Growth Arrest‐Specific 6 (GAS6) Promotes Prostate Cancer Survival by G1 Arrest/S Phase Delay and Inhibition of Apoptosis During Chemotherapy in Bone Marrow

Prostate cancer (PCa) is known to develop resistance to chemotherapy. Growth arrest‐specific 6 (GAS6), plays a role in tumor progression by regulating growth in many cancers. Here, we explored how GAS6 regulates the cell cycle and apoptosis of PCa cells in response to chemotherapy. We found that GAS6 is sufficient to significantly increase the fraction of cells in G1 and the duration of phase in PCa cells. Importantly, the effect of GAS6 on G1 is potentiated during docetaxel chemotherapy. GAS6 altered the levels of several key cell cycle regulators, including the downregulation of Cyclin B1 (G2/M phase), CDC25A, Cyclin E1, and CDK2 (S phase entry), while the upregulation of cell cycle inhibitors p27 and p21, Cyclin D1, and CDK4. Importantly, these changes became further accentuated during docetaxel treatment in the presence of GAS6. Moreover, GAS6 alters the apoptotic response of PCa cells during docetaxel chemotherapy. Docetaxel induced PCa cell apoptosis is efficiently suppressed in PCa cell culture in the presence of GAS6 or GAS6 secreted from co‐cultured osteoblasts. Similarly, the GAS6‐expressing bone environment protects PCa cells from apoptosis within primary tumors in vivo studies. Docetaxel induced significant levels of Caspase‐3 and PARP cleavage in PCa cells, while GAS6 protected PCa cells from docetaxel‐induced apoptotic signaling. Together, these data suggest that GAS6, expressed by osteoblasts in the bone marrow, plays a significant role in the regulation of PCa cell survival during chemotherapy, which will have important implications for targeting metastatic disease. J. Cell. Biochem. 117: 2815–2824, 2016. © 2016 Wiley Periodicals, Inc.We explored how GAS6, expressed by osteoblasts, regulates the cell cycle and apoptosis in PCa cells during chemotherapy in the bone marrow. We demonstrate that GAS6 significantly increases the number of G1 arrested cells by altering signaling networks associated with G1 arrest and S phase delay. Our results suggest that GAS6 contributes to the regulation of PCa cell survival during chemotherapy in the bone marrow microenvironment.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/134410/1/jcb25582_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/134410/2/jcb25582.pd

Mer Tyrosine Kinase Regulates Disseminated Prostate Cancer Cellular Dormancy

Many prostate cancer (PCa) recurrences are thought to be due to reactivation of disseminated tumor cells (DTCs). We previously found a role of the TAM family of receptor tyrosine kinases TYRO3, AXL, and MERTK in PCa dormancy regulation. However, the mechanism and contributions of the individual TAM receptors is largely unknown. Knockdown of MERTK, but not AXL or TYRO3 by shRNA in PCa cells induced a decreased ratio of Pâ Erk1/2 to Pâ p38, increased expression of p27, NR2F1, SOX2, and NANOG, induced higher levels of histone H3K9me3 and H3K27me3, and induced a G1/G0 arrest, all of which are associated with dormancy. Similar effects were also observed with siRNA. Most importantly, knockdown of MERTK in PCa cells increased metastasis free survival in an intraâ cardiac injection mouse xenograft model. MERTK knockdown also failed to inhibit PCa growth in vitro and subcutaneous growth in vivo, which suggests that MERTK has specificity for dormancy regulation or requires a signal from the PCa microenvironment. The effects of MERTK on the cell cycle and histone methylation were reversed by p38 inhibitor SB203580, which indicates the importance of MAP kinases for MERTK dormancy regulation. Overall, this study shows that MERTK stimulates PCa dormancy escape through a MAP kinase dependent mechanism, also involving p27, pluripotency transcription factors, and histone methylation. J. Cell. Biochem. 118: 891â 902, 2017. © 2016 Wiley Periodicals, Inc.Escape from cellular dormancy is the process where previously dormant single disseminated tumor cells reactivate to form cancer microâ metastases, which continue to grow and ultimately make the disease incurable. Here, were show that Mer tyrosine kinase is important for prostate cancer dormancy escape through a mechanism involving MAP kinases, cell cycle inhibitors, epigenetics, and transcription factors associated with pluripotent cells.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/136285/1/jcb25768_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/136285/2/jcb25768.pd

Detection and isolation of disseminated tumor cells in bone marrow of patients with clinically localized prostate cancer

BackgroundDisseminated tumor cells (DTCs) have been reported in the bone marrow (BM) of patients with localized prostate cancer (PCa). However, the existence of these cells continues to be questioned, and few methods exist for viable DTC isolation. Therefore, we sought to develop novel approaches to identify and, if detected, analyze localized PCa patient DTCs.MethodsWe used fluorescence‐activated cell sorting (FACS) to isolate a putative DTC population, which was negative for CD45, CD235a, alkaline phosphatase, and CD34, and strongly expressed EPCAM. We examined tumor cell content by bulk cell RNA sequencing (RNA‐Seq) and whole‐exome sequencing after whole genome amplification. We also enriched for BM DTCs with α‐EPCAM immunomagnetic beads and performed quantitative reverse trancriptase polymerase chain reaction (qRT‐PCR) for PCa markers.ResultsAt a threshold of 4 cells per million BM cells, the putative DTC population was present in 10 of 58 patients (17%) with localized PCa, 4 of 8 patients with metastatic PCa of varying disease control, and 1 of 8 patients with no known cancer, and was positively correlated with patients’ plasma PSA values. RNA‐Seq analysis of the putative DTC population collected from samples above (3 patients) and below (5 patients) the threshold of 4 putative DTCs per million showed increased expression of PCa marker genes in 4 of 8 patients with localized PCa, but not the one normal donor who had the putative DTC population present. Whole‐exome sequencing also showed the presence of single nucleotide polymorphisms and structural variants in the gene characteristics of PCa in 2 of 3 localized PCa patients. To examine the likely contaminating cell types, we used a myeloid colony formation assay, differential counts of cell smears, and analysis of the RNA‐Seq data using the CIBERSORT algorithm, which most strongly suggested the presence of B‐cell lineages as a contaminant. Finally, we used EPCAM enrichment and qRT‐PCR for PCa markers to estimate DTC prevalence and found evidence of DTCs in 21 of 44 samples (47%).ConclusionThese data support the presence of DTCs in the BM of a subset of patients with localized PCa and describe a novel FACS method for isolation and analysis of viable DTCs.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/151343/1/pros23896.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151343/2/pros23896_am.pd

Evolution of Disparities in Prostate Cancer Treatment: Is This a New Normal?

Despite notable screening, diagnostic, and therapeutic advances, disparities in prostate cancer incidence and outcomes remain prevalent. Although commonly discussed in the context of men of African descent, disparities also exist based on socioeconomic level, education level, and geographic location. The factors in these disparities span systemic access issues affecting availability of care, provider awareness, and personal patient views and mistrust. In this review, we will discuss common themes that patients have noted as impediments to care. We will review how equitable access to care has helped improve outcomes among many different groups of patients, including those with local disease and those with metastatic castration-resistant prostate cancer. Even with more advanced presentation, challenges with recommended screening, and lower rates of genomic testing and trial inclusion, Black populations have benefited greatly from various modalities of therapy, achieving comparable and at times superior outcomes with certain types of immunotherapy, chemotherapy, androgen receptor–based inhibitors, and radiopharmaceuticals in advanced disease. We will also briefly discuss access to genomic testing and differences in patterns of gene expression among Black patients and other groups that are traditionally underrepresented in trials and genomic cohort studies. We propose several strategies on behalf of providers and institutions to help promote more equitable care access environments and continued decreases in prostate cancer disparities across many subgroups

Reduction of two histone marks, H3k9me3 and H3k27me3 by epidrug induces neuroendocrine differentiation in prostate cancer

Neuroendocrine prostate cancer (NE PCa) is an aggressive malignancy, often presenting with advanced metastasis. We previously reported that reduction of histone marks regulated by DNMT1 following epidrug (5‐Azacitidine, 5‐Aza) treatment controls induction of epithelial to mesenchymal (EMT) and a cancer stem cell (CSC) phenotype, which facilitates tumorigenesis in PCa cells. Here, we use the epidrug 5‐Aza as a model for how histone marks may regulate the reprogramming of prostate adenocarcinoma into NE phenotypic cells. First, we observed that 5‐Aza treatment of PCa cells in vitro induces a neuron‐like phenotype. In addition, significant increases in the expression of the NE markers N‐Myc downstream regulated gene 1 (NDRG1), enolase‐2 (ENO2), and synaptophysin were observed. Critically, a high density of NE cells with synaptophysin expression was found in tumors generated by 5‐Aza pretreatment of PCa cells. Importantly, induction of NE differentiation of PCa cells was associated with an enhancement of NDRG1 expression by reduction of two histone marks, H3K9me3 and H3K27me3. Further, more NDRG1 expression was detected in the subset of PCa cells with reduced expression of H3K9me3 or H3K27me3 in the tumors generated by 5‐Aza pretreated PCa cells and critically, these biological differences are also observed in small cell carcinoma in advanced stage of human primary PCa tumors. Our results suggest that reduction of histone marks regulated by the epidrug 5‐Aza may control induction of a NE phenotype, which facilitates PCa progression. These studies suggest a strong rationale for developing therapeutics, which target epigenetic regulation.Reduction of histone marks induces an neuroendocrine phenotype in PCa.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142487/1/jcb26586.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/142487/2/jcb26586_am.pd

Additional file 2 of SNHG1 opposes quiescence and promotes docetaxel sensitivity in prostate cancer

Additional file 2. SNHG1silencing is protective against DTX in LNCaPandDU-145 cells. (A) Representative histograms of PI stained LNCaPandDU-145 with or without SNHG1knockdown and with or without treatment with20 nMDTX. (B) Results of quantitation of apoptotic, G0/G1,S, and G2/Mpopulations in untreated or DTX treated, SNHG1silenced or not, cells.Data represent mean ±SD, N=4. Statistical analysis was done usingStudent’s t test: ns, not significant; *P<0.05; **, P<0.01

Additional file 3 of SNHG1 opposes quiescence and promotes docetaxel sensitivity in prostate cancer

Additional file 3. SNHG1silencing results in reduced G2phaseand apoptosis markers in DU-145 cells after DTX treatment. Western blotsshowing the apoptosis marker, cleaved caspase 3 and cleaved PARP1, and the G2marker,cyclin B1. β-actin is loading control. In (A), displayed is the sameblot reprobedfor different proteins. In (B), displayed are differentexposures of the same blot probed for caspase 3 (full length and cleavagefragment) and β-actin. Numbers indicate band density versus siCTRL, normalizedto β-actin

Additional file 4 of SNHG1 opposes quiescence and promotes docetaxel sensitivity in prostate cancer

Additional file 4. Original,full-length images from Western blotting, corresponding to cropped images shownin Fig. 7. SNHG1silencing results in reduced G2phaseand apoptosis markers after DTX treatment. Western blots showing the apoptosismarkers, cleaved caspase 3 and cleaved PARP1, and the G2marker,cyclin B1. β-actin is loading control. For PC3 (A), two separate blotsare shown. For C4-2B (B), a single blot was probed for multipleproteins. Numbers indicate band density versus siCTRL, normalized to β-actin

Additional file 1 of SNHG1 opposes quiescence and promotes docetaxel sensitivity in prostate cancer

Additional file 1. Knockdownof SNHG1reduces DNA synthesis in LNCaPand DU-145 cells. (A) Assessmentof SNHG1knockdown in LNCaPand DU-145 cells. (B) Flow cytometryplot of EdUlabeled cells with or without SNHG1knockdown. (C) Quantitationof the proportion of EdU+cells after transfection with siCTRLor siSNHG1.All graphs depict mean±SD, N=3. Statistical analysis done using Studentt test: *P<0.05; **, P<0.01

CXCL12γ induces human prostate and mammary gland development

BACKGROUND: Epithelial stem cells (ESCs) demonstrate a capacity to maintain normal tissues homeostasis and ESCs with a deregulated behavior can contribute to cancer development. The ability to reprogram normal tissue epithelial cells into prostate or mammary stem-like cells holds great promise to help understand cell of origin and lineage plasticity in prostate and breast cancers in addition to understanding normal gland development. We previously showed that an intracellular chemokine, CXCL12γ, induced cancer stem cells (CSCs) and neuroendocrine characteristics in both prostate and breast adenocarcinoma cell lines. However, its role in normal prostate or mammary epithelial cell fate and development remains unknown. Therefore, we sought to elucidate the functional role of CXCL12γ in the regulation of ESCs and tissue development. METHODS: Prostate epithelial cells (PNT2) or mammary epithelial cells (MCF10A) with overexpressed CXCL12γ was characterized by qRT-PCR, Western blots, and immunofluorescence for lineage marker expression, and FACS analyses, and sphere formation assays to examine stem cell surface phenotype and function. Xenotransplantation animal models were used to evaluate gland or acini formation in vivo. RESULTS: Overexpression of CXCL12γ promotes the reprograming of cells with a differentiated luminal phenotype to a non-luminal phenotype in both prostate (PNT2) and mammary (MCF10A) epithelial cells. The CXCL12γ-mediated non-luminal type cells results in an increase of epithelial stem-like phenotype including the subpopulation of EPCAM(Lo)/CD49f(Hi)/CD24(Lo)/CD44(Hi) cells capable of sphere formation. Critically, overexpression of CXCL12γ promotes the generation of robust gland-like structures from both prostate and mammary epithelial cells in in vivo xenograft animal models. CONCLUSIONS: CXCL12γ supports the reprogramming of epithelial cells into non-luminal cell-derived stem cells, which facilitates gland development