Murine Model Of Prostate Cancer Research Articles

In vivo imaging reveals prostate pathology in the PTEN knockout murine model of prostate cancer

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

MOR209/ES414, a Novel Bispecific Antibody Targeting PSMA for the Treatment of Metastatic Castration-Resistant Prostate Cancer.

Treatment of metastatic, castration-resistant prostate cancer (mCRPC) remains a highly unmet medical need and current therapies ultimately result in disease progression. Immunotherapy is a rapidly growing approach for treatment of cancer but has shown limited success to date in the treatment of mCRPC. We have developed a novel humanized bispecific antibody, MOR209/ES414, built on the ADAPTIR (modular protein technology) platform, to redirect T-cell cytotoxicity toward prostate cancer cells by specifically targeting T cells through CD3ε to prostate cancer cells expressing PSMA (prostate-specific membrane antigen). In vitro cross-linking of T cells with PSMA-expressing tumor cells by MOR209/ES414 triggered potent target-dependent tumor lysis and induction of target-dependent T-cell activation and proliferation. This activity occurred at low picomolar concentrations of MOR209/ES414 and was effective at low T-effector to tumor target cell ratios. In addition, cytotoxic activity was equivalent over a wide range of PSMA expression on target cells, suggesting that as few as 3,700 PSMA receptors per cell are sufficient for tumor lysis. In addition to high sensitivity and in vitro activity, MOR209/ES414 induced limited production of cytokines compared with other bispecific antibody formats. Pharmacokinetic analysis of MOR209/ES414 demonstrated a serum elimination half-life in NOD/SCID γ (NSG) mice of 4 days. Administration of MOR209/ES414 in murine xenograft models of human prostate cancer significantly inhibited tumor growth, prolonged survival, and decreased serum prostate-specific antigen levels only in the presence of adoptively transferred human T cells. On the basis of these preclinical findings, MOR209/ES414 warrants further investigation as a potential therapeutic for the treatment of CRPC. Mol Cancer Ther; 15(9); 2155-65. ©2016 AACR.

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer.

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information about this disease. The human orthotopic prostate cancer xenograft mouse model provides a useful alternative approach for understanding the specific interactions between genetically and molecularly altered tumor cells, their organ microenvironment, and for evaluation of efficacy of therapeutic regimens. This is a well characterized model designed to study the molecular events of primary tumor development and it recapitulates the early events in the metastatic cascade prior to embolism and entry of tumor cells into the circulation. Thus it allows elucidation of molecular mechanisms underlying the initial phase of metastatic disease. In addition, this model can annotate drug targets of clinical relevance and is a valuable tool to study prostate cancer progression. In this manuscript we describe a detailed procedure to establish a human orthotopic prostate cancer xenograft mouse model.

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer

Abstract 897: Methionine restriction alters functional polarization of macrophages in a murine model of prostate cancer

Abstract Background: Epidemiological studies link prostate cancer (CaP) to dietary intake. Recent research has shown that individuals consuming a Western diet, high in protein, have higher circulating IGF-1 (insulin-like growth factor 1) which feeds into the nutrient sensing mTOR pathway. This evolutionarily conserved pathway is central to the development of advanced stage and castration resistant CaP. Our previous published studies have shown that dietary protein restriction inhibits tumor growth via mTOR pathway alteration and reduces circulating IGF-1 levels in vivo. The current study hypothesizes that dietary methionine restriction will alter the functionality of immune cells enhancing the ability of the immune system to respond to cancerous insults. Methods: Mice were fed low (7% protein) or control protein (21% protein) diets. In a prevention study mice consumed modified diets for four weeks prior to being inoculated with CaP tumors. In a simultaneous intervention study mice were inoculated with CaP tumors first then four weeks later were placed on the modified diets. Results: In the low protein diet group, we observed that pro-tumor M2 polarized macrophages were significantly reduced in the tumor microenvironment (TME) for both prevention (p = 0.012) and intervention diet (p = 0.0003) studies as compared to the control diet, in addition to observation of mTOR inhibition and IGF-1 reduction. Besides protein content in diet, we also analyzed the amino acid composition. Multiple groups have shown that methionine restriction (MR) inhibits mTOR activation, alters the immune system, and prolongs the survival of rodents. The mechanism by which MR impacts the innate immune system is still poorly understood. In our preliminary in vitro studies, we observed that modification of a single amino acid (AA), such as methionine, is sufficient to inhibit activation of both the mTOR pathway and M2 polarization (Arginase1 qRT-PCR of two samples in quadruplicate (p = 0.0732)), while increasing the presence of M1 macrophage marker iNOS (p = 0.2689) in bone marrow derived macrophages (BMDMs). Thus, we hypothesize that modulating specific amino acids in the diet is sufficient to alter macrophage expression of M1/M2 characteristics, and their functions in the TME. Conclusions: Preliminarily data shows that altering one specific AA intricately linked to the mTOR pathway is sufficient to change macrophage polarization status both at the protein and gene expression levels, suggesting that dietary alteration of a single AA is capable of altering macrophage function. The results of the study provide the basis for translational use of dietary means to alter the immune system and improve the therapeutic effects of immunotherapies. Citation Format: Ashley Orillion, Remi Adelaiye-Ogala, Li Shen, Eric Ciamporcero, May Elbanna, Sreenivasulu Chintala, Sreevani Arisa, Ben Elzey, Chinghai Kao, Luigi Fontana, Roberto Pili. Methionine restriction alters functional polarization of macrophages in a murine model of prostate cancer. [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 897.

Abstract 2407: Neuroendocrine and luminal progenitors drive cancer progression in prostate cancer

Abstract Neuroendocrine differentiation of prostate cancer (PCa) occurs frequently during the development of castration resistance and rarely in primary tumors. Using the transgenic mouse model of prostate cancer (TRAMP) in a B6/C57 background, we were able to detect, isolate and further characterize basal, luminal and neuroendocrine subsets of cancer cells. We performed allograft experiments in NSG mice to ensure cellular stem and progenitor properties as well as metastatic potential, and thus created a murine model of neuroendocrine prostate cancer. Whilst all three previously described cell populations were present in approximately three quarters of primary TRAMP tumors, some tumors lacked basal stem cells and showed a more aggressive phenotype. These tumors, mainly consisting of small cancer cells, expressed markers of neuroendocrine differentiation such as synaptophysin and chromogranin A as shown by immunohistochemistry and are further referred to as neuroendocrine carcinomas (NECs), as ruled by an experienced uropathologist of our institution. Adenocarcinoma-like tumors (ACs), in contrast, showed high expression of cytokeratins and retained glandular histology. Using fluorescence-activated cell sorting (FACS) against newly discovered NEC markers within the TRAMP model, we found a relative increase of neuroendocrine progenitors in prostate NECs compared to ACs (approximately 75 vs. 33%, respectively). In ACs, on the other hand, luminal progenitors were found to be the predominant drivers of cancer progression. To further evaluate this, we transplanted single-cell-suspensions into NSG mice without androgen supplementation and observed successful engraftment of both non-basal cell populations. Additionally, we were able to passage the resulted tumors for at least two generations and observed maintenance of histology and biological features for tumors of both luminal and neuroendocrine origin. Based on a RNA sequencing, we were able to define gene signatures for neuroendocrine and luminal progenitors, uncovering a number of novel potential therapeutic targets. Ultimately, we were able to evaluate the prognostic value of the signatures obtained from mice in the human disease by in-silico analyses of publically available gene expression profile databases. In conclusion, we created and characterized a murine model of neuroendocrine prostate cancer using flow cytometry and murine allografts. Further, we established gene expression signatures of luminal and neuroendocrine progenitors and translated them to the human disease. Our findings foster the understanding of neuroendocrine differentiation in prostate cancer and may help in developing new targeted approaches in this entity. Citation Format: Maximilian Marhold, Erwin Tomasich, Simon Udovica, Gerwin Heller, Corinna Altenberger, Andreas Spittler, Reinhard Horvat, Peter Horak, Michael Krainer. Neuroendocrine and luminal progenitors drive cancer progression in prostate cancer. [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 2407.

Unique pattern of PMN-MDSC migration in cancer

Abstract Myeloid-derived suppressor cells (MDSC) are pathologically activated immature myeloid cells that contribute to an immunosuppressive tumor microenvironment. In order to exert their effects at either the primary tumor site or in metastases, PMN-like MDSC (PMN-MDSC) must first migrate to these sites. There is some evidence to suggest that chemokine gradients can mediate their recruitment. We have observed that human PMN-MDSC accumulation in colon and lung tumors strongly correlates with the expression of S100A9, CXCL1 and CXCL8 in those tumors. Moreover, we have found that, in comparison to PMN, patient PMN-MDSC have an increased chemotactic response to CXCL8 in spite of decreased cell surface CXCR2 expression. Similarly, in murine transgenic models of melanoma, pancreatic and prostate cancer, we have found that PMN-MDSC migrate more than PMN when stimulated with CXCL1, in spite of similar levels of cell surface CXCR2 expression. Interestingly, murine PMN-MDSC are more migratory even without stimulation, and have greater speed, mean squared displacement and random motility coefficient than PMN. It is possible that in addition to specific factors secreted in the primary and metastatic tumor sites, PMN-MDSC recruitment to these sites is regulated by an altered migratory behavior of the cells themselves.

Dynamic nuclear polarization of biocompatible (13)C-enriched carbonates for in vivo pH imaging.

A hyperpolarization technique using carbonate precursors of biocompatible molecules was found to yield high concentrations of hyperpolarized (13)C bicarbonate in solution. This approach enabled large signal gains for low-toxicity hyperpolarized (13)C pH imaging in a phantom and in vivo in a murine model of prostate cancer.

Abstract PR003: Hypoxia is an essential driver of immune suppression in the tumor microenvironment

Abstract Tumor hypoxia predicts poor outcomes across all cancers and has long been recognized as a critical source of resistance to both chemotherapy and radiotherapy. Despite the success of T cell immune checkpoint blockade in treating melanoma, aggressive adenocarcinomas of the prostate and pancreas are largely resistant to CTLA-4 and PD-1 antibody therapy in the mouse and in man. We find that hypoxic zones of these tumors resist infiltration by T cells even in the context of robust infiltration of normoxic areas of the same tumor (e.g. in the context of T cell checkpoint blockade). Beyond this lack of accessibility to tumor-specific T cells, hypoxia drives the establishment of a highly interdependent network of immunosuppressive stromal cells. Among these, we find myeloid-derived suppressor cells and myofibroblasts to be the critical populations which act together to suppress T cell responses and mediate immunotherapy resistance. Using the hypoxia-specific prodrug Evofosfamide, we show that disruption of hypoxia in both transplantable and genetically-engineered murine models of prostate cancer sensitizes them to antibody blockade of CTLA-4 and PD-1. Loss of immune resistance is a consequence of re-oxygenation of hypoxia zones which results in 1) loss of myeloid suppressors, 2) reduced capacity to suppressively polarize new myeloid immigrants, and 3) loss of suppressive activation of myofibroblasts. This combination of hypoxia disruption and T cell checkpoint blockade has immense potential to render some of the most therapeutically resistant cancers sensitive to immunotherapy. Citation Format: Midan Ai, Pratha Budhani, Michael A. Curran. Hypoxia is an essential driver of immune suppression in the tumor microenvironment. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr PR003.

Abstract A157: Oncolytic poliovirus mediated immune events

Abstract Introduction: Tumor-targeted therapies that efficiently eliminate malignant cells and in the process engage the innate and adaptive immune system are desirable for preventing cancer recurrence. We have pioneered an oncolytic poliovirus therapy, PVSRIPO, which selectively targets and eliminates malignant cells. PVSRIPO is a recombinant polio:rhinovirus chimera, engineered to eliminate neuronal competence and is devoid of neuropathogenicity after intracerebral inoculation in non-human primates and in humans. PVSRIPO has cancer tropism due to widespread ectopic expression of the poliovirus receptor, CD155, in solid cancers. PVSRIPO kills tumor cells with no effect on normal cells. Unlike other oncolytic viruses, PVSRIPO is effective in the presence of neutralizing antibodies and an innate antiviral response. A first-in-human Phase-1 study with PVSRIPO at Duke University has shown remarkable promise in patients with recurrent glioblastoma (GBM), a uniformly lethal disease. PVSRIPO tumor cell killing is associated with the induction of danger- and pathogen-associated molecular patterns (DAMPs and PAMPs) via antiviral type I interferons (IFNs) and simultaneous non-lethal infection of antigen-presenting cells (APCs) such as monocytes and dendritic cells (DCs) via CD155. Type I IFNs are known to inhibit tumor proliferation, activate innate immune cells and bridge innate and adaptive immunity among other functions. Methods: To understand immune events associated with poliovirus infection of APCs, we examined the effects of PVSRIPO treatment on the human macrophage cell line (THP1) and primary human monocyte-derived DCs. PVSRIPO-treated DCs were evaluated for expression of maturation/activation markers and compared to untreated immature and mature DCs. To investigate if DCs exposed to PVSRIPO-induced dying tumor cells are capable of presenting tumor antigens to T cells, we performed an in vitro human immunotherapy assay. Human DCs generated from HLA-A2+ donor cells were incubated with PVSRIPO-induced tumor cell lysate and then used to stimulate autologous T cells in vitro followed by a cytotoxic T lymphocyte (CTL) assay. All tumor cell lines used for the human immunotherapy experiments were positive for HLA-A2 expression. The following tumor cell lines were used for this analysis: DM6 (MART+) melanoma cell line, MDA-MB231 (CEA+) triple-negative breast cancer cell line, SUM149 (EGFR+) inflammatory breast cancer cell line and LNCaP (PSA+) prostate cancer cell line. Results: PVSRIPO infection of THP1 macrophages induces Mda5/Stat1 and Stat1 phosphorylation as well as TNFα and IFNβ release. Coculturing of DCs with PVSRIPO-induced tumor cell lysate stimulates DC activation and IL12 production. Human DCs loaded with PVSRIPO-induced tumor cell lysate are capable of stimulating tumor antigen-specific cytotoxic T cells in vitro. Autologous DCs transfected with RNA that encodes for CEA, EGFR, MART or PSA (tumor antigen-expressing cells) and human tumor cell lines were used as targets to assess tumor antigen-specificity of T cells. Conclusion: Our data suggests that along with destruction of the primary tumor, oncolytic poliovirus mediates immune events. Importantly, we demonstrate that human DCs co-incubated with PVSRIPO-induced tumor cell lysate stimulate tumor antigen-specific cytotoxic T cells in an in vitro human immunotherapy assay. In ongoing studies we are analyzing oncolytic poliovirus mediated immune events in syngeneic, immunocompetent murine models of breast cancer and prostate cancer. Citation Format: Michael C. Brown, Eda K. Holl, David Boczkowski, Darell D. Bigner, Matthias Gromeier, Smita K. Nair. Oncolytic poliovirus mediated immune events. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A157.

Tumor hypoxia drives immune suppression and immunotherapy resistance

Tumor hypoxia predicts poor outcomes across all cancers and has long been recognized as a critical source of resistance to both chemotherapy and radiotherapy. Despite the success of T cell immune checkpoint blockade in treating melanoma, aggressive adenocarcinomas of the prostate and pancreas are largely resistant to CTLA-4 and PD-1 antibody therapy in the mouse and in man. We find that hypoxic zones of these tumors resist infiltration by T cells even in the context of robust infiltration of T cells in normoxic areas of the same tumor (e.g. in the context of T cell checkpoint blockade). Beyond this lack of accessibility to tumor-specific T cells, hypoxia drives the establishment of a highly interdependent network of immunosuppressive stromal cells. Among these, we find myeloid-derived suppressor cells and myofibroblasts to be the critical populations which act together to suppress T cell responses and mediate immunotherapy resistance. Evofosfamide is a hypoxia-specific chemotherapeutic pro-drug which is activated only in the hypoxic cores of tumors and thus can be co-administered with immunotherapy. We find that Evofosfamide-driven disruption of hypoxia zones sensitizes prostate cancer to antibody blockade of CTLA-4 and PD-1 in both transplantable and genetically-engineered murine models of prostate cancer. Co-administration of Evofosfamide and α-CTLA-4/α-PD-1 promotes tumor rejection in a significantly larger percentage of mice than either therapy alone. Mechanistic studies reveals that loss of immune resistance is a consequence of re-oxygenation of hypoxia zones which results in 1) loss of active myeloid suppressor cells, 2) reduced suppressive capacity of new myeloid immigrants, 3) loss of suppressive activation of myofibroblasts, and 4) enhanced infiltration of effector T cells. Therefore, this combination of hypoxia disruption and T cell checkpoint blockade has immense potential to render some of the most therapeutically resistant cancers sensitive to immunotherapy.

Targeted radiotherapy potentiates the cytotoxicity of a novel anti-human DR5 monoclonal antibody and the adenovirus encoding soluble TRAIL in prostate cancer

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) induces a death signal following binding to death receptors (DR4, DR5). We have developed a novel anti-human DR-5 monoclonal antibody (TRA-8) and adenoviral encoding TRAIL (Ad/TRAIL). Herein, we are testing the combined effect of radiotherapy and TRA-8 or Ad TRAIL in prostate cancer cells. Human prostate cancer cell lines LnCap, PC-3 and DU145 were used in this study. Cells were treated either with TRA-8 alone or Ad/TRAIL, radiation alone, or a combination of each at different doses and intervals. Cell survival using the MTS assay and colony forming assay were used to determine radiosensitization. Immunohistochemistry was used to detect bax and bcl-2. Real-time PCR was performed on mRNA of treated prostate cancer cell lines. Finally, a murine model of subcutaneous prostate cancer was used to evaluate the in vivo effect. Cell survival assays detected by MTS assay showed that prostate cell lines treated with a combination of radiation and TRA-8 showed significantly lower survival than cells treated with either radiation or TRA-8 alone. Colony forming assay and cell proliferation assays showed increased killing after combination treatment with TRA-8 or Ad/TRAIL and radiation, than either single agent alone. Mechanistic studies showed that the killing effect was due to induction of apoptosis mostly by increased expression of bax in TRA-8 or Ad/TRAIL treated cells. Additionally, RT-PCR showed an increased copy number of bax in most cells treated with TRA-8 and radiation. It is concluded that radiation and TRA-8 or Ad/TRAIL produced a synergistic effect in refractory prostrate cancer.

The Nuclear Orphan Receptor NR2F6 Is a Central Checkpoint for Cancer Immune Surveillance.

SummaryNuclear receptor subfamily 2, group F, member 6 (NR2F6) is an orphan member of the nuclear receptor superfamily. Here, we show that genetic ablation of Nr2f6 significantly improves survival in the murine transgenic TRAMP prostate cancer model. Furthermore, Nr2f6−/− mice spontaneously reject implanted tumors and develop host-protective immunological memory against tumor rechallenge. This is paralleled by increased frequencies of both CD4+ and CD8+ T cells and higher expression levels of interleukin 2 and interferon γ at the tumor site. Mechanistically, CD4+ and CD8+ T cell-intrinsic NR2F6 acts as a direct repressor of the NFAT/AP-1 complex on both the interleukin 2 and the interferon γ cytokine promoters, attenuating their transcriptional thresholds. Adoptive transfer of Nr2f6-deficient T cells into tumor-bearing immunocompetent mice is sufficient to delay tumor outgrowth. Altogether, this defines NR2F6 as an intracellular immune checkpoint in effector T cells, governing the amplitude of anti-cancer immunity.

A paclitaxel-loaded recombinant polypeptide nanoparticle outperforms Abraxane in multiple murine cancer models.

Packaging clinically relevant hydrophobic drugs into a self-assembled nanoparticle can improve their aqueous solubility, plasma half-life, tumor specific uptake and therapeutic potential. To this end, here we conjugated paclitaxel (PTX) to recombinant chimeric polypeptides (CPs) that spontaneously self-assemble into ~60-nm diameter near-monodisperse nanoparticles that increased the systemic exposure of PTX by 7-fold compared to free drug and 2-fold compared to the FDA approved taxane nanoformulation (Abraxane®). The tumor uptake of the CP-PTX nanoparticle was 5-fold greater than free drug and 2-fold greater than Abraxane. In a murine cancer model of human triple negative breast cancer and prostate cancer, CP-PTX induced near complete tumor regression after a single dose in both tumor models, whereas at the same dose, no mice treated with Abraxane survived for more than 80 days (breast) and 60 days (prostate) respectively. These results show that a molecularly engineered nanoparticle with precisely engineered design features outperforms Abraxane, the current gold standard for paclitaxel delivery.

Abstract 5201: Antimetastatic effect of a Dializable Leucocyte Extract in a murine model of prostate cancer

Abstract Prostate cancer remains the most common non-dermatological cancer in the United States and the second leading cause of cancer death amongst men. The majority of men with progressive prostate cancer develop metastases, being the skeleton the most prevalent metastatic site. Dialyzable leukocyte extracts (DLEs) are heterogeneous mixtures of low-molecular-weight (<10 kDa) peptides that improve clinical responses in various diseases. Our group has shown that DLEs change cytokine serum concentrations in patients and reduce experimental metastasis in a melanoma model. In this work we analyzed the in vitro effects of a human DLE produced under good manufacturing procedures and facilities [1] and evaluated its possible antimetastatic effect in vivo. We employed PC-3 cells and murine Src-transformed Prostate Epithelial Cells (PEC-Src) to assess the direct effects of DLE in prostate cancer cells. Both cell lines were exposed in vitro to the DLE (1 pg/mL- 1 μg/mL) in proliferating (+FBS) or non-proliferating (-FBS) conditions, and cell number was estimated by MTS assays. The effects on cell motility and cell invasion were evaluated by wound healing assays and invasion from collagen-embedded spheroids, respectively. Since DLEs are immunomodulators, we used an experimental metastasis model that consistently produces bone and brain metastasis in inmunocompetent mice [2]. PEC-Src expressing Luciferase2-RFP were intracardiacally injected to FVB mice and metastatic growth in DLE-treated (1, 5, or 25 μg/kg p.o.) animals was quantified weekly by bioluminescence imaging. In vitro exposure to a wide range of DLE concentrations did not alter proliferation or survival in PC-3 or PEC-Src cells, indicating that the evaluated DLE does not has direct antineoplastic effect. Similarly, treatments did not alter prostate cancer cell motility or invasion. In contrast, DLE (1 μg/kg) reduced metastatic tumor burden in brain and bones in inmunocompetent mice. The absence of direct effect on prostate cancer cells suggests that DLE-treatment may be changing cellular or soluble facilitators in the metastatic microenvironments. Supported by PAPIIT-UNAM IN219613. [1] Medina-Rivero E, et al. J Pharma Biomed Anal 88: 289-294, 2014 [2] Sicoli D, et al. Cancer Res 74: 7103-14, 2014 Citation Format: Miguel A. Hernandez-Esquivel, Sonia M. Perez-Tapia, Richard G. Pestell, Marco A. Velasco-Velazquez. Antimetastatic effect of a Dializable Leucocyte Extract in a murine model of prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5201. doi:10.1158/1538-7445.AM2015-5201

Abstract 267: Combining androgen deprivation with immune checkpoint blockade delays the development of castration resistance in a murine model of prostate cancer

Abstract Androgen deprivation therapy induces immune cell infiltration in human prostate cancer. These findings suggest that immunotherapy may be most efficacious when administered concurrently with androgen deprivation, early in disease progression. We used a subcutaneous allograft model of murine prostate cancer (Myc-Cap), which mimics the development of human castration-resistant prostate cancer (CRPC) progression to study the anti-tumor effects of concurrent hormonal/immunotherapy. Implanted Myc-Cap tumors initially respond to androgen deprivation (degarelix acetate or bilateral orchiectomy), but mice eventually progress with CRPC. To test the hypothesis that the combination of androgen deprivation and immune checkpoint blockade could mediate pre-clinical benefit, we treated mice with either anti-PD-1, a depleting anti-CTLA-4 antibody (IgG2A), a non-depleting anti-CTLA-4 antibody (IgG1 D265A) or antibody combinations in the peri-castration period, then followed mice for the development of castration-resistant disease. Interestingly, the depleting anti-CTLA-4 antibody with/without anti-PD-1 antibody was strikingly effective in preventing the emergence of castration-resistant disease. The median castration-resistance free survival was 22 days in mice treated with androgen deprivation alone versus 32 days in mice treated with androgen deprivation and depleting anti-CTLA-4 antibody (P<0.05, compared to androgen deprivation alone) versus 30 days in mice treated with androgen deprivation, depleting anti-CTLA-4 and anti-PD-1 (P<0.05, compared to androgen deprivation alone; non-significant, compared to androgen deprivation and depleting anti-CTLA-4 antibody). Immunologically, we found that castration increases intratumoral infiltration of helper, cytotoxic and regulatory T cells (Tregs), natural killer cells, and macrophages, as well as effector cytokine production of T cells. We also found up-regulated expression of CTLA-4 and PD-1 as well as their respective ligands. Mechanistic studies showed that androgen deprivation combined with depleting anti-CTLA-4 /anti-PD-1 significantly reduces intratumoral Tregs and increases interferon-γ- or tumor necrosis factor-α- producing T cells in the tumor and its draining lymph node. In conclusion, while androgen deprivation renders the tumor microenvironment more immunogenic; the combination of androgen deprivation and depleting anti-CTLA-4 antibody with/without anti-PD-1 can significantly delay the development of CRPC. Citation Format: Ying-Chun Shen, Christina Kochel, Brian J. Francica, Angela Alme, Christopher Nirschl, Thomas Nirschl, Zoila Areli Lopez Bujanda, Maria A. Carrera H, Mark Selby, Alan Korman, Charles G. Drake. Combining androgen deprivation with immune checkpoint blockade delays the development of castration resistance in a murine model of prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 267. doi:10.1158/1538-7445.AM2015-267

Abstract LB-293: MicroRNA-96 targets RARγ and associates with prostate cancer progression

Abstract Nuclear receptors (NRs) integrate dietary and steroidal signals to control cell fate and are strong chemoprevention and chemotherapy targets, for example in prostate cancer (PCa). We mined TCGA and MSKCC PCa cohorts using bootstrap approaches revealing that NRs mRNA was significantly down-regulated more than predicted by chance (p = 7.1e-4). Loss of expression did not reflect somatic mutations or CNVs suggesting that epigenetic mechanisms drive this expression. Using prediction algorithms we identified NR targeting miRNA and revealed a number were significantly elevated, associated with significant negative correlations with specific NR. For example, RARG, which encodes retinoic acid receptor gamma (RARγ), was amongst the most frequently reduced NR, reflecting the prostatic metaplasia in RARγ knockout mice reported by others. Amongst the most significantly and commonly elevated miRNA was miR-96 which is predicted to target RARG and associated with worse disease free survival (p = 1.3e-4). Mining of published microarray and ChIP-Seq data revealed that miR-96 was downstream of androgen receptor (AR) signaling and supported a negative feed-forward relationship between AR and RARγ. Expression analyses revealed inverse correlations of RARG and targeting miR-96 in 14 different PCa cell lines of increasing aggressiveness and along the time course of tumor development in TRAMP and Pten(-/-) murine models of PCa. In RWPE-1 non-malignant prostate cells and in LNCaP PCa cells we validated miR-96 regulation of RARγ expression through miRNA mimic transfection and luciferase assays with miR-96 target sites from the RARG-3′UTR. Functional analyses revealed that modulating miR-96 levels affected the cellular response to the natural ligand for RARγ, all-trans retinoic acid. To capture all genes targeted by miR-96 in RWPE-1 and LNCaP cells we undertook transfection with biotin-labeled miR-96 followed by streptavidin pull-down and microarray analyses. 389 genes were significantly enriched in LNCaP cells and subsequent in silico analyses revealed significant enrichment for known and predicted targets for miR-96 (q-value 1.45e-25), down-regulation of enriched targets, and genes with annotated functions involved in hormone and apoptotic responses (q-value 5.1e-33). In LNCaP cells, approximately 10% of the targets were other transcription factors (e.g. CEBPA, FOXN2), co-regulators (e.g. TBLIX) or epigenetic regulators (e.g. ARID3B), many of which have established roles in the co-regulation of retinoid and androgen signaling. We propose that AR-driven miR-96 disrupts an interdependent network of RARγ and interacting transcription factors that control cell fate. Tumor or serum measurements of miR-96 have clinical potential to be exploited in targeted chemoprevention approaches to predict RARγ signaling status and progression risk. Citation Format: Mark D. Long, Prashant K. Singh, Moray J. Campbell. MicroRNA-96 targets RARγ and associates with prostate cancer progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-293. doi:10.1158/1538-7445.AM2015-LB-293

Ablation of B7-H3 but Not B7-H4 Results in Highly Increased Tumor Burden in a Murine Model of Spontaneous Prostate Cancer.

The costimulatory molecules B7-H3 and B7-H4 are overexpressed in a variety of human tumors and have been hypothesized as possible biomarkers and immunotherapeutic targets. Despite this potential, the predominating uncertainty about their functional implication in tumor-host interaction hampers their evaluation as a target for cancer therapy. By means of a highly physiologic, spontaneous tumor model in mice, we establish a causal link between B7-H3 and host tumor control and found B7-H4 to be redundant.

Identification of Androgen Receptor Splice Variants in the Pten Deficient Murine Prostate Cancer Model.

Androgen receptor (AR) variants are associated with resistance to anti androgen therapy both in human prostate cancer cell lines and clinical samples. These observations support the hypothesis that AR isoform accumulation is a consequence of selective therapeutic pressure on the full length AR. The Pten deficient prostate cancer model proceeds with well-defined kinetics including progression to castration resistant prostate cancer (CRPC). While surgical castration and enzalutamide treatments yield an initial therapeutic response, Pten-/-epithelia continue to proliferate yielding locally invasive primary tumor pathology. That most epithelium remains AR positive, but ligand independent, suggests the presence of oncogenic AR variants. To address this hypothesis, we have used a panel of recently described Pten-/- tumor cell lines derived from both from hormone intact (E4, E8) and castrated Pten mutants (cE1, cE2) followed by RACE PCR to identify and characterize three novel truncated, amino terminus containing AR variants (mAR-Va, b, c). Variants appear not only conserved throughout progression but are correlated with nearly complete loss of full length AR (AR-FL) at castrate androgen levels. The overexpression of variants leads to enhanced transcriptional activity of AR while knock down studies show reduced transcriptional output. Collectively, the identification of truncated AR variants in the conditional PTEN deletion model supports a role for maintaining the CRPC phenotype and provides further therapeutic applications of this preclinical model.

Tracking and Functional Characterization of Epithelial-Mesenchymal Transition and Mesenchymal Tumor Cells during Prostate Cancer Metastasis.

The epithelial-mesenchymal transition (EMT) has been postulated as a mechanism by which cancer cells acquire the invasive and stem-like traits necessary for distant metastasis. However, direct in vivo evidence for the role of EMT in the formation of cancer stem-like cells (CSC) and the metastatic cascade remains lacking. Here we report the first isolation and characterization of mesenchymal-like and EMT tumor cells, which harbor both epithelial and mesenchymal characteristics, in an autochthonous murine model of prostate cancer. By crossing the established Pb-Cre(+/-);Pten(L/L);Kras(G12D) (/+) prostate cancer model with a vimentin-GFP reporter strain, generating CPKV mice, we were able to isolate epithelial, EMT, and mesenchymal-like cancer cells based on expression of vimentin and EpCAM. CPKV mice (but not mice with Pten deletion alone) exhibited expansion of cells with EMT (EpCAM(+)/Vim-GFP(+)) and mesenchymal-like (EpCAM(-)/Vim-GFP(+)) characteristics at the primary tumor site and in circulation. These EMT and mesenchymal-like tumor cells displayed enhanced stemness and invasive character compared with epithelial tumor cells. Moreover, they displayed an enriched tumor-initiating capacity and could regenerate epithelial glandular structures in vivo, indicative of epithelia-mesenchyme plasticity. Interestingly, while mesenchymal-like tumor cells could persist in circulation and survive in the lung following intravenous injection, only epithelial and EMT tumor cells could form macrometastases. Our work extends the evidence that mesenchymal and epithelial states in cancer cells contribute differentially to their capacities for tumor initiation and metastatic seeding, respectively, and that EMT tumor cells exist with plasticity that can contribute to multiple stages of the metastatic cascade.