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

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

Transforming the Healthcare Response to Intimate Partner Violence and Taking Best Practices to Scale

BACKGROUND: Intimate partner violence (IPV) is prevalent among adolescent and adult women, with significant physical, sexual, and mental health consequences. In 2011, the Institute of Medicine\u27s Clinical Preventive Services for Women consensus report recommended universal screening for violence as a component of women\u27s preventive services; this policy has been adopted by the Health Resources and Services Administration (HRSA). These policy developments require that effective clinic-based interventions be identified, easily implemented, and taken to scale. METHODS: To foster dialogue about implementing effective interventions, we convened a symposium entitled Responding to Violence Against Women: Emerging Evidence, Implementation Science, and Innovative Interventions, on May 21, 2012. Drawing on multidisciplinary expertise, the agenda integrated data on the prevalence and health impact of IPV violence, with an overview of the implementation science framework, and a panel of innovative IPV screening interventions. Recommendations were generated for developing, testing, and implementing clinic-based interventions to reduce violence and mitigate its health impact. RESULTS: The strength of evidence supporting specific IPV screening interventions has improved, but the optimal implementation and dissemination strategies are not clear. Implementation science, which seeks to close the evidence to program gap, is a useful framework for improving screening and intervention uptake and ensuring the translation of research findings into routine practice. CONCLUSIONS: Findings have substantial relevance to the broader research, clinical, and practitioner community. Our conference proceedings fill a timely gap in knowledge by informing practitioners as they strive to implement universal IPV screening and guiding researchers as they evaluate the success of implementing IPV interventions to improve women\u27s health and well-being

Drug–gene and drug–drug interactions associated with tramadol and codeine therapy in the INGENIOUS trial

Background: Tramadol and codeine are metabolized by CYP2D6 and are subject to drug-gene and drug-drug interactions. Methods: This interim analysis examined prescribing behavior and efficacy in 102 individuals prescribed tramadol or codeine while receiving pharmaco-genotyping as part of the INGENIOUS trial (NCT02297126). Results: Within 60 days of receiving tramadol or codeine, clinicians more frequently prescribed an alternative opioid in ultrarapid and poor metabolizers (odds ratio: 19.0; 95% CI: 2.8-160.4) as compared with normal or indeterminate metabolizers (p = 0.01). After adjusting the CYP2D6 activity score for drug-drug interactions, uncontrolled pain was reported more frequently in individuals with reduced CYP2D6 activity (odds ratio: 0.50; 95% CI: 0.25-0.94). Conclusion: Phenoconversion for drug-drug and drug-gene interactions is an important consideration in pharmacogenomic implementation; drug-drug interactions may obscure the potential benefits of genotyping

Lessons Learned: It Takes a Village to Understand Inter-Sectoral Care Using Administrative Data across Jurisdictions

Cancer care is complex and exists within the broader healthcare system. The CanIMPACT team sought to enhance primary cancer care capacity and improve integration between primary and cancer specialist care, focusing on breast cancer. In Canada, all medically-necessary healthcare is publicly funded but overseen at the provincial/territorial level. The CanIMPACT Administrative Health Data Group’s (AHDG) role was to describe inter-sectoral care across five Canadian provinces: British Columbia, Alberta, Manitoba, Ontario and Nova Scotia. This paper describes the process used and challenges faced in creating four parallel administrative health datasets. We present the content of those datasets and population characteristics. We provide guidance for future research based on ‘lessons learned’. The AHDG conducted population-based comparisons of care for breast cancer patients diagnosed from 2007-2011. We created parallel provincial datasets using knowledge from data inventories, our previous work, and ongoing bi-weekly conference calls. Common dataset creation plans (DCPs) ensured data comparability and documentation of data differences. In general, the process had to be flexible and iterative as our understanding of the data and needs of the broader team evolved. Inter-sectoral data inconsistencies that we had to address occurred due to differences in: 1) healthcare systems, 2) data sources, 3) data elements and 4) variable definitions. Our parallel provincial datasets describe the breast cancer diagnostic, treatment and survivorship phases and address ten research objectives. Breast cancer patient demographics reflect inter-provincial general population differences. Across provinces, disease characteristics are similar but underlying health status and use of healthcare services differ. Describing healthcare across Canadian jurisdictions assesses whether our provincial healthcare systems are delivering similar high quality, timely, accessible care to all of our citizens. We have provided a description of our experience in trying to achieve this goal and include a list of ‘lessons learned’ and a study process checklist for future use

Structure–activity relationships of substituted 1H-indole-2-carboxamides as CB1 receptor allosteric modulators

A series of substituted 1-indole-2-carboxamides structurally related to compounds Org27569 (), Org29647 () and Org27759 () were synthesized and evaluated for CB1 allosteric modulating activity in calcium mobilization assays. Structure–activity relationship studies showed that the modulation potency of this series at the CB1 receptor was enhanced by the presence of a diethylamino group at the 4-position of the phenyl ring, a chloro or fluoro group at the C5 position and short alkyl groups at the C3 position on the indole ring. The most potent compound () had an IC value of 79 nM which is ∼2.5 and 10 fold more potent than the parent compounds and , respectively. These compounds appeared to be negative allosteric modulators at the CB1 receptor and dose-dependently reduced the of agonist CP55,940. These analogs may provide the basis for further optimization and use of CB1 allosteric modulators

Pol II phosphorylation regulates a switch between transcriptional and splicing condensates

The synthesis of pre-mRNA by RNA polymerase II (Pol II) involves the formation of a transcription initiation complex, and a transition to an elongation complex. The large subunit of Pol II contains an intrinsically disordered C-terminal domain that is phosphorylated by cyclin-dependent kinases during the transition from initiation to elongation, thus influencing the interaction of the C-terminal domain with different components of the initiation or the RNA-splicing apparatus. Recent observations suggest that this model provides only a partial picture of the effects of phosphorylation of the C-terminal domain. Both the transcription-initiation machinery and the splicing machinery can form phase-separated condensates that contain large numbers of component molecules: hundreds of molecules of Pol II and mediator are concentrated in condensates at super-enhancers, and large numbers of splicing factors are concentrated in nuclear speckles, some of which occur at highly active transcription sites. Here we investigate whether the phosphorylation of the Pol II C-terminal domain regulates the incorporation of Pol II into phase-separated condensates that are associated with transcription initiation and splicing. We find that the hypophosphorylated C-terminal domain of Pol II is incorporated into mediator condensates and that phosphorylation by regulatory cyclin-dependent kinases reduces this incorporation. We also find that the hyperphosphorylated C-terminal domain is preferentially incorporated into condensates that are formed by splicing factors. These results suggest that phosphorylation of the Pol II C-terminal domain drives an exchange from condensates that are involved in transcription initiation to those that are involved in RNA processing, and implicates phosphorylation as a mechanism that regulates condensate preference