Leandro Venturutti

Stat3 regulates ErbB-2 expression and co-opts ErbB-2 nuclear function to induce miR-21 expression, PDCD4 downregulation and breast cancer metastasis.

Membrane overexpression of the receptor tyrosine kinase ErbB-2 (MErbB-2) accounts for a clinically aggressive breast cancer (BC) subtype (ErbB-2-positive) with increased incidence of metastases. We and others demonstrated that nuclear ErbB-2 (NErbB-2) also plays a key role in BC and is a poor prognostic factor in ErbB-2-positive tumors. The signal transducer and activator of transcription 3 (Stat3), another player in BC, has been recognized as a downstream mediator of MErbB-2 action in BC metastasis. Here, we revealed an unanticipated novel direction of the ErbB-2 and Stat3 interaction underlying BC metastasis. We found that Stat3 binds to its response elements (GAS) at the ErbB-2 promoter to upregulate ErbB-2 transcription in metastatic, ErbB-2-positive BC. We validated these results in several BC subtypes displaying metastatic and non-metastatic ability, highlighting Stat3 general role as upstream regulator of ErbB-2 expression in BC. Moreover, we showed that Stat3 co-opts NErbB-2 function by recruiting ErbB-2 as its coactivator at the GAS sites in the promoter of microRNA-21 (miR-21), a metastasis-promoting microRNA (miRNA). Using an ErbB-2 nuclear localization domain mutant and a constitutively activated ErbB-2 variant, we found that NErbB-2 role as a Stat3 coactivator and also its direct role as transcription factor upregulate miR-21 in BC. This reveals a novel function of NErbB-2 as a regulator of miRNAs expression. Increased levels of miR-21, in turn, downregulate the expression of the metastasis-suppressor protein programmed cell death 4 (PDCD4), a validated miR-21 target. Using an in vivo model of metastatic ErbB-2-postive BC, in which we silenced Stat3 and reconstituted ErbB-2 or miR-21 expression, we showed that both are downstream mediators of Stat3-driven metastasis. Supporting the clinical relevance of our results, we found an inverse correlation between ErbB-2/Stat3 nuclear co-expression and PDCD4 expression in ErbB-2-positive primary invasive BCs. Our findings identify Stat3 and NErbB-2 as novel therapeutic targets to inhibit ErbB-2-positive BC metastasis.

Progesterone receptor activation downregulates GATA3 by transcriptional repression and increased protein turnover promoting breast tumor growth

IntroductionThe transcription factor GATA3 is involved in mammary gland development and is crucial for the maintenance of the differentiated status of luminal epithelial cells. The role of GATA3 in breast cancer as a tumor suppressor has been established, although insights into the mechanism of GATA3 expression loss are still required.MethodsChromatin immunoprecipitation assays were conducted to study progestin modulation of recruitment of transcription factors to GATA3 promoter. We performed western blot and reverse RT-qPCR experiments to explore progestin regulation of GATA3 protein and mRNA expression respectively. Confocal microscopy and in vitro phosphorylation studies were conducted to examine progestin capacity to induce GATA3 serine phosphorylation in its 308 residue. GATA3 participation in progestin-induced breast cancer growth was addressed in in vitro proliferation and in vivo tumor growth experiments.ResultsIn this study, we demonstrate that progestin-activated progesterone receptor (PR) reduces GATA3 expression through regulation at the transcriptional and post-translational levels in breast cancer cells. In the former mechanism, the histone methyltransferase enhancer of zeste homolog 2 is co-recruited with activated PR to a putative progesterone response element in the GATA3 proximal promoter, increasing H3K27me3 levels and inducing chromatin compaction, resulting in decreased GATA3 mRNA levels. This transcriptional regulation is coupled with increased GATA3 protein turnover through progestin-induced GATA3 phosphorylation at serine 308 followed by 26S proteasome-mediated degradation. Both molecular mechanisms converge to accomplish decreased GATA3 expression levels in breast cancer cells upon PR activation. In addition, we demonstrated that decreased GATA3 levels are required for progestin-induced upregulation of cyclin A2, which mediates the G1 to S phase transition of the cell cycle and was reported to be associated with poor prognosis in breast cancer. Finally, we showed that downregulation of GATA3 is required for progestin stimulation of both in vitro cell proliferation and in vivo tumor growth.ConclusionsIn the present study, we reveal that progestin-induced PR activation leads to loss of GATA3 expression in breast cancer cells through transcriptional and post-translational regulation. Importantly, we demonstrate that GATA3 downregulation is required for progestin-induced upregulation of cyclin A2 and for progestin-induced in vitro and in vivo breast cancer cell growth.

Targeting ErbB-2 nuclear localization and function inhibits breast cancer growth and overcomes trastuzumab resistance.

Membrane overexpression of ErbB-2/HER2 receptor tyrosine kinase (membrane ErbB-2 (MErbB-2)) has a critical role in breast cancer (BC). We and others have also shown the role of nuclear ErbB-2 (NErbB-2) in BC, whose presence we identified as a poor prognostic factor in MErbB-2-positive tumors. Current anti-ErbB-2 therapies, as with the antibody trastuzumab (Ttzm), target only MErbB-2. Here, we found that blockade of NErbB-2 action abrogates growth of BC cells, sensitive and resistant to Ttzm, in a scenario in which ErbB-2, ErbB-3 and Akt are phosphorylated, and ErbB-2/ErbB-3 dimers are formed. Also, inhibition of NErbB-2 presence suppresses growth of a preclinical BC model resistant to Ttzm. We showed that at the cyclin D1 promoter, ErbB-2 assembles a transcriptional complex with Stat3 (signal transducer and activator of transcription 3) and ErbB-3, another member of the ErbB family, which reveals the first nuclear function of ErbB-2/ErbB-3 dimer. We identified NErbB-2 as the major proliferation driver in Ttzm-resistant BC, and demonstrated that Ttzm inability to disrupt the Stat3/ErbB-2/ErbB-3 complex underlies its failure to inhibit growth. Furthermore, our results in the clinic revealed that nuclear interaction between ErbB-2 and Stat3 correlates with poor overall survival in primary breast tumors. Our findings challenge the paradigm of anti-ErbB-2 drug design and highlight NErbB-2 as a novel target to overcome Ttzm resistance.

TNFα-Induced Mucin 4 Expression Elicits Trastuzumab Resistance in HER2-Positive Breast Cancer

Purpose: Although trastuzumab administration improved the outcome of HER2-positive breast cancer patients, resistance events hamper its clinical benefits. We demonstrated that TNFα stimulation in vitro induces trastuzumab resistance in HER2-positive breast cancer cell lines. Here, we explored the mechanism of TNFα-induced trastuzumab resistance and the therapeutic strategies to overcome it. Experimental Design: Trastuzumab-sensitive breast cancer cells, genetically engineered to stably overexpress TNFα, and de novo trastuzumab-resistant tumors, were used to evaluate trastuzumab response and TNFα-blocking antibodies effectiveness respectively. Immunohistochemistry and antibody-dependent cell cytotoxicity (ADCC), together with siRNA strategy, were used to explore TNFα influence on the expression and function of its downstream target, mucin 4 (MUC4). The clinical relevance of MUC4 expression was studied in a cohort of 78 HER2-positive breast cancer patients treated with adjuvant trastuzumab. Results: TNFα overexpression turned trastuzumab-sensitive cells and tumors into resistant ones. Histopathologic findings revealed mucin foci in TNFα-producing tumors. TNFα induced upregulation of MUC4 that reduced trastuzumab binding to its epitope and impaired ADCC. Silencing MUC4 enhanced trastuzumab binding, increased ADCC, and overcame trastuzumab and trastuzumab-emtansine antiproliferative effects in TNFα-overexpressing cells. Accordingly, administration of TNFα-blocking antibodies downregulated MUC4 and sensitized de novo trastuzumab-resistant breast cancer cells and tumors to trastuzumab. In HER2-positive breast cancer samples, MUC4 expression was found to be an independent predictor of poor disease-free survival (P = 0.008). Conclusions: We identified TNFα-induced MUC4 expression as a novel trastuzumab resistance mechanism. We propose MUC4 expression as a predictive biomarker of trastuzumab efficacy and a guide to combination therapy of TNFα-blocking antibodies with trastuzumab. Clin Cancer Res; 23(3); 636–48. ©2016 AACR.

Abstract 1195: TNFα induces multiresistance to HER2-targeted TNFα induces multiresistance to HER2-targeted therapies in HER2-positive breast cancer

HER2 positive (HER2+) is a breast cancer (BC) subtype characterized by HER2 overexpression/amplification that affects nearly 15% of BC patients and correlates with poor prognosis. These patients receive trastuzumab (T), an anti-HER2 monoclonal antibody, but resistance events (40-60%) hamper its clinical benefit. Previously we have demonstrated that TNFα (TNF) induced mucin 4 (MUC4) expression and turned T-sensitive cell lines and tumors into resistant ones. Nowadays, new anti-HER2 therapies are being used in the clinical setting, such as lapatinib (a dual inhibitor of EGFR and HER2), and antibodies like T-DM1 (combines TZ with the anti-microtubule agent emtansine), and pertuzumab (P) that impeds HER2 dimerization. The aim of this work was to study the role of TNF in resistance to the new HER2-targeted therapies. We used BT-474-C (control cells) and BT-474-T2, engineered in our lab to stably overexpress TNF, and were proven to be sensitive and resistant to T, respectively. We performed dose-response curves for T-DM1, they show that inhibits proliferation of BT-474- C cells at 0.01 μg/ml. On the other hand, BT-474-T2 cells were resistant in the same experimental conditions and they exhibited reduced T-DM1 binding with respect to BT-474-C. BT-474-C cells were sensitive to low concentrations of T-DM1 with 0.51 nmol/L, but in BT-474-T2 cells T-DM1 was ~10 times less potent than control cells (IC 50 3.34 nmol/L). When we abrogated MUC4 expression, BT-474-T2 cells were sensitized to T-DM1, showing that TNF-induced MUC4 expression is responsible for T-DM1 resistance in this cell line. We assessed the effect of lapatinib performing a dose-response curve. Results shown a similar IC50 for BT-474 C and T2 cells (0.26 μM and 0.28 μM, respectively). When we studied P effect, we observed that the combination of T+P was more effective inhibiting proliferation in BT-474-C cells than T alone, despite these results binding of the antibody showed no change between the cell lines. In BT-474-T2 cells proliferation was slightly inhibited by the combined treatment. In vivo experiments showed that BT-474-C tumors were sensitive to T and the combination of T+P, but BT-474-T2 tumors did not respond to any of these treatments. These results suggest that TNF plays an important role in multiresistance to HER2-targeted therapies, specifically T-DM1 and P, but not in lapatinib resistance. We propose TNF as an attractive target and we suggest that HER2+ patients resistant to T could be eligible for a combination of HER2-targeted therapies and a TNF-blocking treatment to overcome resistance. Citation Format: Maria F. Mercogliano, Mara De Martino, Sofia Bruni, Leandro Venturutti, Martin Rivas, Matias Amasino, Cecilia J. Proietti, Patricia V. Elizalde, Roxana Schillaci. TNFα induces multiresistance to HER2-targeted TNFα induces multiresistance to HER2-targeted therapies in HER2-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1195. doi:10.1158/1538-7445.AM2017-1195

Invasive micropapillary carcinoma of the breast overexpresses MUC4 and is associated with poor outcome to adjuvant trastuzumab in HER2-positive breast cancer

BackgroundInvasive micropapillary carcinoma of the breast (IMPC) is a histological tumor variant that occurs with low frequency characterized by an inside-out formation of tumor clusters with a pseudopapillary arrangement. IMPC is an aggressive tumor with poor clinical outcome. In addition, this histological subtype usually expresses human epidermal growth factor receptor 2 (HER2) which also correlates with a more aggressive tumor. In this work we studied the clinical significance of IMPC in HER2-positive breast cancer patients treated with adjuvant trastuzumab. We also analyzed mucin 4 (MUC4) expression as a novel biomarker to identify IMPC.MethodsWe retrospectively studied 86 HER2-positive breast cancer patients treated with trastuzumab and chemotherapy in the adjuvant setting. We explored the association of the IMPC component with clinicopathological parameters at diagnosis and its prognostic value. We compared MUC4 expression in IMPC with respect to other histological breast cancer subtypes by immunohistochemistry.ResultsIMPC, either as a pure entity or associated with invasive ductal carcinoma (IDC), was present in 18.6% of HER2-positive cases. It was positively correlated with estrogen receptor expression and tumor size and inversely correlated with patient’s age. Disease-free survival was significantly lower in patients with IMPC (hazard ratio = 2.6; 95%, confidence interval 1.1–6.1, P = 0.0340). MUC4, a glycoprotein associated with metastasis, was strongly expressed in all IMPC cases tested. IMPC appeared as the histological breast cancer subtype with the highest MUC4 expression compared to IDC, lobular and mucinous carcinoma.ConclusionIn HER2-positive breast cancer, the presence of IMPC should be carefully examined. As it is often not informed, because it is relatively difficult to identify or altogether overlooked, we propose MUC4 expression as a useful biomarker to highlight IMPC presence. Patients with MUC4-positive tumors with IMPC component should be more frequently monitored and/or receive additional therapies.

Abstract 2305: miR-16 is a tumor suppressor in progestin-induced breast cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Breast cancer is the most common type of cancer among women in the US and the second leading cause of death associated to cancer. microRNA (miRNA) are short ribonucleic acids with important regulatory functions and with an increasingly acknowledged role in cancer. We recently found that progestins modulate miRNA expression in a progestin-dependent murine breast cancer model. In particular, miR-16 was found to be downregulated by progestins. We here demonstrate that the treatment of T47D and BT-474 human breast cancer cells with the synthetic progestin medroxyprogesterone acetate (MPA) results in a significant miR-16 decrease and a concomitant cell proliferation augment. Interestingly, MPA treatment of T47D-Y cells, a variant of T47D lacking progesterone receptor (PR) expression, did not modify miR-16 levels. Furthermore, transfection of T47D-Y with PR-mPRO, a mutant PR which lacks the ability to activate nongenomic signaling pathways (but retains the transcriptional activity of PR), or with PR-C587A, a variant with the capacity to activate nongenomic pathways but uncapable of binding to DNA, did not recover the ability of progestins to downregulate miR-16, suggesting that both PR actions are necessary for miR-16 dowregulation by progestins. In addition, MPA induced a potent increase in the levels of the oncogenic transcription factor c-Myc, a previously reported suppressor of miR-16. Silencing of the signal transducer and activator of transcription 3 (Stat3) by siRNA, abrogated MPA capacity to induce c-Myc expression. Cyclin E, a cell cycle promoter with a major role in breast cancer progression, has been shown to be a miR-16 target. In this study, we not only demonstrate that MPA induces Cyclin E upregulation (both at the mRNA and protein level), but also that this can be prevented by the inhibition of Stat3 and/or c-Myc. We then wondered whether this regulation would occur in vivo. Thus, we injected 2 x 107 BT-474 cells in nude mice carrying a 17α-Estradiol (E2) pellet, in the absence of matrigel. As previously reported, the tumors first grew but started to regress soon afterwards. At that time point (7 days post-injection), half of the animals were administered MPA. The tumors in the group treated with MPA started to grow at a higher rate than the group supplied with E2 alone. Seven days after MPA supply, we excised some of the tumors and measured miR-16 levels, which showed that the treatment with MPA in vivo, produced a profound downregulation of miR-16. Furthermore, these tumors exhibited a higher level of cyclin E by western blot, validating cyclin E as a target of miR-16 in vivo. Our results are the first ones to identify miR-16 as a tumor suppressor modulated by progestins in human breast cancer cells, demonstrating as well that this regulation is relevant for the proliferative biological effect of progestins. 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 2305. doi:1538-7445.AM2012-2305