Sudeep K. Bose

PAX2 oncogene negatively regulates the expression of the host defense peptide human beta defensin-1 in prostate cancer

Human beta defensin-1 (hBD1) is a component of the immune system which links the innate and adaptive immune responses. We have demonstrated that hBD1 induces rapid cytolysis of prostate cancer cells and that it may also possess tumor suppressive abilities. In addition, there is a high frequency of cancer-specific loss of hBD1 expression which further suggests its potential role in tumor progression. However, the factors responsible for the loss of hBD1 expression are not known. PAX2, a transcriptional regulator normally expressed during early development, has been implicated as an oncogene in carcinomas of the kidney, prostate, breast and ovary. It is known that expression of PAX2 in these tumor cells mediates the evasion of cell death through the suppression of cell death pathways involving the p53 tumor suppressor. However, we have demonstrated that knock-down of PAX2 expression results in cell death independent of p53 status, thus suggesting that additional cell death pathways are negatively regulated by PAX2. Here we describe a novel pathway in which PAX2 represses hBD1 expression through binding of the PAX2 homeodomain to the hBD1 promoter. Furthermore, knock-down of PAX2 expression results in the re-expression of hBD1, and subsequently prostate cancer cell death. These findings are the first to demonstrate that the PAX2 oncogene suppresses hBD1 expression in cancer and further implicate PAX2 as a novel therapeutic target for prostate cancer treatment.

Episodes of Prolactin Gene Expression in GH3 Cells Are Dependent on Selective Promoter Binding of Multiple Circadian Elements

Prolactin (PRL) gene expression in mammotropes occurs in pulses, but the mechanism(s) underlying this dynamic process remains obscure. Recent findings from our laboratory of an E-box in the rat PRL promoter (E-box133) that can interact with the circadian factors, circadian locomoter output cycles kaput (CLOCK) and brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein (BMAL)-1, and was necessary for pulse activity raised the intriguing possibility that the circadian system may be central to this oscillatory process. In this study, we used serum-shocked GH(3) cells, established previously to synchronize PRL pulses between cells in culture, to reveal that pulses of PRL mRNA are linked temporally to the expression of bmal1, cry1, per1, and per3 mRNA in these cells. Moreover, we found that each of these circadian factors binds to the rat PRL promoter by chromatin immunoprecipitation analysis. Using EMSA analysis, we observed that two sites present in the proximal promoter region, E-box133 and E-box10, bind circadian factors differentially (E-box133 interacted with BMAL1, cryptochrome-1, period (PER)-1, and PER3 but not PER2 and E-box10 bound BMAL1, cryptochrome-1, PER2, PER3 but not PER1). More importantly, down-regulation of any factor binding E-box133 significantly reduced PRL mRNA levels during pulse periods. Our results demonstrate clearly that certain circadian elements binding to the E-box133 site are required for episodes of PRL mRNA expression in serum-shocked GH(3) cultures. Moreover, our findings of binding-related differences between functionally distinct E-boxes demonstrate not only that E-boxes can bind different components but suggest that the number and type of circadian elements that bind to an E-box is central in dictating its function.

Angiotensin II up‐regulates PAX2 oncogene expression and activity in prostate cancer via the angiotensin II type I receptor

Paired homeobox 2 gene (PAX2) is a transcriptional regulator, aberrantly expressed in prostate cancer cells and its down‐regulation promotes cell death in these cells. The molecular mechanisms of tumor progression by PAX2 over‐expression are still unclear. However, it has been reported that angiotensin‐II (A‐II) induces cell growth in prostate cancer via A‐II type 1 receptor (AT1R) and is mediated by the phosphorylation of mitogen activated protein kinase (MAPK) as well as signal transducer and activator of transcription 3 (STAT3).

Oncogenic Role of Engrailed-2 (En-2) in Prostate Cancer Cell Growth and Survival

Prostate cancer is the second leading cause of cancer death among men in the United States of America. However, the molecular mechanisms underlying the disease remain largely unknown. Therefore, the identification of tumor specific molecules that serve as targets for the development of new cancer drugs is considered to be a major goal in cancer research. The mouse Engrailed-2 (En-2) gene, which is a homeobox-containing transcription factor was recently identified as a candidate oncogene in breast cancer. Here, we demonstrate that En-2 is over-expressed in human prostate cancer cells as compared to normal prostate epithelial cells. In addition, our data suggests that EN2 expression may be positively modulated by PAX2 transcription factor. Furthermore, down-regulation of EN2 expression by siRNA resulted in a decrease in PAX2 expression. We also provide evidence that down-regulation of EN2 expression causes a dramatic decrease in prostate cancer cell proliferation. Therefore, from our studies we conclude that En-2 is a candidate oncogene in prostate cancer and its PAX2-regulated expression contributes to prostate cancer cell growth.

Administration of connexin43 siRNA abolishes secretory pulse synchronization in GnRH clonal cell populations

GnRH is released from hypothalamic neurons in coordinated pulses, but the cellular basis for this process is poorly understood. Previously, we found that secretory pulses from GT1-7 cells became synchronized with time in culture. Using this culture model, we investigated whether the gap junction proteins connexin43 (Cx43) and/or connexin26 (Cx26) are involved in this synchronization. Our results reveal that cytoplasmic densities immunoreactive for Cx43, and mRNA or protein for Cx43 increase with time in culture. Also, microinjection of day-3 cultures with siRNA for Cx43 abolished synchronized activity at day 7. Interestingly, cytoplasmic plaques, mRNA, or protein for Cx26 remained stable with culture time and Cx26 siRNA administration did not alter secretory activity. Our findings demonstrate that Cx43, but not Cx26 is necessary for synchronized secretory activity in these GT1-7 cultures and raise the possibility that Cx43-related gap junctions may be important in GnRH neuronal coordination in the hypothalamus.

Specific GATA-Binding Elements in the GnRH Promoter Are Required for Gene Expression Pulse Activity: Role of GATA-4 and GATA-5 in This Intermittent Process

Recent evidence reveals that several GATA factors act as versatile transcriptional modulators in neuroendocrine gene expression. The rat GnRH promoter is expressed in an episodic fashion that requires a portion of the promoter termed the neuron-specific enhancer (NSE) for activity. In this study, we examined whether certain GATA regulatory elements in the NSE are necessary for this intermittent activity. When injected into individual living GT1-7 cells, luciferase reporter constructs containing mutations of either GATA-A- or GATA-B-binding sites resulted in a marked reduction in gene expression pulse frequency, while mutations of both sites virtually abolished pulses. In subsequent studies, RT-PCR and western blot analysis revealed for the first time that GATA-5 and GATA-6 were expressed in GT1-7 cells, but electrophoretic mobility shift assays demonstrated further that GATA-5 bound to one of these GATA sites: GATA-A. Chromatin immunoprecipitation analysis revealed that all three factors, GATA-4, GATA-5, and GATA-6, were associated with the GnRH promoter in vivo. Interestingly though, immunoneutralization of GATA-5 or GATA-4 (reported to bind GATA-B) abolished gene expression pulses, but injection of GATA-6 antibody did not, indicating that of these factors just GATA-5 and GATA-4 are critical for intermittent activity. Finally, gel shift competition experiments revealed an interaction between proteins binding at the GATA-A site and those associating with an adjacent OCT1 site, previously shown to be necessary for pulse formation. These findings indicate that episodic GnRH gene expression pulses are mediated by GATA-5 and GATA-4, likely acting through the GATA-binding sites in the GnRH NSE region. Moreover, our observations that factors associated with GATA sites may also interact with OCT1 sites and that both are critical for pulse activity raise the intriguing possibility that GnRH pulse elaboration is a highly complex process that may require the coordinated interaction of several NSE-binding elements of the GnRH promoter.

Regulation of nucleolin expression by miR-194, miR-206, and HuR

Nucleolin is a proliferation-associated protein that is overexpressed in multiple types of cancer. The mechanisms leading to overexpression of nucleolin in specific cancers are not fully understood. This study found that nucleolin is notably elevated in breast cancer cell lines MCF-7 and MDA-231 compared to nonmalignant breast epithelial MCF-10A cells. In silico analyses revealed the presence of putative binding sites for microRNAs miR-194 and miR-206 in the 3′-untranslated region (3′-UTR) of Ncl mRNA. Transfection of the three cell lines with pre-miR-194 or pre-miR-206 specifically decreased the Ncl mRNA and protein expression. Treatments of the cells with antagomiR-194 or antagomiR-206 upregulated nucleolin expression ~2- to 3-fold. Co-transfection of cells with a reporter vector containing the Ncl 3′-UTR downstream from the Renilla luciferase gene and pre-miR-194 or pre-miR-206 led to a ~3-fold decrease in Renilla/firefly luciferase activity. Cytoplasmic levels of the RNA-binding protein HuR were higher in MCF-7 and MDA-231 cells than those in MCF-10A cells. RNA immunoprecipitation assays demonstrated that HuR binds to Ncl mRNA in all the three cell types. ShRNA-mediated knock-down of HuR induced a decrease in nucleolin expression, while exogenous expression of HuR led to upregulation of nucleolin expression. Analysis of the polysome–monosome distribution of Ncl mRNA in HuR knock-down cells demonstrated that HuR enhances the translation efficiency of Ncl mRNA. These findings demonstrate that nucleolin expression is down-regulated by miR-194 and miR-206 and upregulated by HuR.

A Pit-1 Binding Site Adjacent to E-box133 in the Rat PRL Promoter is Necessary for Pulsatile Gene Expression Activity

Recent evidence reveals that prolactin gene expression (PRL-GE) in mammotropes occurs in pulses, but the molecular process(es) underlying this phenomenon remains unclear. Earlier, we have identified an E-box (E-box133) in the rat PRL promoter that binds several circadian elements and is critical for this dynamic process. Preliminary analysis revealed a Pit-1 binding site (P2) located immediately adjacent to this E-box133 raising the possibility that some type of functional relationship may exist between these two promoter regions. In this study, using serum shocked GH3 cell culture system to synchronize PRL-GE activity, we determined that Pit-1 gene expression occurred in pulses with time phases similar to that for PRL. Interestingly, EMSA analysis not only confirmed Pit-1 binding to the P2 site, but also revealed an interaction with factor(s) binding to the adjacent E-box133 promoter element. Additionally, down-regulation of Pit-1 by siRNA reduced PRL levels during pulse periods. Thus, using multiple evidences, our results demonstrate clearly that the Pit-1 P2 site is necessary for PRL-GE elaboration. Furthermore, the proximity of this critical Pit-1 binding site (P2) and the E-box133 element coupled with the evidences of a site-to-site protein interactions suggest that the process of PRL-GE pulse activity might involve more dynamic and intricate cross-talks between promoter elements that may span some, or all, of the proximal region of the PRL promoter in driving its pulsatile expression.