Protein kinase D1 induces G1-phase cell-cycle arrest independent of Checkpoint kinases by phosphorylating Cell Division Cycle Phosphatase 25.

Protein Kinase D1 (PrKD1) functions as a tumor and metastasis suppressor in several human cancers by influencing cell-cycle progression. However, the exact mechanism of cell-cycle regulation by PrKD1 is unclear. Overexpression and ectopic expression of PrKD1 induces G1 arrest in cancer cell lines. Because checkpoint kinases (CHEKs) are known to play a role in progression through the G1 phase, we downregulated CHEK1, which did not overcome the G1 arrest induced by PrKD1. Using in vitro phosphorylation and Western blot assays, we showed that PrKD1 phosphorylates all CDC25 isoforms (known substrates of CHEK kinases), independent from CHEK kinases, suggesting that direct phosphorylation of CDC25 by PrKD1 may be an alternate mechanism of G1 arrest. The study has identified a molecular mechanism for the influence of PrKD1 in cell-cycle progression.

Beta-catenin represses protein kinase D1 gene expression by non-canonical pathway through MYC/MAX transcription complex in prostate cancer.

Down regulation of Protein Kinase D1 (PrKD1), a novel serine threonine kinase, in prostate, gastric, breast and colon cancers in humans leads to disease progression. While the down regulation of PrKD1 by DNA methylation in gastric cancer and by nuclear beta-catenin in colon cancer has been shown, the regulatory mechanisms in other cancers are unknown. Because we had demonstrated that PrKD1 is the only known kinase to phosphorylate threonine 120 (T120) of beta-catenin in prostate cancer resulting in increased nuclear beta-catenin, we explored the role of beta-catenin in gene regulation of PrKD1. An initial CHIP assay identified potential binding sites for beta-catenin in and downstream of PrKD1 promoter and sequencing confirmed recruitment of beta-catenin to a 166 base pairs sequence upstream of exon 2. Co-transfection studies with PrKD1-promoter-reporter suggested that beta-catenin represses PrKD1 promoter. Efforts to identify transcription factors that mediate the co-repressor effects of beta-catenin identified recruitment of both MYC and its obligate heterodimer MAX to the same binding site as beta-catenin on the PrKD1 promoter site. Moreover, treatment with MYC inhibitor rescued the co-repressor effect of beta-catenin on PrKD1 gene expression. Prostate specific knock out of PrKD1 in transgenic mice demonstrated increased nuclear expression of beta-catenin validating the in vitro studies. Functional studies showed that nuclear translocation of beta-catenin as a consequence of PrKD1 down regulation, increases AR transcriptional activity with attendant downstream effects on androgen responsive genes. In silico human gene expression analysis confirmed the down regulation of PrKD1 in metastatic prostate cancer correlated inversely with the expression of MAX, but not MYC, and positively with MXD1, a competing heterodimer of MAX, suggesting that the dimerization of MAX with either MYC or MXD1 regulates PrKD1 gene expression. The study has identified a novel auto-repressive loop that perpetuates PrKD1 down regulation through beta-catenin/MYC/MAX protein complex.

Cell line modeling to study biomarker panel in prostate cancer.

BACKGROUND: African-American men with prostate cancer (PCa) present with higher-grade and -stage tumors compared to Caucasians. While the disparity may result from multiple factors, a biological basis is often strongly suspected. Currently, few well-characterized experimental model systems are available to study the biological basis of racial disparity in PCa. We report a validated in vitro cell line model system that could be used for the purpose. METHODS: We assembled a PCa cell line model that included currently available African-American PCa cell lines and LNCaP (androgen-dependent) and C4-2 (castration-resistant) Caucasian PCa cells. The utility of the cell lines in studying the biological basis of variance in a malignant phenotype was explored using a multiplex biomarker panel consisting of proteins that have been proven to play a role in the progression of PCa. The panel expression was evaluated by Western blot and RT-PCR in cell lines and validated in human PCa tissues by RT-PCR. As proof-of-principle to demonstrate the utility of our model in functional studies, we performed MTS viability assays and molecular studies. RESULTS: The dysregulation of the multiplex biomarker panel in primary African-American cell line (E006AA) was similar to metastatic Caucasian cell lines, which would suggest that the cell line model could be used to study an inherent aggressive phenotype in African-American men with PCa. We had previously demonstrated that Protein kinase D1 (PKD1) is a novel kinase that is down regulated in advanced prostate cancer. We established the functional relevance by over expressing PKD1, which resulted in decreased proliferation and epithelial mesenchymal transition (EMT) in PCa cells. Moreover, we established the feasibility of studying the expression of the multiplex biomarker panel in archived human PCa tissue from African-Americans and Caucasians as a prelude to future translational studies. CONCLUSION: We have characterized a novel in vitro cell line model that could be used to study the biological basis of disparity in PCa between African-Americans and Caucasians.

Novel In Vivo model for combinatorial fluorescence labeling in mouse prostate.

BACKGROUND: The epithelial layer of prostate glands contains several types of cells, including luminal and basal cells. Yet there is paucity of animal models to study the cellular origin of normal or neoplastic development in the prostate to facilitate the treatment of heterogenous prostate diseases by targeting individual cell lineages. METHODS: We developed a mouse model that expresses different types of fluorescent proteins (XFPs) specifically in prostatic cells. Using an in vivo stochastic fluorescent protein combinatorial strategy, XFP signals were expressed specifically in prostate of Protein Kinase D1 (PKD1) knock-out, K-Ras(G) (12) (D) knock-in, and Phosphatase and tensin homolog (PTEN) and PKD1 double knock-out mice under the control of PB-Cre promoter. RESULTS: In vivo XFP signals were observed in prostate of PKD1 knock-out, K-Ras(G) (12) (D) knock-in, and PTEN PKD1 double knock-out mice, which developed normal, hyperplastic, and neoplastic prostate, respectively. The patchy expression pattern of XFPs in neoplasia tissue indicated the clonal origin of cancer cells in the prostate. CONCLUSIONS: The transgenic mouse models demonstrate combinatorial fluorescent protein expression in normal and cancerous prostatic tissues. This novel prostate-specific fluorescent labeled mouse model, which we named Prorainbow, could be useful in studying benign and malignant pathology of prostate.

Genetic and epigenetic stability of human spermatogonial stem cells during long-term culture.

OBJECTIVE: To determine the genetic and epigenetic stability of human spermatogonial stem cells (SSCs) during long-term culture. DESIGN: Experimental basic science study. SETTING: Reproductive biology laboratory. PATIENT(S): Cryopreserved human testicular tissue from two prostate cancer patients with normal spermatogenesis. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Testicular cells before and 50 days after culturing were subjected to ITGA6 magnetic-activated cell sorting to enrich for SSCs. Individual spermatogonia were analyzed for aneuploidies with the use of single-cell 24-chromosome screening. Furthermore, the DNA methylation statuses of the paternally imprinted genes H19, H19-DMR (differentially methylated region), and MEG3 and the maternally imprinted genes KCNQ1OT1 and PEG3 were identified by means of bisulfite sequencing. RESULTS(S): Aneuploidy screening showed euploidy with no chromosomal abnormalities in all cultured and most noncultured spermatogonia from both patients. The methylation assays demonstrated demethylation of the paternally imprinted genes H19, H19-DMR, and MEG3 of 11%-28%, 43%-68%, and 18%-26%, respectively, and increased methylation of the maternally imprinted genes PEG 3 and KCNQ1OT of 13%-50% and 30%-38%, respectively, during culture. CONCLUSION(S): In the current culture system for human SSCs propagation, genomic stability is preserved, which is important for future clinical use. Whether the observed changes in methylation status have consequences on functionality of SSCs or health of offspring derived from transplanted SSCs requires further investigation.

Enrichment of spermatogonial stem cells from long-term cultured human testicular cells.

OBJECTIVE: To evaluate the degree of enrichment of spermatogonial stem cells (SSCs) from human testicular cell cultures by ITGA6+, HLA-/ITGA6+, GPR125+, and HLA-/GPR125+ magnetic-assisted cell sorting (MACS). DESIGN: Experimental basic science study. SETTING: Reproductive biology laboratory. PATIENT(S): Multiple samples of cryopreserved human testicular cells from two prostate cancer patients with normal spermatogenesis. INTERVENTION(S): Cultured human testicular cells subjected to four sorting strategies based on MACS and xenotransplanted to the testes of mice to determine the enrichment for SSCs. MAIN OUTCOME MEASURE(S): Enrichment for human spermatogonia and SSCs tested by expression analysis of spermatogonial markers ITGA6, GPR125, ZBTB16, UCHL1, and ID4 using quantitative real-time polymerase chain reaction (qPCR) and by xenotransplantation into the testes of mice, respectively. RESULT(S): Compared with the nonsorted cultured testicular cells, only the ITGA6+ and HLA-/GPR125+ sorted cells showed enrichment for ID4. No difference in expression of ZBTB16 and UCHL1 was observed. Xenotransplantation of the sorted cell fractions showed a 7.1-fold enrichment of SSCs with ITGA6+. CONCLUSION(S): Magnetic-assisted cell sorting of cultured human testicular cells using ITGA6 allows for enrichment of SSCs, which aids in further molecular characterization of cultured human SSCs and enhances testicular colonization upon transplantation in future clinical settings.

Y chromosome TSPY copy numbers and semen quality.

OBJECTIVE: To determine whether variation in testis-specific protein Y-encoded (TSPY) gene copy number affects semen quality. DESIGN: Nested case-control study. SETTING: University hospital. PATIENT(S): From a consecutive cohort of 1,016 male partners of subfertile couples, unselected for sperm counts, we selected as cases 100 men with the lowest total number of progressively motile sperm (TMC) and as controls, 100 men with the highest total number of progressively motile sperm. INTERVENTION(S): Quantitative real-time polymerase chain reaction (PCR) and Southern blot to determine TSPY copy number. MAIN OUTCOME MEASURE(S): TSPY copy number. RESULT(S): The quantitative PCR method showed excellent agreement with the Southern blot analysis. Cases had a median TSPY copy number of 35 (range 20-73), whereas controls had a median TSPY copy number of 34 (range 26-76). This difference was not statistically significant. CONCLUSION(S): We found no association between TSPY copy numbers and severe spermatogenic failure. The observed variation in TSPY copy number therefore appears to have no functional consequences for semen quality.