Clinically relevant humanized mouse models of metastatic prostate cancer facilitate therapeutic evaluation.

There is tremendous need for improved prostate cancer (PCa) models. The mouse prostate is anatomically and developmentally different from the human prostate and does not spontaneously form tumors. Genetically engineered mouse models lack the heterogeneity of human cancer and rarely establish metastatic growth. Human xenografts are an alternative but must rely on an immunocompromised host. Therefore, we generated PCa murine xenograft models with an intact human immune system (huNOG and huNOG-EXL mice) to test whether humanizing tumor-immune interactions would improve modeling of metastatic PCa and the impact of androgen receptor-targeted and immunotherapies. These mice maintain multiple human immune cell lineages, including functional human T-cells and myeloid cells. Implications: To our knowledge, results illustrate the first model of human PCa that has an intact human immune system, metastasizes to clinically relevant locations, responds appropriately to standard-of-care hormonal therapies, and can model both an immunosuppressive and checkpoint-inhibition responsive immune microenvironment.

Myeloid differentiation factor-2/LY96, a potential predictive biomarker of metastasis and poor outcomes in prostate cancer: clinical implications as a potential therapeutic target.

Prostate cancer (CaP) is the most diagnosed cancer in males and the second leading cause of cancer deaths. Patients with localized tumors are generally curable. However, no curative treatment exists for patients with advanced and metastatic disease. Therefore, identifying critical proteins involved in the metastatic process would help to develop new therapeutic options for patients with advanced and aggressive CaP. We provide strong evidence that Myeloid differentiation factor-2 (MD2) plays a critical role in metastasis and CaP progression. Analysis of tumor genomic data showed that amplifications of MD2 and increased expression are associated with poor outcomes in patients. Immunohistochemistry analysis of tumor tissues showed a correlation between the expression of MD2 and cancer progression. The Decipher-genomic test validated the potential of MD2 in predicting metastasis. In vitro studies demonstrated that MD2 confers invasiveness by activating MAPK and NF-kB signaling pathways and inducing epithelial-mesenchymal transition. Furthermore, we show that metastatic cells release MD2 (sMD2). We measured serum-sMD2 in patients and found that the level is correlated to disease extent. We determined the significance of MD2 in metastasis in vivo and as a therapeutic target, showing that the molecular and pharmacological targeting of MD2 significantly inhibited metastasis in murine models. We conclude that MD2 predicts metastatic behavior, and serum-MD2 could be studied as a potential non-invasive biomarker for metastasis, whereas MD2 presence on prostate biopsy predicts adverse disease outcome. We suggest MD2-targeted therapies could be developed as potential treatments for aggressive metastatic disease.

Clinically relevant humanized mouse models of metastatic prostate cancer to evaluate cancer therapies.

There is tremendous need for improved prostate cancer (PCa) models. The mouse prostate does not spontaneously form tumors and is anatomically and developmentally different from the human prostate. Engineered mouse models lack the heterogeneity of human cancer and rarely establish metastatic growth. Human xenografts represent an alternative but rely on an immunocompromised host. Accordingly, we generated PCa murine xenograft models with an intact human immune system (huNOG and huNOG-EXL mice) to test whether humanizing tumor-immune interactions would improve modeling of metastatic PCa and the impact of hormonal and immunotherapies. These mice maintain multiple human cell lineages, including functional human T-cells and myeloid cells. In 22Rv1 xenografts, subcutaneous tumor size was not significantly altered across conditions; however, metastasis to secondary sites differed in castrate huNOG vs background-matched immunocompromised mice treated with enzalutamide (enza). VCaP xenograft tumors showed decreases in growth with enza and anti-Programed-Death-1 treatments in huNOG mice, and no effect was seen with treatment in NOG mice. Enza responses in huNOG and NOG mice were distinct and associated with increased T-cells within tumors of enza treated huNOG mice, and increased T-cell activation. In huNOG-EXL mice, which support human myeloid development, there was a strong population of immunosuppressive regulatory T-cells and Myeloid-Derived-Suppressor-Cells (MDSCs), and enza treatment showed no difference in metastasis. Results illustrate, to our knowledge, the first model of human PCa that metastasizes to clinically relevant locations, has an intact human immune system, responds appropriately to standard-of-care hormonal therapies, and can model both an immunosuppressive and checkpoint-inhibition responsive immune microenvironment.

Anti-S100A4 Antibody Therapy Is Efficient in Treating Aggressive Prostate Cancer and Reversing Immunosuppression: Serum and Biopsy S100A4 as a Clinical Predictor.

S100A4 oncoprotein plays a critical role during prostate cancer progression and induces immunosuppression in host tissues. We hypothesized that S100A4-regulated oncogenic activity in immunosuppressed prostate tumors promotes growth of neoplastic cells, which are likely to become aggressive. In the current study, we investigated whether biopsy-S100A4 gene alteration independently predicts the outcome of disease in patients and circulatory-S100A4 is druggable target for treating immunosuppressive prostate cancer. Aided by DECIPHER-genomic test, we show biopsy-S100A4 overexpression as predictive of (i) poor ADT response and (ii) high risk of mortality in 228 radical prostatectomy-treated patients. Furthermore, analysis of tumor genome data of more than 1,000 patients with prostate cancer (PRAD/SU2C/FHCRC studies) validated the association of S100A4-alteration to poor survival and metastasis. We show that increased serum-S100A4 levels are associated to the prostate cancer progression in patients. The prerequisite for metastasis is the escape of tumor cells via vascular system. We show that extracellular-S100A4 protein as a growth factor induces vascular transmigration of prostate cancer cells and bone demineralization thus forms an ideal target for therapies for treating prostate cancer. By employing surface plasmon resonance and isothermal titration calorimetry, we show that mab6B12 antibody interacts with and neutralizes S100A4 protein. When tested for therapeutic efficacy, the mab6B12 therapy reduced the (i) osteoblastic demineralization of bone-derived MSCs, (ii) S100A4-target (NFκB/MMP9/VEGF) levels in prostate cancer cells, and (iii) tumor growth in a TRAMPC2 syngeneic mouse model. The immuno-profile analysis showed that mAb6B12-therapy (i) shifted Th1/Th2 balance (increased Stat4+/T-bet+ and decreased GATA2+/CD68+/CD45+/CD206+ cells); (ii) modulated cytokine levels in CD4+ T cells; and (iii) decreased levels of IL5/6/12/13, sTNFR1, and serum-RANTES. We suggest that S100A4-antibody therapy has clinical applicability in treating immunosuppressive prostate cancer in patients.

Identifying and treating ROBO1-ve /DOCK1+ve prostate cancer: An aggressive cancer subtype prevalent in African American patients.

BACKGROUND: There is a need to develop novel therapies which could be beneficial to patients with prostate cancer (CaP) including those who are predisposed to poor outcome, such as African-Americans. This study investigates the role of ROBO1-pathway in predicting outcome and race-based disparity in patients with CaP. METHODS AND RESULTS: Aided by RNA sequencing-based DECIPHER-testing and immunohistochemical (IHC) analysis of tumors we show that ROBO1 is lost during the progressive stages of CaP, a prevalent feature in African-Americans. We show that the loss of ROBO1 predicts high-risk of recurrence, metastasis and poor outcome of androgen-deprivation therapy in radical prostatectomy-treated patients. These data identified an aggressive ROBO1deficient /DOCK1+ve sub-class of CaP. Combined genetic and IHC data showed that ROBO1 loss is accompanied by DOCK1/Rac1 elevation in grade-III/IV primary-tumors and Mets. We observed that the hypermethylation of ROBO1-promoter contributes to loss of expression that is highly prevalent in African-Americans. Because of limitations in restoring ROBO1 function, we asked if targeting the DOCK1 could be an ideal strategy to inhibit progression or treat ROBO1deficient metastatic-CaP. We tested the pharmacological efficacy of CPYPP, a selective inhibitor of DOCK1 under in vitro and in vivo conditions. Using ROBO1-ve and ROBO1+ve CaP models, we determined the median effective concentration of CPYPP for growth. DOCK1-inhibitor treatment significantly decreased the (a) Rac1-GTP/ß-catenin activity, (b) transmigration of ROBO1deficient cells across endothelial lining, and (c) metastatic spread of ROBO1deficient cells through the vasculature of transgenicfl Zebrafish model. CONCLUSION: We suggest that ROBO1 status forms as predictive biomarker of outcome in high-risk populations such as African-Americans and DOCK1-targeting therapy has a clinical potential for treating metastatic-CaP.

COMMD3:BMI1 Fusion and COMMD3 Protein Regulate C-MYC Transcription: Novel Therapeutic Target for Metastatic Prostate Cancer.

Gene rearrangement is reported to be associated to the aggressive phenotype and poor prognosis in prostate cancer. We identified a gene fusion between a transcription repressor (BMI1) and transcriptional factor (COMMD3) in human prostate cancer. We show that COMMD3:BMI1 fusion expression is significantly increased in prostate cancer disease in an order: normal tissue < primary < metastatic tumors (Mets). Although elevated TMPRSS-ERG/ETV fusion is reported in prostate cancer, we identified a subtype of Mets exhibiting low TMPRSS:ETV and high COMMD3:BMI1 We delineated the mechanism and function of COMMD3 and COMMD3:BMI1 in prostate cancer. We show that COMMD3 level is elevated in prostate cancer cell models, PDX models (adenocarcinoma, NECaP), and Mets. The analysis of TCGA/NIH/GEO clinical data showed a positive correlation between increased COMMD3 expression to the disease recurrence and poor survival in prostate cancer. We show that COMMD3 drives proliferation of normal cells and promotes migration/invasiveness of neoplastic cells. We show that COMMD3:BMI1 and COMMD3 regulate C-MYC transcription and C-MYC downstream pathway. The ChIP analysis showed that COMMD3 protein is recruited at the promoter of C-MYC gene. On the basis of these data, we investigated the relevance of COMMD3:BMI1 and COMMD3 as therapeutic targets using in vitro and xenograft mouse models. We show that siRNA-mediated targeting of COMMD3:BMI1 and COMMD3 significantly decreases (i) C-MYC expression in BRD/BET inhibitor-resistant cells, (ii) proliferation/invasion in vitro, and (iii) growth of prostate cancer cell tumors in mice. The IHC analysis of tumors confirmed the targeting of COMMD3-regulated molecular pathway under in vivo conditions. We conclude that COMMD3:BMI1 and COMMD3 are potential progression biomarkers and therapeutic targets of metastatic prostate cancer.

Characterization of Novel Murine and Human PDAC Cell Models: Identifying the Role of Intestine Specific Homeobox Gene ISX in Hypoxia and Disease Progression.

Therapy failure and metastasis-associated mortality are stumbling blocks in the management of PDAC in patients. Failure of therapy is associated to intense hypoxic conditions of tumors. To develop effective therapies, a complete understanding of hypoxia-associated changes in genetic landscape of tumors during disease progression is needed. Because artificially immortalized cell lines do not rightly represent the disease progression, studying genetics of tumors in spontaneous models is warranted. In the current study, we generated a spectrum of spontaneous human (UM-PDC1; UM-PDC2) and murine (HI-PanL, HI-PancI, HI-PanM) models representing localized, invasive, and metastatic PDAC from a patient and transgenic mice (K-rasG12D/Pdxcre/Ink4a/p16-/). These spontaneous models grow vigorously under hypoxia and exhibit activated K-ras signaling, progressive loss of PTEN, and tumorigenicity in vivo. Whereas UM-PDC1 form localized tumors, the UM-PDC2 metastasize to lungs in mice. In an order of progression, these models exhibit genomic instability marked by gross chromosomal rearrangements, centrosome-number variations, Aurora-kinase/H2AX colocalization, loss of primary cilia, and α-tubulin acetylation. The RNA sequencing of hypoxic models followed by qRT-PCR validation and gene-set enrichment identified Intestine-Specific Homeobox factor (ISX)-driven molecular pathway as an indicator PDAC aggressivness. TCGA-PAAD clinical data analysis showed high ISX expression correlation to poor survival of PDAC patients, particularly women. The functional studies showed ISX as a regulator of i) invasiveness and migratory potential and ii) VEGF, MMP2, and NFκB activation in PDAC cells. We suggest that ISX is a potential druggable target and newly developed spontaneous cell models are valuable tools for studying mechanism and testing therapies for PDAC.

BMI1 Drives Metastasis of Prostate Cancer in Caucasian and African-American Men and Is A Potential Therapeutic Target: Hypothesis Tested in Race-specific Models.

PURPOSE: Metastasis is the major cause of mortality in prostate cancer patients. Factors such as genetic makeup and race play critical role in the outcome of therapies. This study was conducted to investigate the relevance of BMI1 in metastatic prostate cancer disease in Caucasian and African-Americans. EXPERIMENTAL DESIGN: We employed race-specific prostate cancer models, clinical specimens, clinical data mining, gene-microarray, transcription-reporter assay, chromatin-immunoprecipitation (ChIP), IHC, transgenic-(tgfl/fl) zebrafish, and mouse metastasis models. RESULTS: BMI1 expression was observed to be elevated in metastatic tumors (lymph nodes, lungs, bones, liver) of Caucasian and African-American prostate cancer patients. The comparative analysis of stage III/IV tumors showed an increased BMI1 expression in African-Americans than Caucasians. TCGA and NIH/GEO clinical data corroborated to our findings. We show that BMI1 expression (i) positively correlates to metastatic (MYC, VEGF, cyclin D1) and (ii) negative correlates to tumor suppressor (INKF4A/p16, PTEN) levels in tumors. The correlation was prominent in African-American tumors. We show that BMI1 regulates the transcriptional activation of MYC, VEGF, INKF4A/p16, and PTEN. We show the effect of pharmacological inhibition of BMI1 on the metastatic genome and invasiveness of tumor cells. Next, we show the anti-metastatic efficacy of BMI1-inhibitor in transgenic zebrafish and mouse metastasis models. Docetaxel as monotherapy has poor outcome on the growth of metastatic tumors. BMI1 inhibitor as an adjuvant improved the taxane therapy in race-based in vitro and in vivo models. CONCLUSIONS: BMI1, a major driver of metastasis, represents a promising therapeutic target for treating advanced prostate cancer in patients (including those belonging to high-risk group).