Dysregulated Estrogen Receptor Signaling in the Hypothalamic-Pituitary-Ovarian Axis Leads to Ovarian Epithelial Tumorigenesis in Mice

<div><p>The etiology of ovarian epithelial cancer is poorly understood, mainly due to the lack of an appropriate experimental model for studying the onset and progression of this disease. We have created a mutant mouse model in which aberrant estrogen receptor alpha (ERα) signaling in the hypothalamic-pituitary-ovarian axis leads to ovarian epithelial tumorigenesis. In these mice, termed ERα^d/d, the ERα gene was conditionally deleted in the anterior pituitary, but remained intact in the hypothalamus and the ovary. The loss of negative-feedback regulation by estrogen (E) at the level of the pituitary led to increased production of luteinizing hormone (LH) by this tissue. Hyperstimulation of the ovarian cells by LH resulted in elevated steroidogenesis, producing high circulating levels of steroid hormones, including E. The ERα^d/d mice exhibited formation of palpable ovarian epithelial tumors starting at 5 months of age with 100% penetrance. By 15 months of age, 80% of ERα^d/d mice die. Besides proliferating epithelial cells, these tumors also contained an expanded population of luteinized stromal cells, which acquire the ability to express P450 aromatase and synthesize E locally. In response to the elevated levels of E, the ERα signaling was accentuated in the ovarian epithelial cells of ERα^d/d mice, triggering increased ERα-dependent gene expression, abnormal cell proliferation, and tumorigenesis. Consistent with these findings, treatment of ERα^d/d mice with letrozole, an aromatase inhibitor, markedly reduced circulating E and ovarian tumor volume. We have, therefore, developed a unique animal model, which serves as a useful tool for exploring the involvement of E-dependent signaling pathways in ovarian epithelial tumorigenesis.</p></div

The ERα^d/d mice form proliferative ovarian tumors.

<p>(<b>A</b>) Gross morphology of ERα^d/d, ERα^f/f, and ERα global KO mouse ovaries at 3, 5, and 8 months of age. (<b>B</b>) Immunohistochemistry of ERα^f/f ovary (panels a, c) and ERα^d/d ovaries (panels b, d) with tumors at 6 months of age using anti-PCNA antibody. Red staining indicates proliferating PCNA positive cells. Arrows point to hyperproliferative OSE (b) and tumor cells (d) in ERα^d/d ovarian tumors. F indicates follicle.</p

ERα localization in the tissues of HPO axis.

<p>(<b>A</b>) Histological sections of ERα^f/f (a) and ERα^d/d (b) hypothalami, ERα^f/f (c) and ERα^d/d (d) pituitaries, ERα^f/f ovary (e, g) and ERα^d/d ovarian tumor (f, h) from adult mice at 6 months of age stained with anti-ERα. Inserts i and j indicate higher magnification depicting ERα positive cells (red staining) in the OSC and ovarian tumor cells. 3 V indicates the third ventricle. A indicates the anterior lobe, I indicates the intermediate lobe, and P indicates the posterior lobe of the pituitaries. Arrows point to OSE cells or theca cells expressing ERα. (<b>B</b>) Ovarian sections obtained from ERα^f/f (left pictures) and ERα^d/d (right pictures) mice were subjected to immunohistochemistry using antibodies against phospho-ERα (S118) (panels a, b) and phospho-Akt (S473) (panels c,d).</p

ERα^d/d ovarian tumor growth is inhibited by P450 aromatase inhibitor.

<p>(<b>A</b>) Ovaries with tumors from ERα^d/d mice treated with letrozole, a P450 aromatase inhibitor, filled silastic capsules or sham empty silastic capsules for 3 months are analyzed by gross morphology and tumor volume. Serum estradiol and LH is assayed by radioimmunoassay. (<b>B</b>) Real-time quantitative PCR was employed to measure mRNA levels of genes associated with ovarian carcinoma, PDGFRα, VCAM, and Wip1, in ovaries with tumors of ERα^d/d mice treated with letrozole-containing or sham empty silastic capsules. (<b>C</b>) Wip1 protein localization in ovaries with tumors of ERα^d/d mice either treated with sham control (a) or treated with letrozole (b). Arrow points to nuclear expression of Wip1. * indicates p<.05. Each treatment group had n = 8 samples.</p

Serum hormone measurements of ERα^f/f and ERα^d/d mice at six months of age.

<p>Hormone levels from 6 mice were measured for each assay.</p

Ablation of uterine <i>Rac1</i> leads to severe female infertility.

<p>^† Some <i>Rac1</i>^<i>d/d</i> females did not deliver any pups during or after the breeding study</p><p>^‡<i>P</i> < 0.0001</p><p>Ablation of uterine <i>Rac1</i> leads to severe female infertility.</p

Abnormal trophoblast proliferation, differentiation and disorganized placentation in <i>Rac1</i> conditional-knockout mouse.

<p>(A & B) Increased population of trophoblast giant cells (TGCs) in the placenta of <i>Rac</i>1^<i>d/d</i> uteri. <b>(A)</b> H and E staining of uterine sections from <i>Rac1</i>^<i>f/f</i> and <i>Rac1</i>^<i>d/d</i> mice on day 10 of pregnancy. TGC and MD indicate trophoblast giant cells and mesometrial decidua, respectively. TGCs are demarcated by dashed lines. Arrows indicate chorionic plate and arrow-heads indicate TGCs. <b>(B)</b> Uterine sections from <i>Rac1</i>^<i>f/f</i> and <i>Rac1</i>^<i>d/d</i> mice on day 10 of pregnancy were subjected to IF using PL1. (<b>C</b>) Uterine sections from <i>Rac1</i>^<i>f/f</i> and <i>Rac1</i>^<i>d/d</i> mice on day 10 of pregnancy were subjected to IF using TPBPA and KRT8 antibodies. (<b>D)</b> H and E staining of uterine sections from <i>Rac1</i>^<i>f/f</i> and <i>Rac1</i>^<i>d/d</i> mice on day 12 of pregnancy. TGC, MD and E indicate trophoblast giant cells, mesometrial decidua, and embryo, respectively.</p

Rac1 regulates vesicular exocytosis in decidual cells by controlling Rab27b.

<p><b>(A & B)</b> Expression of Rab27b mRNA and protein is downregulated in Rac1-null stromal cells. <b>(A)</b> qPCR was performed to monitor the expression of <i>Rab27a</i> and <i>Rab27b</i> in the uteri of <i>Rac</i>1^<i>f/f</i> and <i>Rac</i>1^<i>d/d</i> mice on day 8 of pregnancy. Data represent mean ± SEM from four separate samples and were analyzed by <i>t</i>-test. Asterisks indicate statistically significant differences (***<i>P < 0</i>.001). <b>(B)</b> IF of RAB27B in <i>Rac1</i>^<i>f/f</i> and <i>Rac1</i>^<i>d/d</i> uteri on day 8 of pregnancy. AMD, MD, and E denote antimesometrial decidua, mesometrial decidua, and embryo respectively. <b>(C & D)</b> Secretions by decidual cells are reduced in the conditioned media of Rac1-null stromal cells. Stromal cells isolated from <i>Rac1</i>^<i>f/f</i> and <i>Rac1</i>^<i>d/d</i> uteri on day 4 of pregnancy were cultured for 96 hours, fixed and subjected to IF using VEGFA (<b>C, Left</b>) and IGFBP4 (<b>D, Left</b>) antibodies. Conditioned media from cultured stromal cells isolated from <i>Rac1</i>^<i>f/f</i> and <i>Rac1</i>^<i>d/d</i> uteri were analyzed for VEGFA (<b>C, Right</b>) and IGFBP4 (<b>D, Right</b>) by ELISA. Data represent mean ± SEM from three separate samples and were analyzed by two-way ANOVA with Bonferroni post-test. Asterisks indicate statistically significant differences (*<i>P < 0</i>.05, **<i>P < 0</i>.01, and ***<i>P < 0</i>.001).</p

Enhanced trophoblast proliferation in <i>Rac1</i> conditional-knockout mouse.

<p><b>(A)</b> Expanded trophoblast cells in <i>Rac</i>1^<i>d/d</i> uteri on day 8 of gestation. <b>a:</b> Hematoxylin and Eosin (H & E) staining of uterine sections from <i>Rac1</i>^<i>f/f</i> and <i>Rac1</i>^<i>d/d</i> mice on day 8 of pregnancy. Three representative images of <i>Rac</i>1^<i>d/d</i> uterine sections are shown. <b>b:</b> Quantitation of embryonic areas in H & E stained uterine sections of <i>Rac1</i>^<i>f/f</i> and <i>Rac1</i>^<i>d/d</i> mice on day 8 of pregnancy. Data represent mean ± SEM from six separate samples and were analyzed by non-parametric <i>t</i>-test. Asterisks indicate statistically significant differences (*<i>P < 0</i>.05). <b>(B)</b> Increased proliferation of trophoblast cells in the ectoplacental cone (EPC) of <i>Rac</i>1^<i>d/d</i> uteri. Uterine sections from <i>Rac1</i>^<i>f/f</i> and <i>Rac1</i>^<i>d/d</i> mice on days 7 (panels a and b) and 8 of pregnancy (panels c and d) were subjected to IF using PCNA and cytokeratin 8 (KRT8) antibodies. AMD, MD, and E denote antimesometrial decidua, mesometrial decidua, and embryo respectively.</p

<i>Cuzd1</i>^(-/-) mammary glands do not undergo alveologenesis in response to hormone treatment.

<p><b>(A) <i>Cuzd1</i></b>^<b>(-/-)</b><b>mammary gland morphology in response to hormonal treatment.</b><i>Cuzd1</i>^(+/+) and <i>Cuzd1</i>^(-/-) mice (n = 5) were treated with a vehicle control (a and b) or E+P+PRL (c and d) for 3d. Whole mount analysis of no. 4 inguinal mammary glands of <i>Cuzd1</i>^(+/+) and <i>Cuzd1</i>^(-/-) mice after 3d of hormone treatment. Magnification 6.3x. <b>(B) Analysis of CUZD1 expression.</b> Mammary tissue sections obtained from <i>Cuzd1</i>^(+/+) (a and c) and <i>Cuzd1</i>^(-/-) (b and d) mice were subjected to IHC using an antibody specific for CUZD1. <b>(C) Activation of STAT5.</b> Mammary tissue sections obtained from <i>Cuzd1</i>^(+/+) (a and c) and <i>Cuzd1</i>^(-/-) (b and d) mice were subjected to IHC using an antibody specific for pSTAT5. <b>(D) Analysis of EREG expression.</b> Mammary tissue sections obtained from <i>Cuzd1</i>^(+/+) (a) and <i>Cuzd1</i>^(-/-) (b) mice were subjected to IHC using an antibody specific for EREG. Magnification 20x.</p