Hou, Xiaonan

Abstract Background: At the time of ovarian cancer platinum resistance, standard therapies have limited activity. New anti-cancer drug combinations may lead to improved clinical outcomes. Activity of topoisomerase I (Top I) inhibitors or poly(ADP-ribose) polymerase (PARP) inhibitors have been reported in ovarian cancer and have demonstrated potentiation in other models. Objective: To investigate the efficacy of the Top I inhibitor etirinotecan pegol (NKTR-102) and the PARP inhibitor rucaparib (CO-338) as single agents and in combination in patient-derived xenograft (PDX) models of ovarian cancer. Methods: Five ovarian cancer PDX models in SCID mice were treated for 28 days with NKTR-102 (10 mg/kg/q7d IV), rucaparib (150 mg/kg/qd PO), or combination NKTR-102 plus rucaparib at the same doses and schedules. Of the five PDX models, four were platinum resistant and one was a platinum-sensitive positive control. Each treatment arm had a total of 10 mice and animals were observed daily for body weight and general condition. Tumor size was assessed weekly by ultrasound. The primary endpoint was change in tumor cross-sectional area (cm2) from pretreatment baseline to final tumor burden. Western blot was performed to investigate potential biomarkers of response. Results: Compared to vehicle-treated controls, statistically significant improvement (p<0.05) was observed in 3 of 5 models in single agent rucaparib treated tumors and in 5 of 5 models in single agent NKTR-102 treated tumors. The combination of NKTR-102 plus rucaparib also demonstrated statistically significant improvement compared to vehicle-treated controls in 5 of 5 models. The combination of NKTR-102 plus rucaparib induced maximal tumor regression in 2 of 5 tumors with a trend towards smaller tumors compared to single agent treatment arms. This was not found to be statistically significant in the 28 day study period under which the experiment was performed. Exploratory analysis to assess candidate drug response determinants (e.g.Top I, PAPR I, and PARP polymer formation levels) was investigated. Conclusion: NKTR-102 and rucaparib are well tolerated in mice and demonstrate activity in platinum-resistant and platinum-sensitive ovarian cancer PDX models. Combination therapy demonstrated a trend towards decreased tumor size when compared to single agent activity during treatment. Current efforts are focused on extending the time on study for the PDX models evaluated to determine if statistical superiority of the NKTR-102/rucaparib combination can be established. Citation Format: Caroline C. Nitschmann, Rachel M. Hurley, Marc A. Becker, Xiaonan Hou, Ute Hoch, Thomas C. Harding, Deborah H. Charych, Scott H. Kaufmann, Paul Haluska, Saravut J. Weroha. Antineoplastic activity of Top I inhibitor etirinotecan pegol (NKTR-102) and PARP inhibitor rucaparib (CO-388) in platinum-resistant high-grade serous BRCA WT ovarian cancer PDX models. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B50.

Abstract Ovarian cancer is the most lethal gynecologic malignancy and the fifth leading cause of cancer deaths among women. The current standard of care post surgical cytoreduction is combination platinum/taxane chemotherapy. The initial response varies widely; subsets of carcinomas demonstrate resistance or sensitivity from the onset. The underlying cause of this response heterogeneity remains incompletely understood. Patient-derived xenografts (PDX) serve as useful in vivo models to study molecular response markers and test the efficacy of targeted therapies. Our group has demonstrated a high engraftment rate (>70%) of ovarian cancer PDXs (Avatars) by injecting treatment naïve patient tumor directly into the peritoneal cavity of an in immunocompromised mouse host in an effort to better mimic the anatomic context by which ovarian cancer naturally develops. We now have 328 engrafted Avatar models representing the wide range of ovarian cancer subtypes, including serous (65%), mixed epithelial (10%), endometrioid (7%), clear cell (4%), mucinous (3%), carcinosarcoma (1%) and other (10%). Using these models, we are performing a massively parallel sequencing strategy referred to as BROCA-HR to detect all mutation classes (e.g. gene rearrangements, copy number variations, etc.) and gene aberrations within the Fanconi Anemia-BRCA homologous recombination (HR), non-homologous end joining (NHEJ), PTEN, and DNA Mismatch repair pathways. To date, 148 Avatar models have undergone BROCA sequencing. Observed deleterious mutations included 12 BRCA1 (8%), 6 BRCA2 (4%), and 4 PIK3CA (3%); additional loss of function mutations were also evident in the following genes: ATM, RAD51C, FANCM, FANCD2, FANCA, CHEK2, PALB2, MHS6, CDK12 and GEN1. These models are representative of mutations seen in the TCGA mutation set. Models harboring said mutations are currently being tested with platinum agents and/or PARP inhibitors. Taken together, the Avatars represent a high throughput model system for pre-clinical testing in vivo that effectively recapitulates ovarian cancer (e.g. mutations in BRCA1, BRCA2 and other DNA damage response proteins). We have been able to establish avatars at a high rate, including those with mutations in BRCA1, BRCA2 and other DNA damage response genes. These models will be a unique resource for future studies of biomarkers and novel therapeutic approaches. This work is funded by the Ovarian Cancer Research Fund (750563), Mayo Ovarian SPORE (CA136393), National Cancer Institute (RO1 CA184502). Note: This abstract was not presented at the meeting. Citation Format: Kunal A. Lodhia, Marc A. Becker, Xiaonan Hou, Kimberly R. Kalli, Maria I. Harrell, Elizabeth M. Swisher, John S. Weroha, Paul Haluska. Characterization of 148 ovarian cancer tumografts (Avatars) using BROCA-HR deep sequencing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1474. doi:10.1158/1538-7445.AM2015-1474

Abstract Bowel obstruction is common sequelae of advanced ovarian cancer. Reports suggest that from 5.5 percent to 51 percent of ovarian cancer patients with Stage III or Stage IV disease develop a malignant bowel obstruction. During the course of the disease, one-third of all ovarian cancer patients also will develop pleural effusions, and three-quarters of these contain malignant cells. Such complications adversely affect both survival and quality of life. Our murine primary patient ovarian cancer intraperitoneal tumorgrafts (Avatars) maintain high fidelity of genetic aberrations from the original patient tumor, thus representing a clinically relevant model system. Expression microarray data of Avatars with preferential metastasis to the bowel has revealed upregulation of IGF2. To determine if IGF2 overexpression was sufficient for metastasis, four patient Avatars were transfected with IGF2/Gluc. In order to minimize the potential for genetic drift sometimes seen with in vitro culture, pre-transplantation tumors were minced and incubated with lentiviruses (Control vector or IGF-2/Gluc expression vector) ex-in vivo for a short time (2 hours), then directly transplanted intraperitoneally into immunodeficient mice (five or six mice in each Avatar model). Total 32 animals were used in this study. Stable transfection and overexpression of human IGF-2 was achieved and maintained after three passages as determined by ELISA on tumor tissue lysates. Only one out of 32 mice did not engraft tumor after three months, and tumor taking rate was 96.875%, similar to our previous passage successful rate at 98% and indicate that our ex-in vivo transfection method and overexpression of human IGF-2 did not substantially affect the tumor taking rate. By using Xenogen IVIS200 imaging system (Xenogen Corp.), pleural fluid was observed in all the Avatar mice with tumors overexpressing IGF-2 or not, and the retrieved pleural fluids were positive for malignant cells. However higher pleural nodules forming rate was more common in mice with IGF-2 overexpressing tumors 77% (n=9) vs 40% (n=6). In model PH003/IGF-2, the tumor had 100% bowel metastasis (n=9) compare to 60% in the control group (n=6). Taken together, our Avatar models not only effectively recapitulate ovarian cancer abdominal peritoneum metastasis, but also can mimic advanced ovarian cancer with malignant pleural fluid. The IGF-2 overexpression enhanced metastatic potential relative to control tumor. These results also indicate that the ex-in vivo transfection method developed in this study may be a viable approach for biological research and therapies. This work is funded by the Mayo Ovarian SPORE (CA136393). Citation Format: Xiaonan Hou, Marc A Becker, Saravut J Weroha, Kristina A Butler, Suzanne M Greiner, Paul Haluksa. Overexpression of IGF-2 in primary patient ovarian cancer tumorgrafts increases bowel and thoracic metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1196. doi:10.1158/1538-7445.AM2014-1196

Abstract Purpose: The therapeutic potential of PARP inhibitors is predicted to extend beyond BRCA mutant phenotypes to homologous recombination (HR)-deficient cancers. Response of individual ovarian cancers to niraparib was characterized using treatment naïve, patient-derived tumorgraft models and correlated with predicted HR status and gene expression profile. Methods: Utilizing a living tumor bank of patient-derived ovarian xenografts (PDXs), niraparib (MK-4827) was evaluated in five high-grade ovarian cancer tumorgrafts (PDX077, PDX087, PDX039, PDX080 and PDX95) as monotherapy, combination with carboplatin ± paclitaxel and maintenance therapy. Intraperitoneal tumorgrafts were monitored for tumor growth with twice-weekly transabdominal ultrasound imaging. In vivo response to niraparib was correlated to massively parallel sequencing data (BROCA) for HR genes and with each source patient's platinum response. Tumorgrafts were accordingly classified into predicted HR-proficient and HR-deficient phenotypes. PARP1 and poly(ADP-ribose) polymer (pADPr) expression as well as gene expression by DNA microarray were compared between treatment and control tumor replicates, and HR-deficient vs. HR-proficient tumors. Results: PDX077 (verified to harbor a somatic BRCA2 mutation (predicted HR deficient)) and PDX039 without any HR mutations detected by BROCA (predicted HR proficient) both responded to niraparib monotherapy and combination therapy in vivo (p=0.002 and 0.009 for treated vs. control groups, respectively). Significant growth attenuation, but not regression, in response to single agent treatment was demonstrated in PDX080 (CDK12 mutation; predicted HR-deficient; p=0.02 vs. control). In comparison, a BRCA2-mutant model (PDX095) failed to respond to niraparib monotherapy (p=0.97). As predicted, niraparib was not active as a single agent or in combination with chemotherapy in a BRCA wild-type, predicted HR-proficient tumorgraft (PDX087; p=not significant). Niraparib enhanced tumor regression with carboplatin but not with paclitaxel/carboplatin in an HR-deficient tumorgraft (PDX077), and maintenance niraparib therapy was effective in prolonging recurrence-free survival. pADPr inhibition was demonstrated in all niraparib-treated tumorgrafts, while PARP1 degrada-tion was evident in tumorgrafts responsive to single agent niraparib. Differential gene expression was seen in niraparib treated vs. control tumors in HR-deficient tumorgrafts in comparison to HR-proficient tumors at the end of 4 weeks of treatment. Clustering of 6 most/least differentially expressed genes was predictive of response to niraparib. Furthermore, RAD51C and PTEN gene expression were diminished in PDX039 (HR proficient by BROCA; niraparib responsive), suggesting a possible mechanism for its sensitivity to niraparib. Conclusion: Response to niraparib was seen in both BRCA-mutant and BRCA-wildtype xenografts. Thus, BRCA or HR gene mutational status alone was not sufficient to predict response to niraparib. Low RAD51C, PTEN and loss of PARP1 expression following treatment were most characteristic of tumors responding to niraparib. Paclitaxel may be detrimental to the sensitizing effects of PARP inhibition on carboplatin, which may have clinical implications for the incorporation of PARP inhibitors in front-line treatment. Our data are supportive of the phase III study of niraparib as maintenance therapy in patients with platinum-sensitive ovarian cancer. This abstract is also presented as Poster A30. Citation Format: Mariam Al Hilli, S. John Weroha, Marc Becker, Xiaonan Hou, Sean Harrington, Sarah McKinstrey, Kimberly Kalli, Karin Goodman, Keith Wilcoxen, Elizabeth Swisher, Scott Kaufmann, Paul Haluska. In vivo antitumor activity of the PARP inhibitor niraparib (MK-4827) in homologous recombination deficient and proficient ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr PR05.

Abstract Purpose: Ovarian cancer has a high recurrence and mortality rate. A barrier to improved outcomes includes a lack of accurate models for preclinical testing of novel therapeutics. Experimental Design: Clinically relevant, patient-derived tumorgraft models were generated from sequential patients and the first 168 engrafted models are described. Fresh ovarian, primary peritoneal, and fallopian tube carcinomas were collected at the time of debulking surgery and injected intraperitoneally into severe combined immunodeficient mice. Results: Tumorgrafts demonstrated a 74% engraftment rate with microscopic fidelity of primary tumor characteristics. Low-passage tumorgrafts also showed comparable genomic aberrations with the corresponding primary tumor and exhibit gene set enrichment of multiple ovarian cancer molecular subtypes, similar to patient tumors. Importantly, each of these tumorgraft models is annotated with clinical data and for those that have been tested, response to platinum chemotherapy correlates with the source patient. Conclusions: Presented herein is the largest known living tumor bank of patient-derived, ovarian tumorgraft models that can be applied to the development of personalized cancer treatment. Clin Cancer Res; 20(5); 1288–97. ©2014 AACR.

Abstract Ovarian cancer mortality ranks highest among all gynecologic cancers with growth factor pathways playing an integral role in tumorigenesis, metastatic dissemination, and therapeutic resistance. The HER and VEGF receptor (VEGFR) are both overexpressed and/or aberrantly activated in subsets of ovarian tumors. While agents targeting either the HER or VEGF pathways alone have been investigated, the impact of these agents have not led to overall survival benefit in ovarian cancer. We tested the hypothesis that cotargeting HER and VEGFR would maximize antitumor efficacy at tolerable doses. To this end, ovarian cancer xenografts grown intraperitoneally in athymic nude mice were tested in response to AC480 (pan-HER inhibitor, “HERi”), cediranib (pan-VEGFR inhibitor “VEGFRi”), or BMS-690514 (combined HER/VEGFR inhibitor “EVRi”). EVRi was superior to both HERi and VEGFRi in terms of tumor growth, final tumor weight, and progression-free survival. Correlative tumor studies employing phosphoproteomic antibody arrays revealed distinct agent-specific alterations, with EVRi inducing the greatest overall effect on growth factor signaling. These data suggest that simultaneous inhibition of HER and VEGFR may benefit select subsets of ovarian cancer tumors. To this end, we derived a novel HER/VEGF signature that correlated with poor overall survival in high-grade, late stage, serous ovarian cancer patient tumors. Mol Cancer Ther; 12(12); 2909–16. ©2013 AACR.

Abstract Despite advances in chemotherapeutic options over the last four decades, ovarian cancer remains the most lethal gynecologic malignancy with a cure rate that is essentially unchanged. A major barrier is lack of treatment-naïve, clinically- relevant in vivo models of epithelial ovarian cancer (EOC). To target this problem, we have developed low-passage in vivo models of EOC derived from patient primary debulking procedures at Mayo Clinic (Rochester, MN). Tumors were obtained from consented patients with suspected ovarian cancer, minced and injected into severe combined immunodeficiency (SCID) mice intraperitoneally for model establishment. To the date, we have heterotransplanted 129 EOC or ovarian malignant mixed mullerian (MMMT) tumors. The most common histologic subtype is serous (70.5%), followed by endometrioid (12.4%), clear cell (6.9%), and others (9.9%). The tumors were injected intraperitoneally into severe combined immunodeficiency (SCID) mice and the average time to moribund (TTM) was 174 +/− 83 days. In general, tumors grew faster with serial passages, TTM for epithelial tumors was 87 +/− 15 days and 19.5 +/− 3 days for MMMT. De novo ascites developed in 17 models and 24 models developed bowel metastasis, two clinically-relevant complications in late-stage ovarian cancer patients. Tumorgrafts were histologically similar to their source patient tumors and were positive for expression epithelial markers (pan-cytokeratin). The engraftment rate was 72.4% (n=87) after ≤ 6 months of lead-time, which is the average time to tumorgraft harvest. Passage of well-established tumorgrafts in SCID mice was successful in 88.6 % (n= 70) of all mice but only 56% (n=16) of athymic nude mice, consistent with prior studies demonstrating improved engraftment rates in more immunodeficient mice. Molecular characterization of tumorgrafts by array comparative genomic hybridization revealed similar gains and losses between the patient, early-passage, and later-passage tumorgraft tissues, indicating the tumorgrafts were genetically similar to their primary tumor. Analysis of tumorgrafts using the Affymetrix Human Plus 2.0 arrays revealed a predominance of previously described molecular subtypes C3, C4, and C5. Current investigations with both standard chemotherapy and novel targeted therapies are ongoing. Taken together, these models are as diverse as the patient population from which they are derived, produce clinically-relevant complications of EOC, maintain genetic fidelity in SCID mice, and are useful preclinical models for testing and development of novel therapeutics for ovarian cancer. High rates of engraftment are feasible. These tumorgrafts may help define predictors of response and eventually, lead to better treatments for patients. This work is funded by the Mayo Ovarian SPORE (CA136393) and the Minnesota Ovarian Cancer Alliance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3274. doi:1538-7445.AM2012-3274

Abstract Background: In estrogen receptor positive (ER+) breast cancer human epidermal growth factor receptor 2 (HER2) expression is associated with resistance to tamoxifen (Tam). Endoxifen is the most abundant active metabolite of Tam, resulting in part from cytochrome P450 2D6 (CYP2D6) metabolism. While Tam has been extensively used to model proliferative ER/HER2 cross-talk, there are no published data regarding endoxifen's effect on ER/HER2 nongenomic nor ER genomic signaling. Methods: Using ER+ cells (MCF7) and ER+/HER2+ cells (MCF7-HER2, BT474) we compared the effects of Tam and endoxifen on proliferation using crystal violet assays, Erk1/2 phosphorylation by western blotting, and E2-stimulated ER-regulated gene expression of amphiregulin (AREG), c-Myc (MYC) and progesterone receptor (PGR) by real-time RT-PCR assays. To examine the anti-proliferative affects of endoxifen compared to Tam in vivo we performed E2-stimulated MCF7-HER2 xenograft studies in athymic nude mice. Endoxifen doses of 25 (low) and 75 mg/kg (high) were used based on our prior pharmacokinetic studies showing achievable steady state levels of 100 nM and 1 uM, respectively. Z-Endoxifen HCl was synthesized by NCI. Results: Endoxifen, unlike Tam, potently inhibits growth in both ER+ and ER+/HER2+ breast cancer cell lines at concentrations observed in human CYP2D6 intermediate and extensive metabolizers (>40 nM). Tam (10-1000 nM) stimulates phosphorylation of Erk1/2 in stark contrast to endoxifen at similar doses. Endoxifen (100-1000 nM) significantly decreases E2-stimulated transcription of ER-regulated genes in MCF7-HER2 cells; AREG 2-3 fold, MYC 1-2 fold, and PGR 2-4 fold. Conversely, Tam (10-1000 nM) maintains or stimulates transcription; AREG 2-3 fold, MYC 0.5-2.5 fold, and PGR 0-3 fold. After 7 weeks of treatment of MCF7-HER2 xenografts, both high (p=0.001) and low dose endoxifen (p=0.01) resulted in greater antitumor activity than Tam, with high dose superior to low dose endoxifen (p=0.024). MCF7-HER2 xenografts that progressed on Tam to 200% growth at 7 weeks were randomized to either continued Tam or endoxifen. Four weeks after randomization endoxifen was superior to Tam (p<0.0001) with tumor size decreased by ≥ 20%. No significant toxicity was observed.Conclusions: We have demonstrated in both ER+ and ER+/HER2+ breast cancer cell lines that in contrast to Tam, endoxifen potently inhibits growth (both in vitro and in vivo), does not activate Erk1/2 signaling, and inhibits E2-stimulated transcription of ER-regulated genes. Endoxifen exhibited potent antitumor activity in MCF7-HER2 xenografts refractory to Tam. These data support the ongoing NCI sponsored phase I studies of Z-endoxifen at Mayo Clinic and NCI. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2283. doi:10.1158/1538-7445.AM2011-2283

Abstract Purpose: To improve the significance of insulin-like growth factor–binding protein 5 (IGFBP-5) as a prognostic and potentially predictive marker in patients with breast cancer. Experimental Design: Increased IGFBP-5 expression was identified in MCF-7 cells resistant (MCF-7R4) to the IGF-1R/insulin receptor (InsR) inhibitor BMS-536924 and its role examined by targeted knockdown and overexpression in multiple experimental models. Protein expression of IGFBP-5 was measured by immunohistochemistry in a cohort of 76 patients with breast cancer to examine correlative associations with invasive tumor fraction and outcome. The use of a combined IGFBP-5/IGFBP-4 (BPR) expression ratio was applied to predict anti-IGF-1R/InsR response in a panel of breast cancer lines and outcome in multiple breast tumor cohorts. Results: IGFBP-5 knockdown decreased BMS-536924 resistance in MCF-7R4 cells, whereas IGFBP-5 overexpression in MCF-7 cells conferred resistance. When compared with pathologically normal reduction mammoplasty tissue, IGFBP-5 expression levels were upregulated in both invasive and histologically normal adjacent breast cancer tissue. In both univariate and multivariate modeling, metastasis-free survival, recurrence free survival (RFS), and overall survival (OS) were significantly associated with high IGFBP-5 expression. Prognostic power of IGFBP-5 was further increased with the addition of IGFBP-4 where tumors were ranked based upon IGFBP-5/IGFBP-4 expression ratio (BPR). Multiple breast cancer cohorts confirm that BPR (high vs. low) was a strong predictor of RFS and OS. Conclusion: IGFBP-5 expression is a marker of poor outcome in patients with breast cancer. An IGFBP-5/IGFBP-4 expression ratio may serve as a surrogate biomarker of IGF pathway activation and predict sensitivity to anti-IGF-1R targeting. Clin Cancer Res; 18(6); 1808–17. ©2012 AACR.

AbstractSik-similar protein (Sik-SP), a small nucleolar ribonucleoprotein, has been shown to be primarily involved in ribosome biogenesis. However, its role in the hormone-directed nuclear receptor signaling is largely unknown. Here, we provide novel evidence that Sik-SP is required for appropriate regulation of estrogen receptor (ER)α-mediated estradiol-17β (E2)-dependent uterine physiologic responses in mice. Studies by Western blotting using the newly developed antibodies for Sik-SP showed that this protein is up-regulated in both the ovariectomized wild-type and ERα null uteri by E2. Immunohistochemical analyses in uterine sections showed that this protein is induced in the epithelial and stromal cells. Coimmunoprecipitation studies revealed that E2 directs molecular interaction between Sik-SP and ERα. Furthermore, gel-mobility shift and chromatin immunoprecipitation analyses provided evidence that Sik-SP is recruited with ERα to estrogen-responsive uterine gene promoters. Overexpression of Sik-SP in vitro demonstrated a role for Sik-SP in cellular growth and viability. In a primary uterine epithelial-stromal coculture system, E2 exhibited early induction of Sik-SP in both the epithelial and stromal cells. Interestingly, suppression of Sik-SP in this coculture model, for the stromal but not epithelial cells, caused perturbation of E2-dependent proliferation in the epithelial cell layer. Similarly, in vivo uterine suppression of Sik-SP also caused inhibition of epithelial cell proliferation and aberrant prolongation of water imbibition in the late phase by E2. Finally, studies showed that Sik-SP is physiologically important during the onset of implantation by E2. In conclusion, Sik-SP, an early E2-responsive nucleolar protein, is necessary to induce E2-dependent ERα-mediated appropriate physiologic responses in the uterus.