Virginia Novaro

Carcinoma-associated fibroblasts activate progesterone receptors and induce hormone independent mammary tumor growth: A role for the FGF-2/FGFR-2 axis.

The mechanisms by which mammary carcinomas acquire hormone independence are still unknown. To study the role of cancer‐associated fibroblasts (CAF) in the acquisition of hormone‐independence we used a hormone‐dependent (HD) mouse mammary tumor and its hormone‐independent (HI) variant, which grows in vivo without hormone supply. HI tumors express higher levels of FGFR‐2 than HD tumors. In spite of their in vivo differences, both tumors have the same hormone requirement in primary cultures. We demonstrated that CAF from HI tumors (CAF‐HI) growing in vitro, express higher levels of FGF‐2 than HD counterparts (CAF‐HD). FGF‐2 activated the progesterone receptors (PR) in the tumor cells, thus increasing cell proliferation in both HI and HD tumors. CAF‐HI induced a higher proliferative rate on the tumor cells and in PR activation than CAF‐HD. The blockage of FGF‐2 in the co‐cultures or the genetic or pharmacological inhibition of FGFR‐2 inhibited PR activation and tumor cell proliferation. Moreover, in vivo, the FGFR inhibitor decreased C4‐HI tumor growth, whereas FGF‐2 was able to stimulate C4‐HD tumor growth as MPA. T47D human breast cancer cells were also stimulated by progestins, FGF‐2 or CAF‐HI, and this stimulation was abrogated by antiprogestins, suggesting that the murine C4‐HI cells respond as the human T47D cells. In summary, this is the first study reporting differences between CAF from HD and HI tumors suggesting that CAF‐HI actively participate in driving HI tumor growth.

Reversal of antiprogestin resistance and progesterone receptor isoform ratio in acquired resistant mammary carcinomas

To explore mechanisms related to hormone resistance, three resistant variants of the MPA mouse breast cancer tumor model with low levels of progesterone receptor (PR) isoform A (PR-A)/high PR-B expression were developed by prolonged selective pressure with antiprogestins. The resistant phenotype of one tumor line was reversed spontaneously after several consecutive passages in syngeneic BALB/c mice or by 17-β-estradiol or tamoxifen treatment, and this reversion was significantly associated with an increase in PR-A expression. The responsive parental tumors disclosed low activation of ERK and high activation of AKT; resistant tumors on the other hand, showed the opposite, and this was associated with a higher metastatic potential, that did not revert. This study shows for the first time in vivo a relationship between PR isoform expression and antiprogestin responsiveness, demonstrating that, whereas acquired resistance may be reversed, changes in kinase activation and metastatic potential are unidirectional associated with tumor progression.

Responsiveness to PI3K and MEK Inhibitors in Breast Cancer. Use of a 3D Culture System to Study Pathways Related to Hormone Independence in Mice

Background A significant proportion of breast cancer patients face failure of endocrine therapy due to the acquisition of endocrine resistance. We have explored mechanisms involved in such disease progression by using a mouse breast cancer model that is induced by medroxyprogesterone acetate (MPA). These tumors transit through different stages of hormone sensitivity. However, when cells from tumor variants were seeded on plastic, all were stimulated by progestins and inhibited by antiprogestins such as RU486. Furthermore, cells from a RU486-resistant tumor variant recovered antiprogestin sensitivity. Hypothesis A three-dimensional (3D) culture system, by maintaining differential cellular organization that is typical of each tumor variant, may allow for the maintenance of particular hormone responses and thus be appropriate for the study of the effects of specific inhibitors of signaling pathways associated with disease progression. Method We compared the behavior of tumors growing in vivo and cancer cells ex vivo (in 3D Matrigel). In this system, we evaluated the effects of kinase inhibitors and hormone antagonists on tumor growth. Principal Findings LY294002, a PI3K/AKT pathway inhibitor, decreased both tumor growth in vivo and cell survival in Matrigel in MPA-independent tumors with higher AKT activity. Induction of cell death by anti-hormones such as ICI182780 and ZK230211 was more effective in MPA-dependent tumors with lower AKT activity. Inhibition of MEK with PD98059 did not affect tumor growth in any tested variant. Finally, while Matrigel reproduced differential responsiveness of MPA-dependent and -independent breast cancer cells, it was not sufficient to preserve antiprogestin resistance of RU486-resistant tumors. Conclusion We demonstrated that the PI3K/AKT pathway is relevant for MPA-independent tumor growth. Three-dimensional cultures were useful to test the effects of kinase inhibitors on breast cancer growth and highlight the need for in vivo models to validate experimental tools used for selective therapeutic targeting.

PI3K/AKT pathway regulates phosphorylation of steroid receptors, hormone independence and tumor differentiation in breast cancer

Using a model of medroxyprogesterone acetate (MPA)-induced mouse mammary tumors that transit through different stages of hormone dependence, we previously reported that the activation of the phosphatidylinositol 3-kinase (PI3K)/AKT (protein kinase B) pathway is critical for the growth of hormone-independent (HI) mammary carcinomas but not for the growth of hormone-dependent (HD) mammary carcinomas. The objective of this work was to explore whether the activation of the PI3K/AKT pathway is responsible for the changes in tumor phenotype and for the transition to autonomous growth. We found that the inhibition of the PI3K/AKT/mTOR (mammalian target of rapamycin) pathway suppresses HI tumor growth. In addition, we were able to induce mammary tumors in mice in the absence of MPA by inoculating HD tumor cells expressing a constitutively active form of AKT1, myristoylated AKT1 (myrAKT1). These tumors were highly differentiated and displayed a ductal phenotype with laminin-1 and cytokeratin 8 expression patterns typical of HI tumors. Furthermore, myrAKT1 increased the tumor growth of IBH-6 and IBH-7 human breast cancer cell lines. In the estrogen-dependent IBH-7 cell line, myrAKT1 induced estrogen-independent growth accompanied by the expression of the adhesion markers focal adhesion kinase and E-cadherin. Finally, we found that cells expressing myrAKT1 exhibited increased phosphorylation of the progesterone receptor at Ser190 and Ser294 and of the estrogen receptor α at Ser118 and Ser167, independently of exogenous MPA or estrogen supply. Our results indicate that the activation of the PI3K/AKT/mTOR pathway promotes tissue architecture remodeling and the activation of steroid receptors.

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.

AKT1 and AKT2 isoforms play distinct roles during breast cancer progression through the regulation of specific downstream proteins

The purpose of this study was to elucidate the mechanisms associated with the specific effects of AKT1 and AKT2 isoforms in breast cancer progression. We modulated the abundance of specific AKT isoforms in IBH-6 and T47D human breast cancer cell lines and showed that AKT1 promoted cell proliferation, through S6 and cyclin D1 upregulation, but it inhibited cell migration and invasion through β1-integrin and focal adhesion kinase (FAK) downregulation. In contrast, AKT2 promoted cell migration and invasion through F-actin and vimentin induction. Thus, while overexpression of AKT1 promoted local tumor growth, downregulation of AKT1 or overexpression of AKT2 promoted peritumoral invasion and lung metastasis. Furthermore, we evaluated The Cancer Genome Atlas (TCGA) dataset for invasive breast carcinomas and found that increased AKT2 but not AKT1 mRNA levels correlated with a worse clinical outcome. We conclude that AKT isoforms play specific roles in different steps of breast cancer progression, with AKT1 involved in the local tumor growth and AKT2 involved in the distant tumor dissemination, having AKT2 a poorer prognostic value and consequently being a worthwhile target for therapy.

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 1387: The ratio of progesterone receptor isoforms predicts progestin and antiprogestin response: Interaction of PR with AIB1 or SMRT.

We have previously demonstrated in a mouse model of breast cancer that mammary carcinomas which are responsive to the antiprogestin mifepristone (MFP) express higher levels of isoform A of progesterone receptor (PRA) than isoform B (PRB). PRB is predominant in tumors with constitutive or acquired resistance to MFP, indicating that the PR isoform ratio might predict antiprogestin responsiveness. The aim of this study was to evaluate the effect of MFP and of medroxyprogesterone acetate (MPA) on the growth of human breast cancer xenografts showing different ratios of PR isoforms. We chose IBH-6 cells since they express hormone receptors and they give rise to tumor xenografts without the need of exogenous hormone supply. IBH-6 tumors express higher levels of PRB than PRA and they are not inhibited by antiprogestin treatment. We stably transfected these cells with PRA, PRB or empty vector and sc inoculated 10 6 transfected cells into the right flank of female nude (nu/nu) mice. When tumors reached a size of 9 mm 2 , animals were treated sc with MFP (10 mg/kg/day), MPA (20 mg depot) or vehicle. Interestingly, only tumors showing higher levels of PRA than PRB were inhibited with MFP treatment (p D1 and MYC expression as determined by western blots (p The effect of MPA was not as clear as that of MFP. A slight growth inhibition of PRB transfected cells was observed (p To evaluate the molecular mechanisms involved in MFP-induced inhibitory or stimulatory effects we used wt T47D cells with equimolar PRA and PRB, as well as T47D-YA and -YB cell lines that only express PRA or PRB, respectively. Using confocal studies we showed that in wt T47D and T47D-YA cells, MPA (10 nM; 30 min) treatment induced nuclear co-localization of PR and the coactivator AIB1, while MFP (10 nM; 30 min) treatment promoted PR co-localization with the corepresor SMRT. Interestingly T47D-YB cells showed the opposite pattern: while MPA treatment promoted PR and SMRT co-localization, MFP treatment induced PR and AIB1 co-localization. Ongoing experiments will determine if these coregulators interact with PR at the MYC or CCND1 promoters. The results obtained herein using a human breast cancer xenograft model support our hypothesis that predicts that only breast cancers expressing high levels of PRA are candidates for antiprogestin treatment. Citation Format: Marina Riggio, Sebastian Giulianelli, Victoria Wargon, Virginia Novaro, Claudia Lanari. The ratio of progesterone receptor isoforms predicts progestin and antiprogestin response: Interaction of PR with AIB1 or SMRT. [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 1387. doi:10.1158/1538-7445.AM2013-1387

Abstract 1403: Early tumor regression after endocrine and kinase inhibitor therapy in an experimental model of breast cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC More than 70% of breast cancers express estrogen and progesterone receptors (ER and PR), and are thus susceptible to adjuvant endocrine therapy. Treatments for these patients are mainly focused on blocking ER function at multiple levels. In the last few years, evidence has linked progestin to breast cancer pathogenesis. According to that, PR would be a valid target for breast cancer therapy. In addition, up to 40% of hormone-receptor positive breast tumors have PI3K activating mutations, and then PI3K/AKT inhibition is becoming a new approach for breast cancer treatment. The aim of this work was to study early stages of tumor regression in an experimental model of breast cancer. We tested the effect of an antiprogestin and a PI3K inhibitor for 48 hs, alone or in combination. We used the medroxyprogesterone acetate (MPA)-induced breast cancer model. Tumors of this model express high levels of ER and PR. Initially, they are MPA-dependent, but after a few passages MPA-independent variants appear spontaneously. These variants may respond to an endocrine therapy or may progress to a hormonal resistant phenotype. We have previously demonstrated that the MPA-independent variant C4-HI regresses after 48 hs administration of the antiprogestin Mifepristone (MFP, 12 mg/kg/day) or the PI3K inhibitor Wortmannin (WN, 1 mg/kg/day), displaying an increase in ductal differentiation. We now explored the mechanisms involved in these inhibitory effects. Proliferation of tumor cells, determined as mitotic and Ki67 indexes, is reduced (p<0.01) similarly by MFP and WN respect to vehicle. Consistently, we detected a downregulation in the expression of cell cycle proteins c-MYC and Cyclin D1 determined by western blot and immunohistochemistry. However, after 48hs only WN increased necrosis, apoptosis (p<0.01) and the BAX/BCL-XL ratio. As expected, only WN decreased pAKT and pS6 levels. Furthermore, when given in combination, MFP and WN improved all the above mentioned parameters. In an antiprogestin resistant tumor variant, PI3K inhibition still suppressed tumor growth. We detected that after 7 days of treatment WN-treated C4-2-HI tumors were smaller than vehicle-treated tumors (p<0.001). However, no evident signs of regression were histologically observed. Ongoing experiments are being conducted to evaluate histologically tissue response at earlier time points (48hs) after WN therapy. Taken together, these results suggest that inhibiting PI3K pathway could represent a useful tool for the treatment of hormone receptor-positive tumors, even of those that have progressed to a hormonal resistant phenotype. Understanding the early mechanisms involved in tumor regression may improve the design and monitoring of cancer therapies. Citation Format: Maria Laura Polo, Marina Riggio, Claudia Lanari, Virginia Novaro. Early tumor regression after endocrine and kinase inhibitor therapy in an experimental model of breast cancer. [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 1403. doi:10.1158/1538-7445.AM2013-1403

Abstract 1319: Early tumor regression after endocrine and kinase inhibitor treatments in mammary carcinomas with different hormonal requirements

Two-thirds of diagnosed breasts cancers express either estrogen receptors (ER), progesterone receptors (PR) or both. Unlike most experimental breast cancer models, in the medroxyprogesterone acetate (MPA) model, MPA induces mammary carcinomas that express high levels of ER and PR. In this model we developed a tumor variant that depends on MPA to grow (C4-HD), and another one that grows without it (C4-HI). Previous studies showed that both types of tumors represent a useful tool to study mechanisms of regression after endocrine therapy, since both regress, albeit differently, after prolonged treatment (10 days) with the antiprogestin mifepristone. While regressive C4-HD tumors display extensive apoptosis, C4-HI tumors regress showing increased glandular differentiation. In order to investigate specific mechanisms of regression and to determine if these mechanisms are tumor specific or antitumor agent specific, we evaluated early steps of tumor regression caused by different agents in C4-HD and C4-HI tumors. We evaluated mifepristone (12mg/kg/day), ER modulators such as ICI182780 (25mg/kg) and tamoxifen (5mg/kg/day), and the PI3K/AKT inhibitor wortmannin (1 mg/kg/day). Mice carrying C4-HD or C4-HI tumors were treated for 48 hours and then sacrificed. Tumors were measured, excised and processed for histopathological and immunohistochemical studies as well as western blotting. We found that after 48 hs of treatment the size of C4-HD tumors was only reduced with mifepristone (p Taken together, these results suggest that each tumor variant has its own mechanism of regression in response to endocrine or kinase inhibitor treatments. Such different mechanism of regression in each tumor type may arise as a result of specific tumor-stromal interactions. Understanding the particular cellular mechanisms that lead to each tumor type regression could provide the basis to develop more specific and selective new antitumor therapies or potentiate therapies that already exist. 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 1319. doi:10.1158/1538-7445.AM2011-1319