BRCA1 Regulates Follistatin Function in Ovarian Cancer and Human Ovarian Surface Epithelial Cells

Follistatin (FST), a folliculogenesis regulating protein, is found in relatively high concentrations in female ovarian tissues. FST acts as an antagonist to Activin, which is often elevated in human ovarian carcinoma, and thus may serve as a potential target for therapeutic intervention against ovarian cancer. The breast cancer susceptibility gene 1 (BRCA1) is a known tumor suppressor gene in human breast cancer; however its role in ovarian cancer is not well understood. We performed microarray analysis on human ovarian carcinoma cell line SKOV3 that stably overexpress wild-type BRCA1 and compared with the corresponding empty vector-transfected clones. We found that stable expression of BRCA1 not only stimulates FST secretion but also simultaneously inhibits Activin expression. To determine the physiological importance of this phenomenon, we further investigated the effect of cellular BRCA1 on the FST secretion in immortalized ovarian surface epithelial (IOSE) cells derived from either normal human ovaries or ovaries of an ovarian cancer patient carrying a mutation in BRCA1 gene. Knock-down of BRCA1 in normal IOSE cells demonstrates down-regulation of FST secretion along with the simultaneous up-regulation of Activin expression. Furthermore, knock-down of FST in IOSE cell lines as well as SKOV3 cell line showed significantly reduced cell proliferation and decreased cell migration when compared with the respective controls. Thus, these findings suggest a novel function for BRCA1 as a regulator of FST expression and function in human ovarian cells

Western blot analysis for IOSE cells.

<p>IOSE 397 (A) and IOSE 7576 (B) cells were transiently transfected either with wtBRCA1 or BRCA1-siRNA in each case. Whole cell lysates from the attached cells were fractionated in 4–12% BT gel, and subsequently immunoblotted with the indicated antibodies. Densitometric analyses of the immunoblots shown above are given in C and D respectively.</p

FST assay with IOSE cell line.

<p>(A) IOSE 7576, IOSE 397 and IOSE 592F were grown in 10 cm tissue culture plates and the culture medium was subjected to FST assay as described under ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037697#s2" target="_blank">Methods</a>’. IOSE 7576 cells (B), IOSE 397 cells (C) and IOSE 592F cells (D) were transiently transfected with wtBRCA1 for 48 hr and then FST assays were performed with the culture medium. Additionally, BRCA1 expression was knocked down in both IOSE 7576 (E.) and IOSE 397 (F) cells and then subjected to FST assays. Error bars are SEMs. <b>*</b> P<0.05 (relative to each control).</p

Effect of FST knock down on cell migration.

<p>(A–F) Cell migration analysis was performed for IOSE 7576, IOSE 397 and SKOV3 cell lines that were transfected with either control siRNA or FST-siRNA. Relative fluorescence units (RFU) measured from all the migrated cells in each sample are shown in A–C, whereas, analysis of the total number cells loaded in the Boyden chamber verses total number of cells migrated towards the chemo attractant (10% FBS) in each case are shown in D–F. (G) Western blot for IOSE 7576, IOSE 397 and SKOV3 cell lines confirming knock-down of FST by FST-siRNA.</p

Effect of ectopic expression of BRCA1 on FST secretion.

<p>(A) Standard curve was generated with purified human FST as indicated in the ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037697#s2" target="_blank">Methods</a>’, which was used to quantitate unknown FST concentrations in the indicated samples. SKOV3 cells were transfected as before, either for BRCA1 overexpression (B) or for BRCA1 underexpression (C), and FST assays (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037697#s2" target="_blank">methods</a>) were performed with the diluted culture medium. The same FST assay was performed with the stable clones as indicated (D) as well as with the stable cells where expression of BRCA1 was knocked down by BRCA1-siRNA (E–F). Error bars are SEMs. <b>*</b>P<0.05 (relative to each control). Expression of BRCA1 in the above samples is given in (G).</p

Cell proliferation and cell migration assay with IOSE cells.

<p>(A) Comparative analysis of cell proliferation for IOSE 7576, IOSE 397 and IOSE 592F cell lines. (B) Western blot analysis showing FST levels for IOSE 7576, IOSE 397 and IOSE 592F cell lines. (C–D) Comparative cell migration analysis for IOSE 7576, IOSE 397 and IOSE 592F cell lines. Relative fluorescence units (RFU) was measured using all of the migrated cells to the feeder tray in each sample (C), whereas, analysis of the total number cells loaded in the Boyden chamber verses total number of cells migrated towards the chemo attractant (10% FBS) in each case is shown in (D).</p

Generation of SKOV3 cell clones.

<p>(A) Several stable lines were created using overexpression of BRCA1 and its background vector, pcDNA3 in SKOV3 ovarian carcinoma cells. Expression levels of BRCA1 are shown in all cell clones. Parental indicates non-transfected SKOV3 cells only. Densitometric analysis from three independent immunoblots is given in (B). Error bars are SEMs. * represents P<0.05 (compared to parental).</p

Proceedings of the 2nd Annual Faculty Senate Research Conference: Higher Education During Pandemics

This proceeding contains articles on the various ideas of the academic community presented at The 2nd Annual Faculty Senate Research Conference organized by the University of the District of Columbia (USA) on 05th February 2021. In February 2021, the exponential spread of the coronavirus (COVID-19) and the effect of systemic racism resulted in dramatic changes in colleges and universities. These changes were extremely difficult for students, requiring faculty and institutions to stand in the gap to help students complete their studies. Moving to a completely online format was especially difficult for students traditionally served by UDC, but the institution and its faculty were better prepared than most to address this challenge. However, COVID-19 issues facing minority communities loomed larger, were more complex, and required a deeper societal dialogue.  The permeations of these issues ranged from vaccine hesitancy to social disparities that created impacts on social justice. As the only public, land-grant university in and for the capital city of the United States of America, and a Historically Black College and University, UDC was uniquely positioned to convene this discussion as a part of its 2021- 2nd Annual Faculty Senate Conference. Conference Title: 2nd Annual Faculty Senate Research Conference: Higher Education During PandemicsConference Date: 05 February 2021Conference Location: University of the District of Columbia, USAConference Organizer: University of the District of Columbi