Gonzalo Ricardo Sequeira

Action of hexachlorobenzene on tumor growth and metastasis in different experimental models.

Hexachlorobenzene (HCB) is a widespread organochlorine pesticide, considered a possible human carcinogen. It is a dioxin-like compound and a weak ligand of the aryl hydrocarbon receptor (AhR). We have found that HCB activates c-Src/HER1/STAT5b and HER1/ERK1/2 signaling pathways and cell migration, in an AhR-dependent manner in MDA-MB-231 breast cancer cells. The aim of this study was to investigate in vitro the effect of HCB (0.005, 0.05, 0.5, 5μM) on cell invasion and metalloproteases (MMPs) 2 and 9 activation in MDA-MB-231 cells. Furthermore, we examined in vivo the effect of HCB (0.3, 3, 30mg/kg b.w.) on tumor growth, MMP2 and MMP9 expression, and metastasis using MDA-MB-231 xenografts and two syngeneic mouse breast cancer models (spontaneous metastasis using C4-HI and lung experimental metastasis using LM3). Our results show that HCB (5μM) enhances MMP2 expression, as well as cell invasion, through AhR, c-Src/HER1 pathway and MMPs. Moreover, HCB increases MMP9 expression, secretion and activity through a HER1 and AhR-dependent mechanism, in MDA-MB-231 cells. HCB (0.3 and 3mg/kg b.w.) enhances subcutaneous tumor growth in MDA-MB-231 and C4-HI in vivo models. In vivo, using MDA-MB-231 model, the pesticide (0.3, 3 and 30mg/kg b.w.) activated c-Src, HER1, STAT5b, and ERK1/2 signaling pathways and increased MMP2 and MMP9 protein levels. Furthermore, we observed that HCB stimulated lung metastasis regardless the tumor hormone-receptor status. Our findings suggest that HCB may be a risk factor for human breast cancer progression.

Progestin and antiprogestin responsiveness in breast cancer is driven by the PRA/PRB ratio via AIB1 or SMRT recruitment to the CCND1 and MYC promoters

There is emerging interest in understanding the role of progesterone receptors (PRs) in breast cancer. The aim of this study was to investigate the proliferative effect of progestins and antiprogestins depending on the relative expression of the A (PRA) and B (PRB) isoforms of PR. In mifepristone (MFP)‐resistant murine carcinomas antiprogestin responsiveness was restored by re‐expressing PRA using demethylating agents and histone deacetylase inhibitors. Consistently, in two human breast cancer xenograft models, one manipulated to overexpress PRA or PRB (IBH‐6 cells), and the other expressing only PRA (T47D‐YA) or PRB (T47D‐YB), MFP selectively inhibited the growth of PRA‐overexpressing tumors and stimulated IBH‐6‐PRB xenograft growth. Furthermore, in cells with high or equimolar PRA/PRB ratios, which are stimulated to proliferate in vitro by progestins, and are inhibited by MFP, MPA increased the interaction between PR and the coactivator AIB1, and MFP favored the interaction between PR and the corepressor SMRT. In a PRB‐dominant context in which MFP stimulates and MPA inhibits cell proliferation, the opposite interactions were observed. Chromatin immunoprecipitation assays in T47D cells in the presence of MPA or MFP confirmed the interactions between PR and the coregulators at the CCND1 and MYC promoters. SMRT downregulation by siRNA abolished the inhibitory effect of MFP on MYC expression and cell proliferation. Our results indicate that antiprogestins are therapeutic tools that selectively inhibit PRA‐overexpressing tumors by increasing the SMRT/AIB1 balance at the CCND1 and MYC promoters.

Progesterone Receptor Isoform Ratio: A Breast Cancer Prognostic and Predictive Factor for Antiprogestin Responsiveness

Background: Compelling evidence shows that progestins regulate breast cancer growth. Using preclinical models, we demonstrated that antiprogestins are inhibitory when the level of progesterone receptor isoform A (PR-A) is higher than that of isoform B (PR-B) and that they might stimulate growth when PR-B is predominant. The aims of this study were to investigate ex vivo responses to mifepristone (MFP) in breast carcinomas with different PR isoform ratios and to examine their clinical and molecular characteristics. Methods: We performed human breast cancer tissue culture assays (n = 36) to evaluate the effect of MFP on cell proliferation. PR isoform expression was determined by immunoblotting (n = 282). Tumors were categorized as PRA-H (PR-A/PR-B ≥ 1.2) or PRB-H (PR-A/PR-B ⩽ 0.83). RNA was extracted for Ribo-Zero-Seq sequencing to evaluate differentially expressed genes. Subtypes and risk scores were predicted using the PAM50 gene set, the data analyzed using The Cancer Genome Atlas RNA-seq gene analysis and other publicly available gene expression data. Tissue microarrays were performed using paraffin-embedded tissues (PRA-H n = 53, PRB-H n = 24), and protein expression analyzed by immunohistochemistry. All statistical tests were two-sided. Results: One hundred sixteen out of 222 (52.3%) PR+ tumors were PRA-H, and 64 (28.8%) PRB-H. Cell proliferation was inhibited by MFP in 19 of 19 tissue cultures from PRA-H tumors. A total of 139 transcripts related to proliferative pathways were differentially expressed in nine PRA-H and seven PRB-H tumors. PRB-H and PRA-H tumors were either luminal B or A phenotypes, respectively (P = .03). PRB-H cases were associated with shorter relapse-free survival (hazard ratio [HR] = 2.70, 95% confidence interval [CI] = 1.71 to 6.20, P = .02) and distant metastasis–free survival (HR = 4.17, 95% CI = 2.18 to 7.97, P < .001). PRB-H tumors showed increased tumor size (P < .001), Ki-67 levels (P < .001), human epidermal growth factor receptor 2 expression (P = .04), high grades (P = .03), and decreased total PR (P = .004) compared with PRA-H tumors. MUC-2 (P < .001) and KRT6A (P = .02) were also overexpressed in PRB-H tumors. Conclusion: The PRA/PRB ratio is a prognostic and predictive factor for antiprogestin responsiveness in breast cancer.

Targeting FGFR with BGJ398 in Breast Cancer: Effect on Tumor Growth and Metastasis

BACKGROUND Endocrine resistance and metastatic dissemination comprise major clinical challenges for breast cancer treatment. The fibroblast growth factor receptor family (FGFR) consists of four tyrosine kinase transmembrane receptors, involved in key biological processes. Genomic alterations in FGFR have been identified in advanced breast cancer and thus, FGFR are an attractive therapeutic target. However, the efficacy of FGFR inhibitors on in vivo tumor growth is still controversial. OBJECTIVE The purpose of this study was to evaluate the role of FGFR in tumor growth and breast cancer progression. METHODS Cell proliferation was assessed by 3H-thymidine uptake and cell counting in primary cultures of endocrine resistant mammary carcinomas and a human cell line, respectively. Tumor transplants and cell injections were used to determine in vivo growth and spontaneous metastasis. FGFR1-3 and αSMA expression were evaluated on primary tumors by immunohistochemistry. RESULTS Antiprogestin resistant murine transplants and a human xenograft express high levels of total FGFR1-3. In vitro treatment with the FGFR inhibitor, BGJ398, impaired cell proliferation of resistant variants versus vehicle. In vivo, versus control, BGJ398 treatment decreased one out of four resistant tumors, however all tumors showed a decreased epithelial/stromal ratio. Finally, in a model of hormone resistant mammary cancer that spontaneously metastasizes to the lung, BGJ398 decreased the number of mice with lung metastasis. CONCLUSION FGFR inhibitors are promising tools that require further investigation to identify sensitive tumors. These studies suggest that targeting FGFR combined with other targeted therapies will be useful to impair breast cancer progression.

PDGFB as a vascular normalization agent in an ovarian cancer model treated with a gamma-secretase inhibitor

Ovarian cancer is the fifth leading cause of cancer‐related deaths in women. In the past 20 years, the canonical types of drugs used to treat ovarian cancer have not been replaced and the survival rates have not changed. These facts show the clear need to find new therapeutic strategies for this illness. Thus, the aim of the present study was to investigate the effect of a gamma‐secretase inhibitor (DAPT) in combination with the Platelet‐derived growth factor B (PDGFB) on an ovarian cancer xenograft model. To achieve this goal, we analyzed the effect of the administration of DAPT alone and the co‐administration of DAPT and recombinant PDGFB on parameters associated with tumor growth and angiogenesis in an orthotopic experimental model of ovarian cancer. We observed that the dose of DAPT used was ineffective to reduce ovarian tumor growth, but showed anticancer activity when co‐administered with recombinant PDGFB. The administration of PDGFB alone normalized tumor vasculature by increasing periendothelial coverage and vascular functionality. Interestingly, this effect exerted by PDGFB was also observed in the presence of DAPT. Our findings suggest that PDGFB is able to improve tumor vascularity and allows the anticancer action of DAPT in the tumor. We propose that this therapeutic strategy could be a new tool for ovarian cancer treatment and deserves further studies.

Increased High Molecular Weight FGF2 in Endocrine-Resistant Breast Cancer

Endocrine resistance may develop as a consequence of enhanced growth factor signaling. Fibroblast growth factor 2 (FGF2) consists of a low and several high molecular weight forms (HMW-FGF2). We previously demonstrated that antiprogestin-resistant mammary carcinomas display lower levels of progesterone receptor A isoforms (PRA) than B isoforms (PRB). Our aim was to evaluate the role of FGF2 isoforms in breast cancer progression. We evaluated FGF2 expression, cell proliferation, and pathway activation in models with different PRA/PRB ratios. We performed lentiviral infections of different FGF2 isoforms using the human hormone-responsive T47D-YA cells, engineered to only express PRA, and evaluated tumor growth, metastatic dissemination, and endocrine responsiveness. We assessed FGF2 expression and localization in 81 human breast cancer samples. Antiprogestin-resistant experimental mammary carcinomas with low PRA/PRB ratios and T47D-YB cells, which only express PRB, displayed higher levels of HMW-FGF2 than responsive variants. HMW-FGF2 overexpression in T47D-YA cells induced increased tumor growth, lung metastasis, and antiprogestin resistance compared to control tumors. In human breast carcinomas categorized by their PRA/PRB ratio, we found nuclear FGF2 expression in 55.6% of tumor cells. No differences were found between nuclear FGF2 expression and Ki67 proliferation index, tumor stage, or tumor grade. In low-grade tumor samples, moderate to high nuclear FGF2 levels were associated to carcinomas with low PRA/PRB ratio. In conclusion, we show that HMW-FGF2 isoforms are PRB targets which confer endocrine resistance and are localized in the nuclei of breast cancer samples. Hence, targeting intracellular FGF2 may contribute to overcome tumor progression.

Isoform Specificity of Progesterone Receptor Antibodies

Progesterone receptors (PR) are prognostic and predictive biomarkers in hormone‐dependent cancers. Two main PR isoforms have been described, PRB and PRA, that differ only in that PRB has 164 extra N‐terminal amino acids. It has been reported that several antibodies empirically exclusively recognize PRA in formalin‐fixed paraffin‐embedded (FFPE) tissues. To confirm these findings, we used human breast cancer xenograft models, T47D‐YA and ‐YB cells expressing PRA or PRB, respectively, MDA‐MB‐231 cells modified to synthesize PRB, and MDA‐MB‐231/iPRAB cells which can bi‐inducibly express either PRA or PRB. Cells were injected into immunocompromised mice to generate tumours exclusively expressing PRA or PRB. PR isoform expression was verified using immunoblots. FFPE samples from the same tumours were studied by immunohistochemistry using H‐190, clone 636, clone 16, and Ab‐6 anti‐PR antibodies, the latter exclusively recognizing PRB. Except for Ab‐6, all antibodies displayed a similar staining pattern. Our results indicate that clones 16, 636, and the H‐190 antibody recognize both PR isoforms. They point to the need for more stringency in evaluating the true specificity of purported PRA‐specific antibodies as the PRA/PRB ratio may have prognostic and predictive value in breast cancer.

Abstract P2-05-27: The ratio of progesterone receptor isoform A to B determines the effect of antiprogestins on preclinical models of breast cancer metastasis

In previous studies using several experimental models expressing different progesterone receptor (PR) isoform ratios, we have shown that only those with high levels of isoform A (PRA) are inhibited by antiprogestins whereas those with high levels of isoform B (PRB) are resistant to antiprogestin therapy. Moreover, results obtained using tissue cultures of breast cancer patients confirmed the data observed in these experimental models (May and Rojas et al., ASCO meeting 2015). The aim of this study was to evaluate the role of progestins and antiprogestins on the outgrowth of spontaneous metastatic foci of mammary carcinomas with different PR isoform ratios. We used metastatic tumors from the murine medroxyprogesterone acetate (MPA)-induced breast cancer model: C7-2-HI (PRA>PRB) and C7-HI (PRA Both antiprogestins had similar effects, MFP induced almost complete regression of the C7-2-HI tumor as already described, and TLP inhibited significantly its growth as compared with control tumors, while no changes in tumor size were observed in MPA-treated mice. Lung or lymph node studies confirmed the lack of metastatic foci in MFP-treated mice while a significant decrease (p In conclusion, we have demonstrated that MFP impedes the metastatic process in tumors with higher levels of PRA than PRB while it might promote metastasis in those with the opposite ratio. We suggest that MFP, through PRB receptors, downregulate the expression of Nm23-H1 and increase the expression of MMP-2. The conclusive effects of TLP and MPA need further investigation. These data underscore the importance of isoform ratio determination before treatment of breast cancer patients with endocrine therapies that target PR. Citation Format: Lanari C, Alvarez M, Giulianelli S, Marina R, Sequeira G, May M, Novaro V, Sahores A, Lamb C. The ratio of progesterone receptor isoform A to B determines the effect of antiprogestins on preclinical models of breast cancer metastasis. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-05-27.

Abstract P5-09-19: Progesterone receptor isoform A as a predictive marker of antiprogestin therapy in breast cancer: A tissue culture method to evaluate drug responsiveness

Two thirds of breast cancers express steroid hormone receptors and are susceptible to an endocrine therapy usually aimed to target the estrogen receptors (ER). There is however compelling evidence suggesting that progesterone receptors (PR) participate together with ER, modulating breast cancer growth. Based on our results obtained in an experimental breast cancer model in which antiprogestins induce complete regression of carcinomas with higher levels of PR isoform A (PR-A) than isoform B (PR-B), we propose that antiprogestins such as mifepristone (MFP) might be a possible therapeutic option for patients expressing high levels of PR-A. The goal of our study is to correlate the ratio of PR isoform expression, with the in vitro responsiveness of human breast cancer samples to MFP. Preliminary studies indicated that primary cultures of breast cancer samples overexpressing PR-A were responsive to MFP (10 nM) in vitro. However, since the rate of successful primary cultures was not encouraging (7%), we decided to develop a tissue culture assay to evaluate MFP responsiveness of breast cancer samples in vitro. Forty five breast cancer samples obtained from the General Pacheco´s Hospital, Buenos Aires, were kept in culture medium at room temperature and in dry ice. The former were cut in slices of 100 μm using a tissue chopper usually used to slice mouse nervous tissue for short incubations, and were incubated on filters in the presence of growth medium (fetal calf serum 10%) with or without MFP (10 nM), for 48 hs at 37o C. The slices were then fixed, paraffin embedded, and processed for Ki-67 staining to evaluate proliferating cells. Frozen samples were processed for Western blotting and the ratio of both PR isoforms was determined in nuclear extracts. In 17/45 (38%) of the samples valuable information was obtained. This was related with the quality (size and amount of viable cells) of the sample. The number of Ki-67 positive cells over the total viable cells was evaluated by a pathologist (M. May) in at least six different control or MFP-treated slices. From nine cases in which the Ki-67 index in control slices doubled the value obtained in MFP-treated slices (p<0.05), 8 were samples in which PR-A was higher than PR-B. MFP increased (p<0.05) the Ki-67 index in one sample with higher levels of PR-B than PR-A. No significant differences were observed in the remaining cases (null, equimolar or higher levels of PR-B than PR-A). We still plan to increase the number of samples in our study. Our data suggest that the tissue culture method developed is much more efficient than the primary cultures to evaluate drug or hormone responsiveness in breast cancer tissue. The results obtained herein support the proposal of PR-A as a predictive marker of antiprogestin therapy in breast cancer patients. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-19.

Abstract 1399: Antiprogestin responsiveness of a human breast cancer xenograft model overexpressing progesterone receptors isoforms A (PRA) or B (PRB).

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Endocrine therapies in breast cancer are designed to target directly or indirectly the estrogen receptors alpha (ERα). There is increasing evidence indicating that progesterone receptors (PR) are involved as ERα modulating breast cancer growth. Antiprogestins had been used in clinical trials in patients who have failed to other therapies; however, the results were not very clear. Our laboratory has developed an experimental murine model of breast cancer in which mammary carcinomas, expressing ER and PR, transit through different stages of hormone dependency. We demonstrated using this model, that only tumors with higher levels of PRA than PRB were those responsive to antiprogestin treatment. Moreover, the antiprogestin mifepristone (MFP) was able to stimulate the growth of tumors overexpressing PRB. The aim of this study was to move into a human in mouse scenario in order to test our hypothesis postulating that only breast cancer patients with higher levels of PRA than PRB are susceptible to MFP treatment. T47D-YA and T47D-YB kindly provided by Dr. K. Horwitz (University of Colorado, Denver), were inoculated sc with matrigel into the right and left flanks respectively of female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice (5/group; 3 experiments) that have been implanted one week before with a sc 17-β-estradiol pellet (0.5mg). After 10 days, when tumors reached a size greater than 20 mm2, the mice were treated with silastic or MFP (6 mg) pellets sc. In other experiments MFP was administered as daily injections of 10 mg/kg/body weight. Animals were followed for 2 weeks. A significant decrease in tumor size was only registered in T47D-YA tumors treated with MFP (p<0.05). Interestingly the T47D-YB tumors growing in the contralateral flank of the same mice increased their size with MFP treatment. They reached a size even larger than control -YB tumors (p<0.05). T47D-YA tumors showed nuclear MYC and Cyclin D1 staining whereas a cytoplasmic staining was observed in MFP-treated tumors 72 hours after treatment (immunohistochemistry). Conversely, an intense nuclear staining of both proteins was observed in T47D-YB tumors growing with MFP treatment. It can be concluded that in this human in mouse model, a specific inhibitory effect of MFP is only obtained in xenografts expressing PRA, while a stimulatory effect may be achieved in tumors expressing only PRB. These results are in agreement with our data in murine tumors and as a whole suggest that antiprogestins may be excellent therapeutic tools for breast cancer patients with higher levels of PRA than PRB. Citation Format: Gonzalo R. Sequeira, Paola Rojas, Maria May, Claudia Lanari. Antiprogestin responsiveness of a human breast cancer xenograft model overexpressing progesterone receptors isoforms A (PRA) or B (PRB). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1399. doi:10.1158/1538-7445.AM2013-1399