BRCA2 inhibition enhances cisplatin-mediated alterations in tumor cell proliferation, metabolism, and metastasis

Tumor cells have unstable genomes relative to non-tumor cells. Decreased DNA integrity resulting from tumor cell instability is important in generating favorable therapeutic indices, and intact DNA repair mediates resistance to therapy. Targeting DNA repair to promote the action of anti-cancer agents is therefore an attractive therapeutic strategy. BRCA2 is involved in homologous recombination repair. BRCA2 defects increase cancer risk but, paradoxically, cancer patients with BRCA2 mutations have better survival rates. We queried TCGA data and found that BRCA2 alterations led to increased survival in patients with ovarian and endometrial cancer. We developed a BRCA2-targeting second-generation antisense oligonucleotide (ASO), which sensitized human lung, ovarian, and breast cancer cells to cisplatin by as much as 60%. BRCA2 ASO treatment overcame acquired cisplatin resistance in head and neck cancer cells, but induced minimal cisplatin sensitivity in non-tumor cells. BRCA2 ASO plus cisplatin reduced respiration as an early event preceding cell death, concurrent with increased glucose uptake without a difference in glycolysis. BRCA2 ASO and cisplatin decreased metastatic frequency invivo by 77%. These results implicate BRCA2 as a regulator of metastatic frequency and cellular metabolic response following cisplatin treatment. BRCA2 ASO, in combination with cisplatin, is a potential therapeutic anti-cancer agent

Estrogen receptors and human disease

Estrogens influence many physiological processes in mammals, including but not limited to reproduction, cardiovascular health, bone integrity, cognition, and behavior. Given this widespread role for estrogen in human physiology, it is not surprising that estrogen is also implicated in the development or progression of numerous diseases, which include but are not limited to various types of cancer (breast, ovarian, colorectal, prostate, endometrial), osteoporosis, neurodegenerative diseases, cardiovascular disease, insulin resistance, lupus erythematosus, endometriosis, and obesity. In many of these diseases, estrogen mediates its effects through the estrogen receptor (ER), which serves as the basis for many therapeutic interventions. This Review will describe diseases in which estrogen, through the ER, plays a role in the development or severity of disease

Compromised fertility disrupts Peg1 but not Snrpn and Peg3 imprinted methylation acquisition in mouse oocytes

Growth and maturation of healthy oocytes within follicles requires bidirectional signaling and intercellular gap junctional communication. Aberrant endocrine signaling and loss of gap junctional communication between the oocyte and granulosa cells leads to compromised folliculogenesis, oocyte maturation and oocyte competency, consequently impairing fertility. Given that oocyte-specific DNA methylation establishment at imprinted genes occurs during this growth phase, we determined whether compromised endocrine signaling and gap junctional communication would disrupt de novo methylation acquisition using ERβ and connexin37 genetic models. To compare mutant oocytes to control oocytes, DNA methylation acquisition was first examined in individual, 20-80 μm control oocytes at three imprinted genes, Snrpn, Peg3 and Peg1. We observed that each gene has its own size-dependent acquisition kinetics, similar to previous studies. To determine whether compromised endocrine signaling and gap junctional communication disrupted de novo methylation acquisition, individual oocytes from Esr2- and Gja4-deficient mice were also assessed for DNA methylation establishment. We observed no aberrant or delayed acquisition of DNA methylation at Snrpn, Peg3 or Peg1 in oocytes from Ers2-deficient females, and no perturbation in Snrpn or Peg3 de novo methylation in oocytes from Gja4-null females. However, Gja4-deficiency resulted in a loss or delay in methylation acquisition at Peg1. One explanation for this difference between the three loci analyzed is the late establishment of DNA methylation at the Peg1 gene. These results indicate that compromised fertility though impaired intercellular communication can lead to imprinting acquisition errors. Further studies are required to determine the effects of subfertility/infertility originating from impaired signaling and intercellular communication during oogenesis on imprint maintenance during preimplantation development

Expression of Extracellular Matrix Components Is Disrupted in the Immature and Adult Estrogen Receptor β-Null Mouse Ovary

<div><p>Within the ovary, Estrogen Receptor β (ERβ) is localized to the granulosa cells of growing follicles. 17β-estradiol (E2) acting via ERβ augments the actions of follicle stimulating hormone in granulosa cells, leading to granulosa cell differentiation and formation of a preovulatory follicle. Adult ERβ-null females are subfertile and possess ovaries with reduced numbers of growing follicles and corpora lutea. Because the majority of E2 production by granulosa cells occurs once puberty is reached, a role for ERβ in the ovary prior to puberty has not been well examined. We now provide evidence that lack of ERβ disrupts gene expression as early as post-natal day (PND) 13, and in particular, we identify a number of genes of the extracellular matrix (ECM) that are significantly higher in ERβ-null follicles than in wildtype (WT) follicles. Considerable changes occur to the ECM occur during normal folliculogenesis to allow for the dramatic growth, cellular differentiation, and reorganization of the follicle from the primary to preovulatory stage. Using quantitative PCR and immunofluorescence, we now show that several ECM genes are aberrantly overexpressed in ERβ-null follicles. We find that Collagen11a1, a protein highly expressed in cartilage, is significantly higher in ERβ-null follicles than WT follicles as early as PND 13, and this heightened expression continues through PND 23–29 into adulthood. Similarly, Nidogen 2, a highly conserved basement membrane glycoprotein, is elevated in ERβ-null follicles at PND 13 into adulthood, and is elevated specifically in the ERβ-null focimatrix, a basal lamina-like matrix located between granulosa cells. Focimatrix laminin and Collagen IV expression were also higher in ERβ-null ovaries than in WT ovaries at various ages. Our findings suggest two novel observations: a) that ERβ regulates granulosa cell gene expression ovary prior to puberty, and b) that ERβ regulates expression of ECM components in the mouse ovary.</p> </div

Collagen 11a1 mRNA and protein levels are higher in granulosa cells and ovaries of ERβ-null mice than in wildtype mice.

<p>A. Granulosa cells were isolated and pooled from ovaries of untreated PND 13 or PND 23–29 wildtype (+/+) or ERβ-null (−/−) mice, and the levels of <i>Col11a1</i> mRNA were determined by quantitative RT-PCR compared to an <i>Rpl7</i> control (± SEM of three independent experiments). Wildtype and ERβ-null average mRNA levels were compared using an unpaired two-tailed Student's t-test. a: p<0.05. B–D. Immunofluorescence with an anti-COL11A1 antibody was used to detect COL11A1 localization and expression in ovaries isolated from wildtype (+/+) and ERβ-null (−/−) mice at (B) PND 13 (a–d), (C) PND 23–29 (a–f; negative controls with secondary antibody only are shown in g and h), and (D) PND 60 (adult). Various magnifications are shown. (B) Scale bar = 100 µM for a–b, and 50 µM for c–d. (C) Scale bar = 200 µM for a–b and g–h, 100 µM for c–d, and 50 µM for e–f; (D) Scale bar = 200 µM for a–b, 100 µM for c–d.</p

Collagen IV expression and localization in immature and adult ERβ-null and wildtype mouse ovaries.

<p>A. Granulosa cells were isolated and pooled from ovaries of untreated PND 23–29 wildtype (+/+) or ERβ-null (−/−) mice, and the levels of <i>Col4</i> mRNA were determined by quantitative RT-PCR compared to an <i>Rpl7</i> control (± SEM of three independent experiments). B. Immunofluorescence with an anti-COL4 antibody was used to detect COL4 localization and expression in ovaries isolated from wildtype (+/+) and ERβ-null (−/−) mice at PND 23–29 (a–d; negative controls with secondary antibody only are shown in e and f). COL4 was localized to the follicular basal lamina (white filled arrowhead), focimatrix (open arrowhead), thecal matrix (asterix), and endothelial basal lamina of stromal blood vessels (square). Scale bar = 200 µM for a–b and e–f, 100 µM for c–d. C. No significant differences in COL4 expression within the focimatrix were observed, as determined by the number of focimatrix “speckles” counted per follicle analyzed by Receiver Operating Characteristic analysis (top panel) and a two-tailed, un-paired Student's t-test (± SEM, bottom panel). Each dot in the scatter plot (top panel) represents one follicle. D. COL4 expression in adult ERβ-null and wildtype mouse ovaries. Immunofluorescence with anti-COL4 antibodies was used to detect COL4 localization and expression in ovaries isolated from adult wildtype (+/+) and ERβ-null (−/−) mice. Two magnifications are shown. Scale bar = 200 µM for a–b, 100 µM for c–d. E. Expression of COL4 in the focimatrix was quantified by counting the number of focimatrix speckles/follicle, and these values were compared between genotypes by Receiver Operating Characteristic analysis (top panel) and a two-tailed, un-paired Student's t-test (± SEM, bottom panel). Each dot in the scatter plot (bottom panel) represents one follicle. d: <i>p</i><0.0001.</p