The ovarian transcriptome of reproductively aged multiparous mice: candidate genes for ovarian cancer protection

In middle-aged women, the decline of ovarian follicle reserve below a critical threshold marks menopause, leading to hormonal, inflammatory, and metabolic changes linked to disease. The highest incidence and mortality of sporadic ovarian cancer (OC) occur at post-menopause, while OC risk is reduced by full-term pregnancies during former fertile life. Herein, we investigate how parity history modulates the ovarian transcriptome related to such declining follicle pool and systemic inflammation in reproductively-aged mice. Female C57BL/6 mice were housed under multiparous and virgin (nulliparous) breeding regimens from adulthood until estropause. The ovaries were then subjected to follicle count and transcriptional profiling, while a cytokine panel was determined in the sera. As expected, the follicle number was markedly decreased just by aging. Importantly, a significantly higher count of primordial and total follicles was observed in aged multiparous relative to aged virgin ovaries. Consistently, among the 65 genes of higher expression in aged multiparous ovaries, 27 showed a follicle count-like pattern, 21 had traceable evidence of roles in follicular/oocyte homeostasis, and 7 were transforming-growth factor beta (TGF-beta)/bone morphogenetic protein (BMP) superfamily members. The remaining genes were enriched in cell chemotaxis and innate-immunity, and resembled the profiles of circulating CXCL1, CXCL2, CXCL5, CSF3, and CCL3, chemokines detected at higher levels in aged multiparous mice. We conclude that multiparity during reproductive life promotes the retention of follicle remnants while improving local (ovarian) and systemic immune-innate surveillance in aged female mice. These findings could underlie the mechanisms by which pregnancy promotes the long-term reduced OC risk observed at post-menopause.FONDECYT, Ministry of Education, Chile: 1130292. United States Department of Health & Human Services National Institutes of Health (NIH) - USA: ZIA CT000260

The 15kDa Selenoprotein and Thioredoxin Reductase 1 Promote Colon Cancer by Different Pathways

Selenoproteins mediate much of the cancer-preventive properties of the essential nutrient selenium, but some of these proteins have been shown to also have cancer-promoting effects. We examined the contributions of the 15kDa selenoprotein (Sep15) and thioredoxin reductase 1 (TR1) to cancer development. Targeted down-regulation of either gene inhibited anchorage-dependent and anchorage-independent growth and formation of experimental metastases of mouse colon carcinoma CT26 cells. Surprisingly, combined deficiency of Sep15 and TR1 reversed the anti-cancer effects observed with down-regulation of each single gene. We found that inflammation-related genes regulated by Stat-1, especially interferon-γ-regulated guanylate-binding proteins, were highly elevated in Sep15-deficient, but not in TR1-deficient cells. Interestingly, components of the Wnt/β-catenin signaling pathway were up-regulated in cells lacking both TR1 and Sep15. These results suggest that Sep15 and TR1 participate in interfering regulatory pathways in colon cancer cells. Considering the variable expression levels of Sep15 and TR1 found within the human population, our results provide insights into new roles of selenoproteins in cancer

Signatures of selected NCI-60 cell lines for Sep15 and Stat-1.

<p>Opposing directions of transcript intensity scores (z-scores) for Sep15 and Stat-1 mRNAs were highly significant (r = -0.646, <i>P<</i>0.01) for human central nervous system (CNS), leukemia and ovarian cancer cell lines. Mean centered average protein activity patterns for Stat-1 (Stat-1_20 antibody) significantly correlated (r = 0.661, <i>P<</i>0.01) with Stat-1 mRNA expression.</p

Possible connection to Wnt/β-catenin signaling pathway in shTR1/Sep15 cells.

<p>mRNA levels of <b>(A)</b> Speg; <b>(B)</b> Ccr1; and <b>(C)</b> Tnc; and (<b>D</b>) Apc in control, shSep15, shTR1, and shTR1/Sep15 cells, as determined by real-time RT-PCR, and expressed relative to <i>Gapdh</i>. Columns, mean (<i>n</i> = 3–6); bars, SE; (<i>**P</i><0.01, <i>***P</i><0.001).</p

Expression of Sep15 and TR1 in CT26 cells.

<p>Cells were stably transfected with the pU6-m3 control, shSep15, shTR1 or shTR1/shSep15 (as indicated). <b>(A)</b> Expression of Sep15 by real-time RT-PCR (upper panel) and Western blotting (lower panel). <b>(B)</b> Expression of TR1 by real-time RT-PCR (upper panel) and Western blotting (lower panel). <b>(C)</b> Thioredoxin reductase activity. Columns, mean (<i>n</i> = 3–6); bars, SE; (<i>*P</i><0.05, <i>**P</i><0.01, compared to control).</p

Anchorage-dependent, -independent growth and formation of lung metastases.

<p>(<b>A</b>) Growth rates of shSep15, shTR1 and shTR1/Sep15 cells compared to controls (<i>n</i> = 6). (<b>B</b>) Anchorage-independent growth in soft agar of shSep15, shTR1 and shTR1/Sep15 cells compared to controls (<i>n</i> = 4–8). (<b>C</b>) Formation of experimental lung metastases after i.v. injection of 5×10⁵ cells (control, shSep15, shTR1, or shTR1/shSep15) into BALB/c mice (<i>n</i> = 10/construct). (<i>***P</i><0.001, compared to control).</p

Cell cycle analysis.

<p>Percent of cells in each cell cycle phase as determined by FACS analysis at (<b>A</b>) 0 h; (<b>B</b>) 6 h; (<b>C</b>) 24 h; and (<b>D</b>) 48 h after release from cell synchronization. mRNA expression of (<b>E</b>) CyclinB1 (<i>Ccnb1</i>), and (<b>F</b>) CyclinB1 Interacting-Protein-1 (<i>Ccnb1ip1</i>), as determined by real-time RT-PCR. Columns, mean (<i>n</i> = 3–9); bars, SE; (<i>*P</i><0.05, <i>**P</i><0.01, <i>***P</i><0.001).</p

Validation of gene expression for genes significantly changed in shSep15 cells.

<p>Expression of <b>(A)</b> Ifi44 mRNA (upper panel) and protein (lower panel); <b>(B)</b> Irf-7 mRNA; <b>(C)</b> Usp18 mRNA; (<b>D</b>) Gbp-6 mRNA; <b>(E)</b> Ifnγ mRNA; (<b>F</b>) Gbp-1 mRNA (upper panel) and protein (lower panel); (<b>G</b>) Stat-1 mRNA (upper panel) and protein (lower panel); (<b>H</b>), Casp6 mRNA; (<b>I</b>) Casp12 mRNA; and (<b>J</b>) Afp mRNA. Expression of mRNA in control, shSep15, shTR1, and shTR1/Sep15 cells was determined by real-time RT-PCR, and expressed relative to <i>Gapdh</i>. Columns, mean (<i>n</i> = 3–6); bars, SE; (<i>*P</i><0.05, <i>**P</i><0.01, <i>***P</i><0.001). Protein expression was determined by Western blotting, and expressed relative to β-actin or α-tubulin, as indicated.</p