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Background HER2-positive (+) and triple negative breast cancer (TNBC) have the worst survival among BC. BC patients are treated with chemotherapy (CT) and/or radiotherapy (RT), and HER2+ BC patients also receive targeted therapies, such as trastuzumab (Tz). The abundance of tumor infiltrating lymphocytes (TILs), in both HER2+ and TNBC, has a major good prognostic value. Thus, indicating that immunological evasion mechanisms are present in the tumor microenvironment (TME) hampering the efficacy of the treatments. We previously showed that soluble tumor necrosis factor α (sTNF) induces upregulation of mucin 4 (MUC4), which shields Tz epitope on HER2 impairing Tz binding and its effects. In preclinical models of de no5vo Tz-resistant tumors, administration of the sTNF blocking agent INB03 (DN) together with Tz inhibited tumor growth. We proved that MUC4 expression is an independent predictor of poor DFS in patients treated with adjuvant Tz. Our goal is to study whether MUC4 plays a role in tumor immune evasion in HER2+ and TNBC. Methods Untreated primary BC samples were assessed for TILs density (H&E) and MUC4 expression by immunohistochemistry. Tumors with TILs ≥30% and >50%, for TNBC and HER2+ BC respectively, and MUC4 scores 2 and 3 (0-3) were deemed positive. A cohort of 56 TNBC and 90 HER2+BC, stage I-III were retrospectively retrieved from Hospital Fernández and Instituto Henry Moore from 2013-2017, and clinicopathological and treatment characteristics were obtained from electronic records. TNBC were treated with adjuvant (41) or neoadjuvant CT +/- RT (15). HER2+BC patients received adjuvant Tz + CT. The association between MUC4 and OS was assessed by Kaplan Meier and log rank test and between MUC4 and TILs using Chi2. JIMT-1 HER2+ BC, de novo resistant tumors to Tz, containing a doxycycline (Dox)-inducible shRNA MUC4 plasmid (JIMT-1shMUC4) growing in nude mice were treated with IgG, Tz, DN or Tz + DN. Tumor growth was measured and macrophages and NK cells were determined in the TME by flow cytometry. Anti-asialo GM1 and clodronate-encapsulated liposomes were used to deplete NK cells and macrophages, respectively. Results We found an inverse relationship between TILs and MUC4 expression in HER2+ and TNBC (P=0.02 and P= 5 x10-5, respectively). Patients with MUC4+ TNBC have a shorter OS (P=0.03) and MUC4 was an independent predictor of OS [P=0.01; HR 4.9 (95%CI 1.4-17.0)]. To study MUC4 involvement in macrophage and NK cells recruitment in a Tz resistant model, nude mice bearing JIMT-1-shMUC4 tumors were treated or not with Dox to abolish MUC4 expression. Both groups received IgG, Tz, DN or DN + Tz. In control groups (without Dox), only Tz + DN administration was able to inhibit tumor growth (75% inhibition, P Citation Format: Roxana Schillaci, Sofia Bruni, Florencia Mauro, María F Mercogliano, Agustina Roldan-Deamicis, Cecilia J Proietti, Rosalía Cordo-Russo, Gloria Inurrigarro, Agustina Dupont, Carla Adami, Daniel Lopez Della Vecchia, Sabrina Barchuck, Silvina Figurelli, Ernesto Gil Deza, Sandra Ares, Felipe G Gercovich, Patricia V Elizalde. Mucin 4 expression in high risk breast cancer: Predicting and overcoming resistance to immunotherapy [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-32.

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.

Supplementary Figure Legends

Supplementary Figure S1. Aggressive features of tumors overexpressing TNFα. A-C, H&E staining of BT-474 T2 tumors showing A, muscle and B, dermis infiltration (depicted by arrows). C, peritumor leukocyte infiltration (arrow). D, histopathological analysis of BT-474 T1 tumors. BT-474 T1 tumors were established and treated as described in Figure 2. First and second lines are H&E stainings and third line depicts HER2 staining by IHC. Arrows point out mitotic figures.

Supplementary Figure S2. Determination and scoring of MUC4 and TNFα expression in breast cancer biopsies by IHC. A, tissue microarrays were used to establish a score of MUC4 by IHC analysis as described by Workman et al (17). Score 0 represents no stain to less than 30% of cells stained faintly, 1+ greater than 30% of cells stained with light to moderate intensity, 2+ greater than 50% of cells stained moderately, 3+ intense staining of majority of the epithelial population. The yellow arrow in the inset of MUC4 (score 0) shows positive staining in endothelial cells, used as an internal control. B, TNFα score was equivalent to that described for MUC4. For both proteins scores of 2+ and 3+ were considered as positive expression.

Supplementary Table S1. Patient baseline characteristics. Supplementary Table S2. Growth rate of TNFα-producing and control BT-474 tumors treated with trastuzumab

Revised Supplementary Data

Supplementary Figure S3. MUC4 expression and outcome of patients with luminal breast cancer respect. Kaplan-Meier analysis of the probability of DFS of patients with luminal breast cancer (HER2-negative, estrogen receptor-positive), based on the expression of MUC4. The clinicopathological characteristics of this cohort are shown in Supplementary Table S1.

The Breast Cancer Revealed initiative was designed and conducted to know the status of breast cancer at each point of breast cancer care, through i) prevention, ii) detection, iii) diagnosis, iv) treatment, and iv) the capacity of our health systems. The expert panel from 11 Latin American countries identified several strategies and proposed high impact priorities, including implementation of prevention policies, improve primary healthcare capacity for breast cancer screening, have adequate infrastructure to make effective and timely diagnoses, have a multidisciplinary team in the treatment process, access to a variety of treatments for all types of patients, have a coordinated and articulated system from primary care to specialized hospital. In a region with limited resources, prioritization in high-impact strategies for breast cancer control could lead to improved clinical outcomes and quality of life for our patients.

Background: Novel strategies aimed to overcome trastuzumab (Tz) resistance of HER2+ breast cancer (BC) are needed. Recently, we demonstrated a novel immune evasion strategy used by BC where tumor necrosis factor alpha (TNF) induces upregulation of the transmembrane glycoprotein mucin 4 (MUC4) via NF-kB activation to impair Tz binding that prevents antibody mediated killing of BC cells. Etanercept, a non-selective inhibitor of soluble and transmembrane TNF (sTNF, tmTNF), downregulated MUC4 expression and sensitized de novo Tz-resistant BC xenografts to Tz. Moreover, we showed that MUC4 expression is an independent predictor of poor disease-free survival in patients treated with Tz in the adjuvant setting (Clin Cancer Res 2017, 23:636). Etanercept is immunosuppressive due to off-target effects on tmTNF while selective inhibition of sTNF improves the immune response to the tumor (Cancer Immunol Res 2016, 4:441). Because of the immunosuppressive properties of etanercept, we wanted study if the dominant negative-TNF protein XPro1595 (DN-TNF; also known as INB03) that neutralizes sTNF without affecting tmTNF is able to downregulate MUC4 to inhibit Tz-resistant tumor growth and improve innate antitumor immune response. Methods: To assess the effect of DN-TNF on Tz-resistant HER2+ tumor growth, JIMT-1 cells were s.c. injected in nude mice. When tumors were established, animals were treated with IgG, DN-TNF, Tz or DN-TNF+Tz, i.p. twice a week for one month. Innate immune response was determined by flow cytometry analysis of NK cells activation and degranulation and myeloid-derived suppressor cells (MDSC) subtypes in tumor microenvironment (TME) and in spleen. Tz-dependent NK cells degranulation was assessed in splenocytes using HER2+, Tz-sensitive cell line BT-474 as the target. MUC4 and phospho NF-kB expression was determined by Western blot. Results: Treatment with Tz or DN-TNF had no impact on JIMT-1 tumor growth. However, co-treatment with DN-TNF and Tz resulted in significantly less growth. At day 21st, tumor volume was 75mm3 in DN-TNF+Tz vs 300mm3 control groups. DN-TNF+Tz treatment showed a decrease in myeloid cell infiltration and MDSC phenotype was enriched in the granulocytic-MDSC vs monocytic-MDSC suggesting a less immunosuppressive TME. DN-TNF+Tz administration significantly increased activation and degranulation of tumor infiltrating NK cells. In addition, spleen NK cells from these animals exhibited enhanced Tz-dependent degranulation vs control groups. MUC4 expression was downregulated in tumors treated with DN-TNF and NF-kB phosphorylation was inhibited (all comparisons p Conclusion: These results suggest that targeting sTNF together with Tz treatment improves antitumor immune response reducing tumor burden. Activated NK cells can more effectively attack the tumor due to a less suppressive TME and decreased MUC4 expression enhancing Tz binding in Tz-resistant HER2+ BC. Patients with increased levels of TNF expressing MUC4 in their tumors could be eligible for a combined therapy with DN-TNF and Tz to overcome/avoid resistance to therapy. These results can be translated quickly into the clinic. Citation Format: Schillaci R, Bruni S, De Martino M, Mercogliano MF, Inurrigarro G, Frahm I, Proietti CJ, Elizalde PV. Neutralizing soluble tumor necrosis factor alpha overcomes trastuzumab-resistant breast cancer immune evasion by downregulating mucin 4, improving NK cell function and decreasing myeloid-derived suppressor cells in tumor microenvironment [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-20-14.

HER2+ is a breast cancer (BC) subtype characterized by the overexpression/amplification of HER2. Patients receive trastuzumab (Tz) but many (27-42%) do not achieve an objective response. We demonstrated that the overexpression of TNFɑ induces Tz resistance in tumors by upregulating the membrane glycoprotein MUC4, which masks Tz epitope on HER2, impairing its binding and reducing its therapeutic effects. We have also proved that blocking the soluble TNFɑ isoform with INB03 (DN) reduces MUC4 expression, overcomes Tz resistance and unleashes an antitumor innate immune response characterized by an increase in NK cell-activation and degranulation and a macrophage (Mφ) polarization to the M1 subtype. This study aims to determine the impact of MUC4 expression on Mφ and NK cells antitumor activity in the presence of Tz or Tz+DN, and on human T-lymphocyte recruitment and differentiation. Tz-resistant HER2+ BC human cell lines, JIMT-1 and KPL-4, were genetically modified to stably express a doxycycline-inducible (Dox) MUC4 shRNA (shMUC4) or a scramble one (shControl), and injected s.c. into female nude mice. After tumor establishment, the animals were randomly divided into a control group (-Dox) or an induced group (+Dox), and treated twice a week i.p. with IgG (5 mg/kg) or Tz+DN (5 and 10 mg/kg each, respectively) (-Dox) and with IgG or Tz (+Dox). Chlodronate or anti-asialo GM1 was used to deplete Mφ or NK cells, respectively. Tumor volume was measured routinely and, at the end point, tumors were processed and infiltrating immune cells were analyzed by flow cytometry. In -Dox tumors, both Mφ and NK cells are needed to achieve Tz+DN antitumor effect (p We conclude that Mφ are key players in the Tz-mediated antitumor innate immune response. Moreover, MUC4 expression impairs T-cell effector function and promotes immunologically cold HER2+ tumors, an additional resistance mechanism of this glycoprotein. We propose that women with HER2+MUC4+ BC could benefit from the combined treatment of Tz+DN to enhance innate and adaptive antitumor immune responses and prevent or overcome Tz resistance. Because MUC4 expression can be easily determined from biopsy tissue, early determination of MUC4 status in HER2+ BC could guide therapeutic choices to improve treatment outcomes. Citation Format: Sofia Bruni, Florencia L. Mauro, Maria F. Mercogliano, Cecilia J. Proietti, Carla Adami, Agustina Dupont, Gloria Inurrigarro, Rosalia Cordo Russo, Patricia V. Elizalde, Roxana Schillaci. MUC4 enables immune tumor evasion in HER2+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2047.

Invasive 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. Fil: Mercogliano, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Inurrigarro, Gloria. Sanatorio Mater Dei Hermanas de María de Schoenstatt; Argentina Fil: de Martino, Mara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Venturutti, Leandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Rivas, Martin Alfredo. Cornell University; Estados Unidos Fil: Cordo Russo, Rosalia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Proietti Anastasi, Cecilia Jazmín. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Fernandez, Elmer Andres. Universidad Católica de Córdoba; Argentina Fil: Frahm, Isabel. Sanatorio Mater Dei Hermanas de María de Schoenstatt; Argentina Fil: Barchuk, Sabrina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos ; Argentina Fil: Allemand, Daniel H.. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos ; Argentina Fil: Figurelli, Silvina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos ; Argentina Fil: Gil Deza, Ernesto. Instituto Oncológico Henry Moore; Argentina Fil: Ares, Sandra. Instituto Oncológico Henry Moore; Argentina Fil: Gercovich, Felipe G.. Instituto Oncológico Henry Moore; Argentina Fil: Cortese, Eduardo. Ministerio de Defensa. Fuerza Aérea Argentina. Hospital Aeronáutico Central ; Argentina Fil: Amasino, Matías. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Guzmán, Pablo. Universidad de La Frontera; Chile Fil: Roa, Juan C.. Universidad de La Frontera; Chile Fil: Elizalde, Patricia Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Schillaci, Roxana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina

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. Fil: Mercogliano, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: de Martino, Mara. Ministerio de Ciencia. Tecnología e Innovación Productiva. Agencia Nacional de Promoción Cientifíca y Tecnológica; Argentina Fil: Venturutti, Leandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Rivas, Martin Alfredo. Weill Cornell Medical College; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Proietti Anastasi, Cecilia Jazmín. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Inurrigarro, Gloria. Sanatorio Mater Dei; Argentina Fil: Frahm, Isabel. Sanatorio Mater Dei; Argentina Fil: Allemand, Daniel H.. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Juan A. Fernández"; Argentina Fil: Gil Deza, Ernesto. Instituto Oncológico Henry Moore; Argentina Fil: Ares, Sandra. Instituto Oncológico Henry Moore; Argentina Fil: Gercovich, Felipe G.. Instituto Oncológico Henry Moore; Argentina Fil: Guzmán, Pablo. Universidad de La Frontera; Chile Fil: Roa, Juan Carlos. Universidad de La Frontera; Chile. Pontificia Universidad Católica de Chile; Chile Fil: Elizalde, Patricia Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Schillaci, Roxana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina

Purpose: To retrospectively measure the mean apparent diffusion coefficient (ADC) with diffusion-weighted magnetic resonance (MR) imaging and the mean metabolic ratio (MET) with three-dimensional (3D) hydrogen 1 (1H) MR spectroscopic imaging in regions of interest (ROIs) drawn over benign and malignant peripheral zone (PZ) prostatic tissue and to assess ADC, MET, and combined ADC and MET for identifying malignant ROIs, with whole-mount histopathologic examination as the reference standard. Materials and Methods: The institutional review board approved this HIPAA-compliant retrospective study and issued a waiver of informed consent. From among 61 consecutive patients with prostate cancer, 38 men (median age, 61 years; range, 42–72 years) who underwent 1.5-T endorectal MR imaging before radical prostatectomy and who fulfilled all inclusion criteria of no prior hormonal or radiation treatment and at least one PZ lesion (volume, >0.1 cm3) at whole-mount pathologic examination were included. ADC maps were gene...

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. Fil: Venturutti, Leandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Romero, Lucía Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Urtreger, Alejandro Jorge. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Chervo, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Cordo Russo, Rosalia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Mercogliano, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Inurrigarro, G.. Sanatorio Mater Dei; Argentina Fil: Pereyra, M. G.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Proietti Anastasi, Cecilia Jazmín. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Izzo, Franco. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Díaz Flaqué, María Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Sundblad, Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Roa, J. C.. Pontificia Universidad Católica de Chile; Chile. Universidad de La Frontera; Chile Fil: Guzmán, P.. Universidad de La Frontera; Chile Fil: Bal, Elisa Dora. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Charreau, Eduardo Hernan. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Schillaci, Roxana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Elizalde, Patricia Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina

Introduction The 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. Methods Chromatin 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. Results In 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. Conclusions In 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. Electronic supplementary material The online version of this article (doi:10.1186/s13058-014-0491-x) contains supplementary material, which is available to authorized users.

To retrospectively measure the mean apparent diffusion coefficient (ADC) with diffusion-weighted magnetic resonance (MR) imaging and the mean metabolic ratio (MET) with three-dimensional (3D) hydrogen 1 ((1)H) MR spectroscopic imaging in regions of interest (ROIs) drawn over benign and malignant peripheral zone (PZ) prostatic tissue and to assess ADC, MET, and combined ADC and MET for identifying malignant ROIs, with whole-mount histopathologic examination as the reference standard.The institutional review board approved this HIPAA-compliant retrospective study and issued a waiver of informed consent. From among 61 consecutive patients with prostate cancer, 38 men (median age, 61 years; range, 42-72 years) who underwent 1.5-T endorectal MR imaging before radical prostatectomy and who fulfilled all inclusion criteria of no prior hormonal or radiation treatment and at least one PZ lesion (volume,0.1 cm(3)) at whole-mount pathologic examination were included. ADC maps were generated from diffusion-weighted MR imaging data, and MET maps of (choline plus polyamine plus creatine)/citrate were calculated from 3D (1)H MR spectroscopic imaging data. ROIs in the PZ identified by matching pathologic slides with T2-weighted images were overlaid on MET and ADC maps. Areas under the receiver operating characteristic curves (AUCs) were used to evaluate accuracy.The mean ADC +/- standard deviation, (1.39 +/- 0.23) x 10(-3) mm(2)/sec, and mean MET (0.92 +/- 0.32) for malignant ROIs differed significantly from the mean ADC, (1.69 +/- 0.24) x 10(-3) mm(2)/sec, and mean MET (0.73 +/- 0.18) for benign ROIs (P.001 for both). In distinguishing malignant ROIs, combined ADC and MET (AUC = 0.85) performed significantly better than MET alone (AUC = 0.74; P = .005) and was also better than ADC alone (AUC = 0.81), although the difference was not statistically significant (P = .09).The combination of ADC and MET performs significantly better than MET for differentiating between benign and malignant ROIs in the PZ.

Metastasis is a complex multistep process, responsible for as much as 90% of cancer-related deaths, yet obtaining successful treatment for these patients remains an elusive challenge. It has long been recognized that the tyrosine kinase receptor ErbB-2 and the signal transducer and activator of transcription 3 (Stat3), two major players in the breast cancer (BC) scenario, are involved in BC metastatic dissemination through a mechanism where Stat3 acts as a downstream effector of ErbB-2 action. In addition, we and others have also disclosed the role of nuclear ErbB-2 (NErbB-2) in BC, whose presence we identified as a poor prognostic factor in MErbB-2-positive tumors. On the other hand, microRNAs are short non-coding endogenous RNAs with regulatory functions. In particular, high levels of microRNA-21 (miR-21), a well-known oncomiR, have been reported to actively promote invasion and metastasis in BC cell lines and tissues. Here, we describe a novel hierarchical interaction between Stat3, ErbB-2 and miR-21, underlying the metastatic phenotype of ErbB-2-positive BC. We disclosed that Stat3 acts as an upstream regulator of ErbB-2 expression and function. In a panel of cell lines corresponding to different BC subtypes we found that Stat3 induced ErbB-2 expression at the transcriptional level through its recruitment to response elements (called GAS) at the ErbB-2 promoter. Furthermore, we demonstrated that Stat3 co-opted NErbB-2 function, recruiting it as a coactivator, to assemble a transcriptional complex at the GAS sites of the miR-21 promoter, leading to miR-21 up-regulation. We showed that the increase in miR-21 levels resulted in the downregulation of the metastasis suppressor protein PDCD4, a well known miR-21 target. In order to assess the physiological relevance of our molecular findings, we developed an in vivo model of ErbB-2-overexpressing metastatic BC, in which Stat3 activation was inhibited by transfection with a Stat3 dominant negative variant (Stat3Y705F) or its expression was silenced by siRNAs. We demonstrated through reconstitution assays that ErbB-2 and miR-21 were necessary downstream mediators of Stat3-induced metastases development. Furthermore, we explored the clinical significance of our findings in a cohort of ErbB-2-positive primary invasive BC patients and demonstrated that Stat3 and ErbB-2 nuclear co-expression was associated with low PDCD4 expression levels, and that this correlated with the presence of nodal metastases. Our present results in experimental models and in the clinic shed light on the molecular mechanisms underlying BC metastasis and highlight targeting either Stat3 or NErbB-2 as novel therapeutic strategies for ErbB-2-positive BC patients. #: 15-A-2849-AACR Citation Format: Leandro Venturutti, Lucía V. Romero, Alejandro J. Urtreger, María F. Chervo, María F. Mercogliano, Rosalía I. Cordo Russo, Matías G. Pereyra, Gloria Inurrigarro, María C. Díaz Flaqué, Victoria Sunblad, Juan C. Roa, Pablo Guzmán, Elisa Bal de Kier-Joffe, Eduardo H. Charreau, Roxana Schillaci, Patricia V. Elizalde. Stat3 and ErbB-2 interaction in breast cancer metastasis. [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 2265. doi:10.1158/1538-7445.AM2015-2265

ErbB-2 is a transmembrane tyrosine kinase receptor overexpressed/amplified in ∼15% of breast cancer patients and its overexpression correlates with poor prognosis. These patients are treated with trastuzumab (T), an anti-ErbB-2 monoclonal antibody, but only 40-60% of them respond when used in combination with chemotherapy. This lack of response is due to de novo or acquired resistance to T. In previous studies we have demonstrated that tumor necrosis factor alpha (TNF) transactivates ErbB-2, and subsequently activates Akt and NF-κB pathways in T-sensitive cell lines, which leads to an increase in cyclin D1 levels and proliferation, even in the presence of T. In this work we explored the role and the underlying mechanism of TNF in T resistance. In JIMT-1 and KPL-4, two de novo T-resistant cell lines, targeting TNF with etanercept (E, TNFR2-human Fc IgG fusion protein) in presence or absence of T did not affect cell growth. Remarkably, combined administration of T plus E decreased in vitro proliferation of both cell lines. Moreover, we observed a reduction in Akt and p65 NF-κB phosphorylation in the combined treatment, measured by Western Blot (WB). Similar results were obtained in vivo: tumor growth inhibition of the E+T group was >50% for JIMT-1 and >70% for KPL-4 vs. control, E or T groups. To explore acquired resistance to T, we engineered the T-sensitive BT-474 cell line to stably overexpress TNF (T2). Our results show that T2 xenografts are resistant to T administration while control tumors expressing empty vector (C2) dramatically regressed under the same treatment. Histopathological studies shed some light to understand the mechanism of TNF-induced T resistance by demonstrating that T2 tumors showed mucinous foci. It has been reported that one of the mechanisms of T resistance in JIMT-1 is due to mucin 4 (MUC4) expression, a transmembrane glycoprotein that masks the ErbB-2 epitope recognized by T. Evaluation of MUC4 by immunohistochemistry (IHC) showed a more intense MUC4 staining in T2 than in C2 tumors, which was confirmed by WB of cell extracts. Treatment with E and E+T in JIMT-1 and KPL-4 tumors showed weak to null staining for MUC4, in contrast to the strong staining obtained in IgG or T-treated tumors by IHC. Interestingly, in vitro treatment of JIMT-1 and KPL-4 cells with E, induced an increase in T binding measured by flow cytometry. In addition, T2 cells exhibited reduced binding of T and reduced antibody-dependent cellular cytotoxicity compared to C2 cells, which was reversed by MUC4 knockdown. Finally, p65 NF-κB knockdown in T2 cells impaired TNF-induced MUC4 expression. These results indicate that TNF plays an important role in T resistance stimulating MUC4 expression through activation of NF-κB pathway. Our data suggest that both TNF and MUC4 could be used as biomarkers of resistance to T. Furthermore, blockage of TNF could be a promising therapy for ErbB-2-positive breast cancer patients with acquired or de novo T resistance. Citation Format: María F. Mercogliano, Mara De Martino, Leandro Venturutti, Martín A. Rivas, Gloria Inurrigarro, Isabel Frahm, Cecilia J. Proietti, Franco Izzo, Patricia V. Elizalde, Roxana Schillaci. TNFα-induced MUC4 elicits trastuzumab resistance in ErbB-2-positive breast cancer. [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 712. doi:10.1158/1538-7445.AM2015-712