progression and bone development. Hence, we determined the association of IGF-1 expression to treatment response, and whether this expression resulted from tumor cells, the microenvironment, or their interactions. Methods: We first measured serum IGF-1 levels in men with CRPC treated with dasatinib plus docetaxel. To investigate the source of IGF-1, we utilized different mouse models harboring human PCa cells, and used species-specific IGF-1 ELISA kits (mouse vs. human). Results: In men with CRPC, an increase in IGF-1 levels after one cycle of treatment is associated with a higher response rate and longer duration of treatment with docetaxel and dasatinib. Xenograft experiments with subcutaneous, and intratibial injection of PCa cells and treatment of mice with dasatinib-based combinations suggest that direct interaction of PCa cells with bone microenvironment is necessary for IGF-1 induction, is entirely host-derived, and occurs only in mice that respond to dasatinib-based therapy. Conclusions: Our results support a role for serum IGF-1 as a potential biomarker for dasatinib-based combination treatments in CRPC. Inoculation of human PCa cells into murine hosts was essential in determining that IGF-1 results from the bone microenvironment. Further studies are warranted to validate these findings in a larger cohort of patients in a prospective manner.

Background: Treatment of metastatic prostate cancer (PCa) with single agents has shown only modest efficacy. We hypothesized dual inhibition of different pathways in PCa results in improved tumor inhibition. The Src family kinases (SFK) and insulin-like growth factor-1 (IGF-1) signaling axes are aberrantly activated in both primary PCa and bone metastases and regulate distinct and overlapping functions in PCa progression. We examined the antitumor effects of combined inhibition of these pathways. Materials and Methods: Src andIGF-1 receptor (IGF-1R) inhibition was achieved in vitro by short hairpin (sh)RNA and in vitro and in vivo by small molecule inhibitors (dasatinib and BMS-754807, against SFK and IGF-1R/Insulin Receptor(IR), respectively). Results: In vitro, inhibition of IGF-1 signaling affected cell survival and proliferation. SFK blockade alone had modest effects on proliferation, but significantly enhanced the IGF-1R blockade. These findings correlated with a robust inhibition of IGF-1induced Akt1 phophorylation by dasatinib, whereas Akt2 phosphorylation was SFK independent and only inhibited by BMS754807. Thus, complete inhibition of both Akt genes, not seen by either drug alone, is likely a major mechanism for the decreased survival of PCa cells. Furthermore, dasatinib and BMS-754807 inhibited in vivo growth of the primary human xenograft MDA PCa 133, with corresponding inhibition of Akt in tumors. Also, both orthotopic and intratibial tumor growth of PC-3 cells were more potently inhibited by dual SFK and IGF-1R/IR blockade compared to either pathway alone, with a corresponding decrease in bone turnover markers. Conclusions: Dual IGF-1R/IR and SFK inhibition may be a rational therapeutic approach in PCa by blocking both independent and complementary processes critical to tumor growth.

61 Background: The Src and IGF-1R axes are aberrantly activated in both PCa and the microenvironment of bone metastases. Dasatinib and BMS-754807 are clinically promising small molecule inhibitors with high potency against Src family kinases (SFK) and IR/IGF-1R, respectively. Based on a phase I/II clinical trial in which 9/19 pts treated with docetaxel + dasatinib were increased in serum IGF-1 levels after one cycle, the aim of this study was to establish potential antitumor cooperativity of inhibiting both IGF-1R and Src in experimental PCa models in vitro and in mice. Methods: Inhibition of Src and IGF-1R pathways was accomplished by pharmacologic agents (dasatinib against Src and BMS-754807 against IR/IGF-1R) as well as by shRNA, in PC3 and LNCaP cells. In vivo studies were done after orthotopic and intratibial injection of PC3 cells in nude mice. Results: SFK inhibition decreased proliferation and migration of PCa cells whereas IGF-1R blockade induced apoptosis. All anti-tumor effects were enhanced by dual blockade. IGF-1 induced phosphorylation of Akt1 and 2. Only Akt 1 phosphorylation was decreased by dasatinib; whereas Akt 1 and 2 phosphorylation were completely abrogated by the combination. Dasatinib and BMS-754807 inhibited orthotopic in vivo tumor growth of PC3 cells more potently than either inhibitor alone. Similarly, intratibial tumor growth and bone destruction was significantly reduced with the drug combination, accompanied by a decrease in serum bone turnover markers alkaline phosphatase and N-telopeptide. Conclusions: Dual inhibition of Src and IGF-1R has greater anti-tumor effect in PCa cells compared to inhibiting either alone. In the presence of IGF-1, dasatinib and BMS-754807 are necessary to inhibit IGF-1-induced phosphorylation of Akt1 and 2 in tumor cells in culture. In intratibial models, decreased bone turnover markers in serum support the concept of targeting both the epithelial and bone microenvironment. The combination of dasatinib and BMS-754807 may be a rational therapeutic approach in PCa by blocking complementary processes of tumor growth and progression.

e15004 Background: The Src and IGF-1R axes are aberrantly activated in both PCa and the microenvironment of bone metastases. Dasatinib and BMS-754807 are clinically promising small molecule inhibit...

21 Background: The Src and IGF-1R axes are aberrantly activated in both PCa and the microenvironment of bone metastases. Dasatinib and BMS-754807 are clinically promising small molecule inhibitors with high potency against Src family kinases and IGF-1R, respectively. The aim of this study was to establish antitumor co-operativity by combining IGF-1R and Src blockade in a preclinical PCa model. Methods: LNCaP and PC3 cells were used as models for androgen-dependent and independent PCa, respectively. Inhibition of Src and IGF-1R pathways was accomplished by pharmacologic agents (dasatinib against Src and BMS-754807 against IGF-1R) as well as by shRNA. Serum IGF-1 levels were measured in patients (pts) with castration-resistant PCa (CRPC) treated with dasatinib and docetaxel in a phase II trial. Results: Src inhibition decreased proliferation of PCa cells, and migration in modified Boyden Chamber and wound assays. In contrast, IGF-1R blockade induced apoptosis (increased Sub-G1 fraction cells, Annexin-V(+) cells and PARP cleavage). Phosphorylation of Akt was partially inhibited by either drug alone and almost completely abrogated by the combination. Intraprostatic injection of shIGF-1R or shSrc PC3 cells in nude mice led to an 83% and 60% decrease in tumor size compared to control shRNA, respectively. In both cell lines, all observed antitumor effects were enhanced when dual blockade was used, relative to blocking the Src or IGF-1R pathway alone. In 9/19 (47%) pts with CRPC, treatment with dasatinib resulted in a compensatory increase of serum IGF-1 levels. Conclusions: Dual inhibition of Src and IGF-1R is effective and complementary in PCa, mediated, in part, through inhibition of the downstream target Akt. In about half of pts treated with dasatinib, an increase in soluble IGF-1 levels was observed, indicating there is a compensatory upregulation of survival pathways that might be abrogated by dual inhibition of Src and IGF-1R. The combination of dasatinib and BMS-754807 may be a rational therapeutic approach in PCa by blocking complementary processes of tumor growth and progression. [Table: see text]