Pilot study assessing gut microbial diversity among sexual and gender minority young adults.

Evidence supports that people identifying as a sexual or gender minority (SGMs) experience minority-related stress resulting from discrimination or expectations of prejudice, and that this is associated with increased mental and physical health problems compared to cisgender heterosexuals. However, the biological mechanisms driving minority-related stress impacts remain unknown, including the role of the gut microbiome. Thus, the aim of this study was to determine the relationship between SGM status and gut microbiome health among young adults attending a 4-year university. To this end, a prospective pilot study was completed in the fall and spring semesters of 2021-22. Self-identified SGMs (N = 22) and cisgender-heterosexuals (CIS-HET, N = 43) completed in-person interviews to provide mental health data and demographic information. Nail and saliva samples were collected at the time of interview to quantify chronic and acute cortisol. Stool samples were collected within 48 hours of interview for microbiome analysis. Assessment of the gut microbiota identified a significant reduction in alpha diversity among the SGM group, even when adjusting for mental health outcome. SGM group showed trends for higher abundance of microbes in phylum Bacteroidetes and lower abundance of microbes in phyla Firmicutes, Actinobacteria, and Proteobacteria compared to the CIS-HET group. These findings support that the gut microbiome could be contributing to negative health effects among the SGM community.

Cks1 regulates cdk1 expression: a novel role during mitotic entry in breast cancer cells.

Cks1, a small protein whose expression is strongly associated with aggressive breast tumors, is a component of cyclin-cdk complexes, as well as the SCF(Skp2) ubiquitin ligase. In these studies, we explored its roles in estrogen receptor-positive breast tumor cells. When exposed to the antiestrogen ICI 182780, these cells accumulate in G(1) by reducing the expression of Cks1, and increasing the levels of p130/Rb2, a cdk2 inhibitor and SCF(Skp2) target. Heregulin beta1 or estradiol abrogate antiestrogen effects by increasing Cks1 expression, down-regulating p130/Rb2 and inducing S phase entry. Depletion of Cks1 in these cells by RNA interference concomitantly decreased Skp2 and up-regulated p130/Rb2 and another SCF(Skp2) target, p27(Kip1). Remarkably, however, Cks1-depleted cells not only exhibit slowed G(1) progression, but also accumulate in G(2)-M due to blocked mitotic entry. Notably, we show that cdk1 expression, which is crucial for M phase entry, is drastically diminished by Cks1 depletion, and that restoration of cdk1 reduces G(2)-M accumulation in Cks1-depleted cells. cdk1 reduction in Cks1-depleted cells is a consequence of a marked decrease in its mRNA and not due to alteration in its proteolytic turnover. Both heregulin beta1 and estradiol could neither restore cdk1 nor sustain cycling in Cks1-depleted cells, although classical estrogen receptor function remained unaltered. Cks1 depletion also decreased Skp2 in human mammary epithelial cells without altering cell cycle progression. Thus, the indispensability of Cks1 to the breast cancer cell cycle, versus its redundancy in normal cells, suggests that Cks1 abrogation could be an effective interventional strategy in breast cancer.

MEK ablation in MCF-7 cells blocks DNA synthesis induced by serum, but not by estradiol or growth factors.

The role of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) signaling in estrogen receptor positive (ER(+)) MCF-7 breast carcinoma cells is not well understood. We depleted MEK by cotransfection of MEK1 and MEK2 siRNA duplexes in a MCF-7 derived line (MCF-7/ lacZ, ML-20) and determined its effect on serum, 17beta-estradiol (E(2)), and growth factor induced DNA synthesis. MEK knockdown did not decrease fetal bovine serum-induced DNA synthesis in ML-20 cells although it did inhibit DNA synthesis induced by estrogen-stripped calf serum (CCS) suggesting that MEK activation plays an important role in growth signaling induced by serum components other than estrogen. Consistent with this notion, MEK knockdown only modestly decreased DNA synthesis induced by E(2)-supplemented CCS medium in ML-20 cells. Similarly, MEK knockdown only caused moderate decreases in DNA synthesis induced by fibroblast growth factor-1 (FGF-1) or heregulin-beta1 (HRGbeta1) in this media. Also, there were only minimal effects of MEK knockdown in cells treated with growth factor-supplemented serum-free medium. Although MEK depletion inhibited ERK1/2 phosphorylation induced by CCS in these cells, that induced by growth factor supplemented CCS media was relatively unaffected. Similarly, ERK1/2 phosphorylation induced by growth factor-supplemented serum-free media was also relatively unaffected by MEK depletion. These results suggest that pathways regulating DNA synthesis induced by serum in MCF-7 cells are significantly more dependent on constitutive MEK levels than that induced by E(2) or growth factors.

siRNA mediated knockdown of fibroblast growth factor receptors 1 or 3 inhibits FGF-induced anchorage-independent clonogenicity but does not affect MAPK activation.

Supplementation with exogenous growth factors such as fibroblast growth factors (FGFs) is essential for anchorage-independent growth of the SW-13 human adrenal adenocarcinoma cell line. We have found that SW-13 cells express mRNAs for FGFRs 1, 3, and 4, but not FGFR2. To assess the roles of individual FGFRs, in anchorage-independent growth, we determined the effects of down-regulation of each FGFR on FGF2- and FGF4-mediated soft agar colony formation in these cells. Using RNAi strategies we found that knockdown of either FGFR1 or FGFR3 leads to inhibition of FGF2- or FGF4-induced soft agar clonogenicity without affecting that induced by heregulin beta1. However, this inhibition is independent of ERK1/2 activation as levels of FGF-induced phospho-ERK 1/2 remain unchanged upon knockdown of either FGFR1 or FGFR3. Conversely, RNAi-mediated knockdown of FGFR4 appeared to have no significant effect on either FGF2- or FGF4-induced anchorage-independent colony formation, or ERK1/2 phosphorylation. These results suggest that constitutive levels of both FGFR1 and FGFR3, but not FGFR4 are essential for FGF-stimulated anchorage-independent growth of SW-13 cells.