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Background. Clinical trials have demonstrated that trastuzumab deruxtecan (T-DXd) provides a durable responses for patients with HER2-positive and HER2 low metastatic breast cancer (BC). With T-DXd treatment, approximately 50% of patients with HER2+ metastatic BC were still alive and progression-free at 24 months (DESTINY-Breast03). We previously shown that mucin 4 (MUC4) expression is an independent predictor of poor response to trastuzumab in HER2-positive BC patients. We also showed that MUC4 is upregulated by soluble TNFα (sTNFα) secreted by the tumor, which confers primary trastuzumab resistance since it hides trastuzumab epitope on the HER2 molecule, reducing its binding and diminishing its therapeutic effects. In preclinical models of de novo trastuzumab-resistant tumors, we proved that administration of the sTNFα blocking agent INB03 (DN) together with trastuzumab inhibited tumor growth and induced an innate immune response in the tumor microenvironment (TME). DN is a dominant-negative inhibitor of sTNFα that is not immunosuppressive because it does not affect transmembrane TNFα. Our goal is to study whether neutralizing sTNFα can improve T-DXd effects in a multiple HER2-targeted therapy-resistant model. Methods JIMT-1 is a HER2-positive BC cell line resistant to trastuzumab, pertuzumab and lapatinib, which expresses MUC4. JIMT-1 tumor-bearing nude mice were treated with (1) IgG 5 mg/kg, (2) T-DXd 5 mg/kg, (3) T-DXd 2.5 mg/kg, (4) T-DXd 1.25 mg/kg, (5) DN 10 mg/kg, (6) T-DXd 5 mg/kg +DN, (7) T-DXd 2.5 mg/kg +DN and (8) T-DXd 1.25 mg/kg +DN. T-DXd and IgG were administered i.v. on days 0, 7 and 14. DN was administered i.p. twice a week for 3 weeks. Tumor growth was monitored regularly. Tumor-infiltrating macrophages, NK cells and myeloid-derived suppressor cells (MDSCs) were evaluated by immunofluorescence and flow cytometry. Results The dose-response curve of T-DXd exhibited tumor growth inhibitions of 83% (5 mg/kg), 61% (2.5 mg/kg) and 37% (1.25 mg/kg) vs IgG-treated tumors. DN alone had no antitumor effect. Combination of T-DXd with DN resulted in a reinforced antitumor effect, as the tumor growth inhibition escalated to 98% (T-DXd 5 mg/kg+ DN), 81% (T-DXd 2.5 mg/kg+DN) and 73% (T-DXd 1.25 mg/kg+DN). Moreover, we observed that addition of DN to T-DXd 1.25 and 5 mg/kg enhances the infiltration of resident macrophages (p < 0.05 and p Citation Format: Sofia Bruni, Florencia Mauro, Cecilia Proietti, Rosalia Cordo-Russo, Mercogliano María Florencia, Roxana Schillaci. Soluble TNFα blockade enhances trastuzumab deruxtecan antitumor effect in HER2-positive breast cancer model [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-11-12.

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.

Clinical trials showed that trastuzumab deruxtecan (T-DXd) provides durable responses for patients with HER2+ and HER2 low metastatic breast cancer (BC), determined by immunohistochemistry. Approximately 50% of patients with HER2+ metastatic BC were still alive and progression-free at 24 months (DESTINY-Breast03). We proved that mucin 4 (MUC4) expression is an independent predictor of poor response to trastuzumab in HER2+ BC patients. In JIMT-1 tumors we proved that soluble TNFα (sTNFα) upregulates MUC4, conferring trastuzumab resistance by hiding its epitope on the HER2 molecule and reducing its binding. Here, we study whether sTNFα blockade with INB03 (DN) plays a role in regulation of innate immunity to enhance T-DXd antitumor effects in a multiple HER2-targeted therapy-resistant model. Nude mice bearing HER2+MUC4+ JIMT-1 tumor, primary resistant to trastuzumab, pertuzumab and lapatinib, were treated with IgG 5 mg/kg, T-DXd 5 mg/kg (T-DXd 5), 2.5 mg/kg (T-DXd 2.5) or 1.25 mg/kg (T-DXd 1.25), DN 10 mg/kg or the combined therapies. T-DXd and IgG were administered i.v. on days 0, 7 and 14. DN was administered i.p. twice a week for 3 weeks. Tumor growth was monitored. Mitotic index was analyzed in H&E tumor sections. The tumor-infiltrating innate cells, macrophages, NK cells and myeloid-derived suppressor cells (MDSCs), were studied by flow cytometry. T-DXd dose-response curves exhibited tumor growth inhibitions of 83% (T-DXd 5), 61% (T-DXd 2.5) and 37% (T-DXd 1.25) vs IgG-treated tumors. DN alone had no antitumor effect. T-DXd+DN reinforced the antitumor effect, as tumor growth inhibition escalated to 98% (T-DXd 5+DN), 81% (T-DXd 2.5+DN) and 73% (T-DXd 1.25+DN). A reduced number of mitotic figures were observed in T-DXd 5, T-DXd 1.25+DN and T-DXd 5+DN. Combining DN with T-DXd 1.25 and 5 enhanced the infiltration of resident macrophages and promoted polarization to the M1-like phenotype The tumor associated macrophages (TAMs) were similar among treatments. However, the combination T-DXd 1.25+DN showed an increase in M1-like tumor associated macrophages (TAMs) and a decrease in M2-like TAMs vs T-DXd 1.25 alone. T-DXd 1.25+DN treatment mimics the increase of infiltrating NK cells observed in the T-DXd 2.5 and 5 doses Finally, adding DN to T-DXd 2.5 and 5 diminishes MDSCs infiltration. Combination therapies were well tolerated without evidence of toxicity. Our results suggest that sTNFα blockade enhances T-DXd effect in a multiple HER2-targeted therapy resistant model. Adding DN allows to lower T-DXd doses to induce a reinforced antitumor innate immune response, reduced tumor cell mitosis and achieve similar tumor inhibition. Since sTNFα and MUC4 expression proved to be important variables in the response to T-DXd, neutralizing this cytokine may open new therapeutic strategies to treat patients with MUC4 expressing tumors or have progression on T-DXd therapy. Citation Format: Sofia Bruni, Florencia L. Mauro, Sofia Naveiro, Maria F. Mercogliano, Roxana Schillaci. Soluble TNFα blockade improves effectiveness of trastuzumab deruxtecan and boosts antitumor potential of macrophages in a HER2+ tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2273.

The tumor microenvironment component hyaluronan (HA) induces tumor progression through the interaction with its cell surface receptor CD44. Accumulation of HA is associated with poor prognosis and resistance to the anti-ErbB-2 agent trastuzumab (TZ) in breast cancer (BC). ErbB-2 receptor is overexpressed in 15-20% of BC patients (ErbB-2+) and constitutes an important therapeutic target. Despite the clinical efficiency of therapies targeting ErbB-2, resistance to these drugs remains a major issue. In addition to its membrane function, ErbB-2 migrates to the nucleus (NErbB-2) where it acts as a transcription factor (TF) or as a coactivator of TF, modulating proliferation, metastasis, and resistance to anti-ErbB-2 therapies in BC. CD44 has also been found in the nucleus (NCD44). Although crosstalk between HA/CD44 and ErbB-2 pathways has been reported, how their molecular interactions mediate TZ resistance remains poorly known. Our in silico studies showed that TZ-resistant cells express higher CD44 levels than TZ-sensitive ones. We previously reported that stimulation with the ErbBs ligand heregulin (HRG) induces NErbB-2 translocation, acquired-TZ resistance and proliferation in the TZ-sensitive SK-BR-3 cell line. Here, we found that HRG also increased CD44 mRNA expression in SK-BR-3 cells. Then, we explored the role of HA in modulating CD44 and ErbB-2 nuclear localization in BC cells. Immunofluorescence and confocal microscopy studies showed that stimulation with exogenous HA induced nuclear translocation of ErbB-2 in T47D cells. Interestingly, we found constitutive presence of CD44 and ErbB-2 protein in nuclear lysates of JIMT-1 cells, a de novo TZ-resistant BC model. HA stimuli further enhanced this nuclear localization. Contrarily, treatment with the chemical inhibitor of HA synthesis 4-methylumbelliferone (4MU) decreased not only HA levels but also NErbB-2 in JIMT-1 cells. Furthermore, 4MU inhibited the proliferation of two TZ-resistant cell lines, JIMT-1 and MDA-MB-453, in a dose-dependent manner. 4MU also inhibited HRG-induced proliferation in SK-BR-3 cells. Even more, wound-healing assays showed that 4MU inhibited migration of JIMT-1 cells. This inhibition was similar to the one observed when ErbB-2 was excluded from the nucleus via transfection with the hErbB-2ΔNLS mutant. In summary, we reveal that HA induces CD44 and ErbB-2 nuclear localization in BC cells, suggesting a novel crosstalk between HA/CD44 and ErbB-2 pathways. These findings also highlight the blockade of HA synthesis with 4MU as a novel therapeutic strategy in TZ-resistant BC. Citation Format: Santiago Madera, Valentina C. Bulian, Sharon S. Merin, María F. Mercogliano, Roxana Schillaci, Rosalia I. Cordo Russo. Exploring a novel crosstalk between hyluronan/CD44 and ErbB-2 pathways in resistance to ErbB-2-targeted therapies in breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3633.

Triple negative breast cancer (TNBC) refers to tumors that do not express clinically significant levels of estrogen and progesterone receptors, and lack membrane overexpression or gene amplification of ErbB-2/HER2, a receptor tyrosine kinase. Transcriptome and proteome heterogeneity of TNBC poses a major challenge to precision medicine. Clinical biomarkers and targeted therapies for this disease remain elusive, so chemotherapy has been the standard of care for early and metastatic TNBC. Our present findings placed ErbB-2 in an unanticipated scenario: the nucleus of TNBC (NErbB-2). Our study on ErbB-2 alternative splicing events, using a PCR-sequencing approach combined with an RNA interference strategy, revealed that TNBC cells express either the canonical (wild-type) ErbB-2, encoded by transcript variant 1, or the non-canonical ErbB-2 isoform c, encoded by alternative variant 3 (RefSeq), or both. These ErbB-2 isoforms function in the nucleus as transcription factors. Evicting both from the nucleus or silencing isoform c only, blocks TN cell and tumor growth. This reveals not only NErbB-2 canonical and alternative isoforms role as targets of therapy in TNBC, but also isoform c dominant oncogenic potential. Furthermore, we validated our findings in the clinic and observed that NErbB-2 correlates with poor prognosis in primary TN tumors, disclosing NErbB-2 as a novel biomarker for TNBC. Our discoveries challenge the present scenario of drug development for personalized BC medicine that focuses on wild-type RefSeq proteins, which conserve the canonical domains and are located in their classical cellular compartments. 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: Petrillo, Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; 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: 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: Bellora, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto Andino Patagónico de Tecnologías Biológicas y Geoambientales. Universidad Nacional del Comahue. Instituto Andino Patagónico de Tecnologías Biológicas y Geoambientales; Argentina Fil: Cenciarini, Mauro Ezequiel. 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: Chiauzzi, Violeta Alicia. 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: Santa María de la Parra, Lucía. 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: Pereyra Matías 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: Güttlein, Leandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Podhajcer, Osvaldo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Daniotti, Jose Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Dupont, Agustina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Juan A. Fernández"; Argentina Fil: Barchuk, Sabrina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Juan A. Fernández"; Argentina Fil: Figurelli, Silvina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Juan A. Fernández"; Argentina Fil: Lopez Della Vecchia, Daniel Edgardo. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Juan A. Fernández"; Argentina Fil: Roa, Juan Carlos. Universidad de La Frontera. Núcleo Científico y Tecnológico en Recursos Naturales; Chile. Pontificia Universidad Católica de Chile; Chile Fil: Guzmán, Pablo. Universidad de La Frontera. Núcleo Científico y Tecnológico en Recursos Naturales; Chile 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: 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

Breast cancer is the most common cancer in women and the leading cause of death. HER2 overexpression is found in approximately 20% of breast cancers and is associated with a poor prognosis and a shorter overall survival. Tratuzumab, a monoclonal antibody directed against the HER2 receptor, is the standard of care treatment. However, a third of the patients do not respond to therapy. Given the high rate of resistance, other HER2-targeted strategies have been developed, including monoclonal antibodies such as pertuzumab and margetuximab, trastuzumab-based antibody drug conjugates such as trastuzumab-emtansine (T-DM1) and trastuzumab-deruxtecan (T-DXd), and tyrosine kinase inhibitors like lapatinib and tucatinib, among others. Moreover, T-DXd has proven to be of use in the HER2-low subtype, which suggests that other HER2-targeted therapies could be successful in this recently defined new breast cancer subclassification. When patients progress to multiple strategies, there are several HER2-targeted therapies available; however, treatment options are limited, and the potential combination with other drugs, immune checkpoint inhibitors, CAR-T cells, CAR-NK, CAR-M, and vaccines is an interesting and appealing field that is still in development. In this review, we will discuss the highlights and pitfalls of the different HER2-targeted therapies and potential combinations to overcome metastatic disease and resistance to therapy.

Enhancer of Zeste homolog 2 (EZH2) is a histone methyltransferase which catalyzes the trimethylation of lysine 27 of histone H3 (H3K27me3), an epigenetic mark associated with chromatin compaction and transcriptional repression of target genes. This constitutes its canonical mechanism of action. Several studies have shown that EZH2 is able to activate gene transcription by forming transcriptional complexes through mechanisms that do not involve histone methylation. Indeed, EZH2 can also act in a non-canonical function by regulating transcription independently of its enzymatic activity. We have previously described that progestins induce the interaction between Progesterone Receptor (PR) and EZH2 in breast cancer (BC) cells, which results in the downregulation of tumor suppressor GATA3 and in the increase in cell proliferation. Since EZH2 has been implicated in the progression of several types of cancer, including those of the breast, and our own previous results indicate that EZH2 is required for progestin-induced breast cancer growth, we hypothesized that EZH2 could function as a mediator in the pro-tumorigenic effects of progestins, targeting specific tumor suppressor and differentiating genes to allow ER/PR-positive BC growth. In the present work we found that progestin treatment of T47D cells induced EZH2 mRNA and protein expression, which was abolished by the use of the progestin antagonist RU486 and when PR expression was blocked by siRNAs. We also studied the participation of EZH2 in the regulation of PR-regulated genes TNFalpha (TNFα), cyclin D1 and TIMP2. By ELISA assays, we observed that progestin treatment of T47D cells for 24 h induced TNFα secretion. This effect was abolished when EZH2 expression was inhibited in the presence of EZH2 siRNAs and when EZH2 enzymatic activity was blocked by the use of GSK126. Regarding TIMP2, we observed that EZH2 canonical activity participates in TIMP2 downregulation exerted by progestin treatment. We showed that EZH2 non-canonical function is also involved in progestin modulation of TIMP2 given that blockage of EZH2 activity did not completely revert progestin effect. Finally, we demonstrated that progestin upregulation of cyclin D1 did not require EZH2 activity. Our results prove that EZH2 engagement in PR target genes may involve its canonical or non-canonical function and therefore support further studies on other progestin-regulated cancer genes implicated in tumor growth. Presentation: No date and time listed

Triple negative breast cancer (TNBC) is clinically defined by the absence of estrogen and progesterone receptors and the lack of membrane overexpression or gene amplification of the receptor tyrosine kinase ErbB-2/HER2. Due to its heterogeneity, clinical biomarkers and targeted therapies for this disease remain elusive, and chemotherapy has been the standard of care for TNBC. ErbB-2 is classically located at the membrane of BC cells, where it triggers signaling cascades and promotes oncogenesis. We previously demonstrated that ErbB-2 is also localized in the nucleus (NErbB-2) of TNBC cells, from where it drives growth (1). We also discovered that TNBC expresses both wild-type ErbB-2 (WTErbB-2) and alternative ErbB-2 isoform c (ErbB-2c) (1). ErbB-2 migrates to the nucleus via retrograde transport. Here, we revealed that Retro-2, an inhibitor of retrograde transport that protects cells form the deleterious effects of toxins and viruses, evicts both WTErbB-2 and ErbB-2c from the nucleus of BC cells. Using BC models from several molecular subtypes, as well as normal breast cells, we demonstrated that Retro-2 specifically halts the proliferation of cells expressing NErbB-2. Moreover, Retro-2 decreased the expression of genes induced by NErbB-2 (i. e. cyclin D1 and Erk5) and promoted cell cycle arrest at G0/G1 phase and apoptosis. In addition to R2 growth inhibitory activity in vitro, we here also demonstrated that its optimized cyclic derivative Retro-2.1 (in particular the (S)-enantiomer) showed improved efficacy both to evict ErbB-2 isoforms from the nucleus and to inhibit proliferation in vitro. Importantly, Retro-2 eviction of both ErbB-2 isoforms from the nucleus resulted in a striking growth abrogation in multiple TNBC preclinical models, including xenografts and tumor explants). Our mechanistic studies demonstrated that Retro-2 induces a differential accumulation of WTErbB-2 at the early endosomes and plasma membrane, and of ErbB-2c at the Golgi, shedding light both on Retro-2 action on endogenous protein cargoes undergoing retrograde transport and on the biology of ErbB-2 splicing variants. Compelling evidence demonstrated that mRNAs 5' and 3' untranslated regions (UTRs) mediate post-transcriptional regulation of gene expression and determine protein levels and fate. While both T1 and T3 have different 5' but the same 3' UTRs sequences, our in silico studies showed that T1 and T3 RNA secondary structures vary in the region containing both their 5' and 3' UTRs. These findings suggest that T3 secondary structure impacts in its cell specific localization. Together, our present discoveries identify R2 as a precision oncology tool to target NErbB-2 retrograde transport. This novel theragnostic approach could greatly improve the outcome of TNBC patients. (1) Chervo MF et al, Oncogene 2020: 39: 6245-62. Presentation: No date and time listed

Triple negative breast cancer (TNBC) has poor prognosis and neither established biomarkers nor therapeutic targets. On the one hand, the androgen receptor (AR), a steroid hormone receptor (SR) which is expressed in 10-53% of TNBC and proved to be critical for BC proliferation, has been proposed as a new target in TNBC. On the other, we and others have shown that membrane ErbB-2 migrates to the nucleus (nuclear ErbB-2, NErbB-2) where it binds DNA at HER-2 associated sequences (HAS) to regulate BC proliferation and migration. Since we have previously shown a functional interplay between growth factors and SR signaling pathways in BC, we propose the existence of an interaction between AR and ErbB-2 which is involved in NErbB-2+/AR+ BC growth. The experimental model used was the human TNBC cell line MDA-MB-453 which displays high expression levels of AR and NErbB-2. We have previously shown that dihydrotestosterone (DHT)-activated AR induces Src-mediated ErbB-2 rapid activation and its migration to the nucleus where it binds to HAS sites in the DNA. By coimmunoprecipitation and WB studies, we have now shown that this crosstalk involves ErbB-2 and AR physical association which is inhibited by the AR antagonist enzalutamide. By microarray and bioinformatics analysis we have also previously identified a set of differentially expressed genes (DEGs) in the presence of DHT and NErbB-2 eviction to define an independent predictor of better clinical outcome in TNBC. By using the Tumor Immune Estimation Resource (TIMER) we found a significant association of the expression of the gene signature and the abundance of tumor immune infiltrates, in accordance with the involvement of the gene signature in the immune response and interferon pathways. We have validated the regulation of the gene signature in MDA-MB-453 cells and found that in the presence of DHT, eviction of NErbB-2 significantly induces the mRNA expression of the genes CXCL10, HLA-A, NMI, STAT1 and TAP1, members of said gene signature. We have also identified AREs and HAS sites in STAT1, NMI and TAP1 and studied AR and ErbB-2 recruitment to the chromatin by ChIP assays. We found that DHT induces ErbB-2 binding to its HAS sites in STAT1 in regulatory regions. Our findings evidence that DHT-activated AR induces ErbB-2 rapid activation and its migration to the nucleus where it binds to HAS sites in regulatory regions of a set of genes involved in the immune response in TNBC. Presentation: No date and time listed

ErbB-2, a member of ErbB family of receptor tyrosine kinases, is a key oncogenic driver in breast cancer. Despite clinical efficiency of ErbB-2-targeted therapies (trastuzumab, TZ), resistance to drugs is a major issue in the clinic. While ErbB-2 is mainly a plasma membrane-bound receptor, it also migrates to the nucleus (NErbB-2) where it can act as a transcription factor or coactivator. We previously reported that NErbB-2 is a major proliferation driver in TZ-resistant breast cancer. To investigate the NErbB-2 dependent transcriptome, RNAseq was performed using a TZ-resistant breast cancer model (JIMT-1 cells) with high constitutive levels of NErbB-2. JIMT-1 cells were transfected with an ErbB-2 nuclear localization domain mutant (hErbB-2ΔNLS), which also acts as a dominant-negative inhibitor of endogenous NErbB-2 migration. Exclusion of ErbB-2 from the nucleus resulted in up-regulation of 280 genes and down-regulation of 33 genes. Functional analysis revealed that NErbB-2 blockade enriched the expression of genes involved in type-I interferon (IFN) signaling pathway. IFNB1 and its downstream effectors OAS2 and TRIM22 were among the top up-regulated genes. In an independent breast cancer model (i. e., HCC-1569 cells), exclusion of NErbB-2 from the nucleus also induced expression of these genes. Blockade of NErbB-2 localization by injection of the hErbB-2ΔNLS mutant into JIMT-1 tumor xenografts significantly inhibited in vivo tumor growth and induced mRNA expression of IFNB1, OAS2 and TRIM22. Interestingly, blockade of NErbB-2 localization by treatment with Retro-2, an inhibitor of the retrograde transport, showed similar effects consistent with modulation of the IFN signaling pathway by NErbB-2. Bioinformatic analyses showed that both the promoter and the coding region of the IFNB1 gene contain ErbB-2 associated sequences (HAS sites). ChIP-PCR analyses revealed ErbB-2 recruitment to the HAS sites of the IFNB1 promoter and coding regions in normal growth conditions. Transfection of JIMT-1 cells with the hErbB-2ΔNLS mutant abolished the recruitment of ErbB-2 at the IFNB1 gene and also caused an increase in histone H4 acetylation, a marker of active gene transcription. NErbB-2 immunostaining in a cohort of 32 primary invasive ErbB-2-positive breast carcinomas treated with TZ revealed that NErbB-2 expression correlated with a poor disease-free survival. While this cohort is small, the findings suggest that NErbB-2 could be used as a biomarker of poor response to TZ in the clinic. In summary, our findings indicate that NErbB-2 drives the growth of TZ-resistant breast cancer cells via transcriptional repression of the IFNB1 signaling pathway, and highlight NErbB-2 as a therapeutic target and biomarker in TZ-resistant breast cancer. Presentation: No date and time listed

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.

Tumor necrosis factor alpha (TNFα) is a pleiotropic cytokine known to have contradictory roles in oncoimmunology. Indeed, TNFα has a central role in the onset of the immune response, inducing both activation and the effector function of macrophages, dendritic cells, natural killer (NK) cells, and B and T lymphocytes. Within the tumor microenvironment, however, TNFα is one of the main mediators of cancer-related inflammation. It is involved in the recruitment and differentiation of immune suppressor cells, leading to evasion of tumor immune surveillance. These characteristics turn TNFα into an attractive target to overcome therapy resistance and tackle cancer. This review focuses on the diverse molecular mechanisms that place TNFα as a source of resistance to immunotherapy such as monoclonal antibodies against cancer cells or immune checkpoints and adoptive cell therapy. We also expose the benefits of TNFα blocking strategies in combination with immunotherapy to improve the antitumor effect and prevent or treat adverse immune-related effects. Fil: Mercogliano, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Bruni, Sofia. 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: Mauro, 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: 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

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.

Triple negative breast cancer (TNBC) is clinically defined by the absence of estrogen and progesterone receptors and the lack of membrane overexpression or gene amplification of the receptor tyrosine kinase ErbB-2/HER2. Due to its heterogeneity, clinical biomarkers and targeted therapies for this disease remain elusive, and chemotherapy has been the standard of care for early and metastatic TNBC. ErbB-2 is classically located at the membrane of BC cells, where it triggers signalling cascades and promotes oncogenesis. However, we have demonstrated that ErbB-2 is also localized in the nucleus (NErbB-2) of TNBC cells and primary tumors, from where it drives growth. We also discovered that TNBC expresses both wild-type ErbB-2 (WTErbB-2) and alternative ErbB-2 isoform c (ErbB-2c). ErbB-2 migrates to the nucleus via retrograde transport. The small molecule Retro-2 is a non-toxic inhibitor of the retrograde transport route that protects cells from the deleterious effects of toxins and viruses. Here, we revealed that Retro-2 evicts both WTErbB-2 and ErbB-2c from the nuclei. Using BC models from several molecular subtypes, we demonstrated that Retro-2 specifically halts the proliferation of cells expressing NErbB-2 in a dose-dependent manner, whilst did not inhibit cell proliferation in the ErbB-2-negative MCF10A normal breast cell line. Additionally, Retro-2 decreased the expression of genes induced by NErbB-2 (cyclin D1 and Erk5) and promoted cell cycle arrest at G0/G1 phase and apoptosis. Even more, in preclinical models (including xenografts and tumor explants), Retro-2 treatment resulted in the eviction of NErbB-2 and abrogation of tumor growth. Our mechanistic studies demonstrated that Retro-2 induces a differential accumulation of WTErbB-2 at the early endosomes and plasma membrane, and of ErbB-2c at the Golgi, further preventing its sorting to the endoplasmic reticulum. These findings shed light both on Retro-2 action on endogenous protein cargoes undergoing retrograde transport and on the biology of ErbB-2 splicing variants. Together, our present discoveries provide evidence for the rational repurposing of Retro-2 as a novel therapeutic agent for TNBC. Citation Format: Santiago Madera, Franco Izzo, Maria F. Chervo, Agustina Dupont, Violeta A. Chiauzzi, Sofia Bruni, Ezequiel Petrillo, Diego Montero, Sharon Merin, Maria F. Mercogliano, Cecilia J. Proietti, Roxana Schillaci, Rosalia I. Cordo Russo, Patricia V. Elizalde. Blockade of retrograde transport in triple negative breast cancer excludes ErbB-2 isoforms from the nucleus and abrogates tumor growth [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 344.

Tamm-Horsfall Protein (THP) is expressed exclusively in the kidney and is the most abundant protein in urine. THP has a role in antibacterial host defense but also in inflammatory disorders of the urogenital tract. THP has been shown to regulate the innate and adaptive immunity in the urinary tract by potently activating macrophages and dendritic cells. Given these immunostimulatory effects, we hypothesize that systemic THP administration can promote an antitumor and antimetastatic response. THP was isolated from human urine of healthy individuals at a purity of >99% with less than < 1 UE/mg de THP of endotoxin. We treated murine J744 macrophages and human monocytes with 10 and 100 µg/ml THP for 18h and TNFα levels were determined by ELISA. In J744 macrophages TNFα levels reached 0.9 and 1.4 ng/ml, respectively and in human monocytes 4.5 and 6 ng/ml, respectively, confirming the macrophage-stimulating properties of THP. We then evaluated proliferation and migration by cell count and wound healing, respectively, in the 4T1 murine triple negative breast cancer (TNBC) model and the MDA-MB-231 human TNBC model. We cultured 4T1 and MDA-MB-231 cells for 4 days with 100 µg/ml THP and observed an inhibition in proliferation of 62% and 40%, respectively. Cell migration was evaluated under treatment with 100 µg/ml THP for 18h. Migration was inhibited by 70% in 4T1 cells and by 30% in MDA-MB-231 cells. These data highlight the antitumoral and antimetastatic effect of THP in TNBC. The in vivo experiments were performed by administering THP 3 times a week and/or docetaxel (Dx) at 15 mg/kg twice a week in BALB/c mice bearing 4T1 tumors (50-75 mm2), and tumor growth, lung metastasis and survival were determined. A dose-response curve (0.3-3 mg/kg) was performed using THP administered either s.c. or i.p. THP s.c. at 1.5 µg/kg and THP i.p. at 3 µg/kg induced 40% and 38% tumor growth inhibition, respectively. THP also reduced metastasis measured as the number of lung nodules, and the addition of Dx deepened the effect even more. In the case of survival studies, the tumors were surgically removed after reaching 50 mm3 and two days later the animals were treated with vehicle, Dx, or Dx+THP. Log rank analysis of Kaplan-Meier plots showed a significant improvement of overall survival in mice treated with Dx+THP (P= 0.011) and only a trend with Dx treatment (P=0.067). Here we show that THP decreases TNBC proliferation, reduces metastasis dissemination and improves Dx's effect. These evidences are especially important in TNBC whose standard of care is only chemotherapy. In conclusion, THP can be a valuable agent to enhance the use of Dx in the chemotherapy treatment of TNBC tumors. However, beneficial effects over other tumors should be confirmed. Citation Format: María Florencia Mercogliano, Sofia Bruni, Mara De Martino, Elena Cavanagh, Liliana Balanian, Emilio Sojo, Felipe Inserra, Jose Groisman, Roxana Schillaci. Tamm-Horsfall protein as a novel antitumor therapy for triple negative 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 1316.

Hormone receptor-positive (HR+, estrogen and/or progesterone receptor-positive) and HER2-negative breast cancer (BC) subtype is a biologically heterogeneous entity that comprises 70% of BCs. This subtype includes both luminal (Lum) A- and B-like subtypes, which have differences in prognosis and sensitivity to endocrine therapies. The development of biomarkers guiding treatment decisions in these settings is required. Tumor suppressor PDCD4 (programmed cell death 4), which can be found both in the nucleus (NPDCD4) or the cytoplasm (CPDCD4), inhibits tumor growth and metastasis, and its loss is associated with poor prognosis in solid tumors. To explore the clinical relevance of PDCD4 in BC, we analyzed its expression by immunohistochemistry in a cohort of 619 patients with primary invasive BC. We found that 34.7% of patients showed NPDCD4 and 21.3% showed CPDCD4. NPDCD4 positivity, but not CPDCD4, was associated with lower clinical stage (P = 0.0003), with presence of more differentiated tumors (P = 6.4x10-6), and with estrogen and progesterone receptor (PR) expression (P = 9.2x10-9 and P = 2.8x10-9, respectively). Kaplan-Meier analysis revealed that NPDCD4 expression was associated with a longer overall survival (OS) and disease-free survival (DFS) in LumA-like (P = 0.008 and P = 0.028, respectively) and LumB-like (P = 0.004 and P = 0.012, respectively) subtypes. Interestingly, patients with LumB-like tumors displaying NPDCD4 presented estimated OS and DFS rates similar to the ones observed in patients with LumA-like tumors also expressing NPDCD4, indicating that its presence improves the clinical outcome of LumB-like patients. Multivariate Cox regression analysis identified NPDCD4 as an independent predictor of good clinical outcome in both LumA-like (HR: 0.45, 95% CI 0.22-0.96, P = 0.038) and LumB-like (HR: 0.28, 95% CI 0.10-0.80, P = 0.018) subtypes. We validated our results by in silico analysis using expression data from the METABRIC cohort. Bioinformatics analysis of BC cells from the Cancer Cell Line Encyclopedia revealed a positive correlation between PDCD4 and PR expression (P = 0.015). Since LumB-like tumors present a higher risk of resistance to endocrine therapy and both PR and PDCD4 levels in this subtype are lower than in the LumA-like one, we postulated that the presence of PR may modulate PDCD4 expression. Silencing of PR expression in HR+ cells decreased PDCD4 protein levels while reconstitution of PR in a PR-null cell line increased them, confirming PR requirement for PDCD4 modulation. In line with PDCD4 physiological function, its knockdown increased cell migration capability of HR+ BC cells, whereas its restoration led to a decrease in cell migration of HR-negative BC models. Our findings identified NPDCD4 positivity as a novel biomarker of clinical outcome in LumA- and B-like subtypes and revealed PDCD4 reconstitution as a novel therapeutic strategy in BC.

The hormone receptor-positive (estrogen and/or progesterone receptor (PR)-positive) and HER2-negative breast cancer (BC) subtype is a biologically heterogeneous entity that includes luminal A-like (LumA-like) and luminal B-like (LumB-like) subtypes. Decreased PR levels is a distinctive biological feature of LumB-like tumors. These tumors also show reduced sensitivity to endocrine therapies and poorer prognosis than LumA-like tumors. Identification of biomarkers to accurately predict disease relapse in these subtypes is crucial in order to select effective therapies. We identified the tumor suppressor PDCD4 (programmed cell death 4), located in the nucleus (NPDCD4), as an independent prognostic factor of good clinical outcome in LumA-like and LumB-like subtypes. NPDCD4-positive LumB-like tumors presented overall and disease-free survival rates comparable to those of NPDCD4-positive LumA-like tumors, indicating that NPDCD4 improves the outcome of LumB-like patients. In contrast, NPDCD4 loss increased the risk of disease recurrence and death in LumB-like compared with LumA-like tumors. This, along with our results showing that LumB-like tumors present lower NPDCD4 positivity than LumA-like tumors, suggests that NPDCD4 loss contributes to endocrine therapy resistance in LumB-like BCs. We also revealed that PR induces PDCD4 transcription in LumB-like BC, providing a mechanistic explanation to the low PDCD4 levels in LumB-like BCs lacking PR. Finally, PDCD4 silencing enhanced BC cell survival in a patient-derived explant model of LumB-like disease. Our discoveries highlight NPDCD4 as a novel biomarker in LumA- and LumB-like subtypes, which could be included in the panel of immunohistochemical markers used in the clinic to accurately predict the prognosis of LumB-like tumors. Fil: Madera, Santiago. 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: 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: Chiauzzi, Violeta Alicia. 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: Pereyra Matías 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: 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: 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: Roldán Deamicis, Agustina. 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. Núcleo Científico y Tecnológico en Recursos Naturales; Chile Fil: Dupont, Agustina. 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, Juan Carlos. Universidad de La Frontera. Núcleo Científico y Tecnológico en Recursos Naturales; Chile. Universidad Católica de Chile; Chile Fil: Cenciarini, Mauro Ezequiel. 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: Barchuk, Sabrina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Juan A. Fernández"; Argentina Fil: Figurelli, Silvina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Juan A. Fernández"; Argentina Fil: Lopez Della Vecchia, Daniel Edgardo. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Juan A. Fernández"; Argentina Fil: Levit, Claudio. Sanatorio Sagrado Corazón. Servicio de Ginecología; Argentina Fil: Lebersztein, Gabriel. Sanatorio Sagrado Corazón. Servicio de Ginecología; Argentina Fil: Anfuso, Fabiana. Sanatorio Sagrado Corazón. Servicio de Ginecología; Argentina Fil: Castiglioni, Teresa. Centro de Patología Dr. Elsner; Argentina Fil: Cortese, Eduardo Mateo. Hospital Aeronáutico Central. Servicio de Ginecología; Argentina Fil: Ares, Sandra. Instituto Oncológico Henry Moore; Argentina Fil: Gil Deza, Ernesto. Instituto Oncológico Henry Moore; Argentina Fil: Gercovich, Felipe G.. Instituto Oncológico Henry Moore; 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: 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: Cordo Russo, Rosalía I.. 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

Triple negative breast cancer (TNBC) refers to tumors that do not express clinically significant levels of estrogen and progesterone receptors, and lack membrane overexpression or gene amplification of ErbB-2 tyrosine kinase receptor. Transcriptome and proteome heterogeneity of TNBC poses a major challenge to precision medicine. Gene expression analyses have categorized TNBC into distinct molecular subtypes. Up to 78% of clinical TNBCs belong to the basal-like (BL) subtype. Here we found ErbB-2 in an unanticipated scenario: the nucleus of TNBC (NErbB-2). Our study on ErbB-2 alternative splicing, using a PCR-sequencing approach combined with RNA interference, revealed that BL TNBC cells express the canonical ErbB-2 (WTErbB-2), encoded by transcript 1, and the non-canonical isoform c, encoded by alternative transcript 3 (T3). The latter was not previously reported in normal or malignant cells. To characterize the isoform c we designed siRNAs targeting T3 (T3 siRNAs), which silenced up to 93% of said isoform. Transfection of T3 siRNAs into BL cells expressing only isoform c or both isoform c and WTErbB-2 was sufficient to decrease cell proliferation. Intratumoral injections of T3 siRNAs into mice bearing BL TN tumors also blocked in vivo growth. To explore whether isoform c growth-promoting effect is due to its functions as a transcriptional regulator, we performed RNA-seq in BL cells expressing only this isoform. We identified a set of genes differentially regulated in BL cells where we evicted isoform c from the nucleus, as compared to control cells. In the up-regulated group, we found enrichment of pro-apoptotic and tumor suppressor genes and in the down-regulated one, genes involved in proliferation and stemness. We used gene set enrichment analysis (GSEA) to identify the biological processes associated with these isoform c-regulated genes. We found a pronounced enrichment of gene sets related to apoptosis, activation of DNA damage pathways and cell cycle arrest in response to eviction of nuclear isoform c. GSEA also revealed negative regulation of gene sets involved in cell motility, cellular differentiation and growth pathways in BL cells lacking nuclear isoform c expression. These results suggest that NErbB-2 function modulates tumor growth and promotes a metastatic phenotype in TNBC. Furthermore, our clinical findings identified NErbB-2 as an independent predictor of shorter OS (HR 2.54; 95% CI 1.22-5.28; P = 0.013), DFS (HR 2.91; 95% CI 1.44-5.87; P = 0.003), and DMFS (HR 2.59; 95% CI 1.20-5.60; P = 0.015) in 99 TN primary tumors. Our discoveries challenge the present scenario of drug development for personalized BC medicine that focuses on wild-type proteins, which conserve the canonical domains and are located in their classical cellular compartments, highlighting the potential of NErbB-2 isoforms as novel therapeutic targets and clinical biomarkers in TNBC.

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

Endometrial cancer (EC) is the second most common gynecological cancer worldwide. Myometrial invasion (MI) is a key event in EC dissemination. This study aimed to evaluate FXYD5/dysadherin (FXYD5/Dys) expression in EC tissue and uterine aspirate (UA) biopsies and to assess molecular/functional changes associated with its expression in cellular models. Fil: Besso, María José. 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: Rosso, Marina. 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: Lapyckyj, Lara. 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: Moiola, Cristian Pablo. Universidad Autonoma de Barcelona. Hospital Vall D' Hebron. Instituto de Investigación Vall D'hebron; España Fil: Matos, María Laura. 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: 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: Reventos, Jaume. Universidad Autonoma de Barcelona. Hospital Vall D' Hebron. Instituto de Investigación Vall D'hebron; España Fil: Colas, Eva. Universidad Autonoma de Barcelona. Hospital Vall D' Hebron. Instituto de Investigación Vall D'hebron; España Fil: Gil Moreno, Antonio. Universidad Autonoma de Barcelona. Hospital Vall D' Hebron. Instituto de Investigación Vall D'hebron; España Fil: Wernicke, Alejandra. Hospital Italiano; Argentina Fil: Orti, Roberto. Hospital Italiano; Argentina Fil: Vazquez, Monica Hebe. 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

Triple negative breast cancer (TNBC) refers to a subtype of tumors with poor prognosis, devoid of hormone receptors and of membrane overexpression or gene amplification of ErbB-2. Due to its molecular heterogeneity, TNBC represents a major clinical challenge. In this regard, clinical biomarkers and targeted therapies remain elusive, and chemotherapy has been the standard of care for early and metastatic TNBC. ErbB-2, a member of the ErbB family of tyrosine kinase receptors, is a major player in the BC scenario. While it is a cell membrane-bound receptor, it migrates to the nucleus (NErbB-2) where it acts as a transcription factor or coactivator. We recently found that both the canonical (wild-type, WT) ErbB-2 and the alternative isoform c are located in the nucleus of TNBC, a scenario with an aggressive oncogenic potential. The route of intracellular transport from the plasma membrane to the trans Golgi network (TGN) and the endoplasmic reticulum (ER) is termed retrograde trafficking, and constitutes the pathway by which ErbB-2 migrates to the nucleus. The retrograde transport route is also hijacked by toxins and viruses to access the ER and exert their deleterious effects. Retro-2, a small molecule inhibitor, was shown to protect cells from toxin and virus effects by blocking their retrograde trafficking. Given the high levels of NErbB-2 in TNBC cells, we explored whether treatment with Retro-2 modulates localization of ErbB-2 and proliferation in TNBC. We found that Retro-2 treatment decreased the levels of both WT ErbB-2 and isoform c in the nucleus of TNBC cells demonstrating that Retro-2 effects are not limited to a particular ErbB-2 isoform. Indeed, immunofluorescence assays revealed accumulation of ErbB-2 in the Golgi after Retro-2 treatment further preventing its sorting to the ER. We previously demonstrated that growth factors induce ErbB-2 migration into the nucleus in ErbB-2-positive BC cells. Consistently, we observed that Retro-2 prevents growth factor-induced NErbB-2 in ErbB-2-positive BC cells. Retro-2 treatment resulted in a dose-dependent decrease in cell proliferation in a panel of TNBC cells, whilst did not inhibit cell proliferation in the ErbB-2-negative MCF10A normal breast cell line. Moreover, disruption of retrograde transport by Retro-2 decreased the expression of cell cycle related NErbB-2 target genes (i.e. Erk5 and cyclin D1) therefore inducing cell cycle arrest at the G0/G1 phase. Most importantly, Retro-2 excluded ErbB-2 from the nucleus and abrogated tumor growth in preclinical models of TNBC. Collectively, our findings reveal Retro-2, a non-toxic inhibitor of the retrograde transport route, as a candidate novel therapeutic agent for TNBC based on its ability to evict ErbB-2 from the nucleus and to abrogate TNBC growth.

Triple negative breast cancer (TNBC) has poor prognosis and neither established biomarkers nor therapeutic targets. On the one hand the androgen receptor (AR), a steroid hormone receptor (SR) which is expressed in 10-53% of TNBC and proved to be critical for BC proliferation, has been proposed as a new target in TNBC. On the other hand, we and others have shown that membrane ErbB-2 migrates to the nucleus (nuclear ErbB-2, NErbB-2) where it binds DNA at HER-2 associated sequences (HAS) to regulate BC proliferation and migration. Since we have previously shown a functional interplay between growth factors and SR signaling pathways in BC, we propose the existence of an interaction between AR and ErbB-2 which is involved in NErbB-2+/AR+ BC growth. The experimental model used was the human TNBC cell line MDA-MB-453 which displays high expression levels of AR and NErbB-2. By Western Blot (WB) we found that dihydrotestosterone (DHT) treatment for short times (minutes) did not regulate ErbB-2 phosphorylation status at residues Tyr1221/1222 and 1248 which were constitutively activated. However, DHT led to an increase in ErbB-2 phosphorylation at residue Tyr877 which we have proved to be required for ErbB-2 nuclear migration. The latter effect was blocked by the AR antagonist enzalutamide (enza). Blockage of Src activity with dasatinib inhibited DHT-induced ErbB-2 phosphorylation at Tyr877. By Immunofluorescence and confocal microscopy analyses and subcellular fractionation studies we demonstrated that DHT induced ErbB-2 nuclear migration which was inhibited by enza. By chIP we found that DHT induced ErbB-2 recruitment to a HAS site in ERK5, a gene involved in BC proliferation, and to a HAS site in FKBP5, a classical AR responsive gene. By WB we demonstrated that transfection with an ErbB-2 mutant which is unable to translocate to the nucleus and functions as a dominant negative inhibitor of ErbB-2 nuclear migration (hErbB-2ΔNLS), inhibited FKBP51 up-regulation by DHT. Finally, by microarray and bioinformatics analysis we identified 315 differentially expressed genes (DEGs) in the presence of DHT and NErbB-2 eviction. Enrichment analyses showed that the DEGs belonged to the immune response and interferon pathways. Kaplan-Meier analysis revealed that the expression of 6 genes was significantly associated with overall survival in TNBC patients from the METABRIC cohort: CXCL10, TAP1, STAT1, NMI, HLA-A and NLRC5. Multivariate Cox regression analysis identified the combined expression of the 6 genes as an independent predictor of better clinical outcome in TNBC (HR: 0.56, 95% CI 0.38-0.82, P = 0.003). In conclusion, our findings evidence that DHT-activated AR induces Src-mediated ErbB-2 rapid activation and its migration to the nucleus where it binds to HAS sites in the DNA. Moreover, based on the DEGs of NErbB-2 eviction in presence of DHT we identified a gene signature associated with favorable outcome in TNBC.

Lapatinib (LAP), a dual EGFR/HER2 tyrosine kinase inhibitor, is used as second-line therapy in women with HER2+ breast cancer (BC), but less than 25% of the patients achieve an objective response. An alternative therapeutic approach is needed to overcome LAP resistance in women with metastatic HER2+ BC, particularly in patients with central nervous system (CNS) metastasis where large biological molecules are not effective. Previously, we reported that women with HER2+ tumors that express transmembrane glycoprotein mucin 4 (MUC4) have worse survival, and that HER2+/MUC4+ cell lines resistant to trastuzumab (T) express higher levels of tumor necrosis factor α (TNF) than T-sensitive cell lines. In addition, we proved that inhibition of soluble TNF (sTNF) decreases the expression of MUC4 and reverses T resistance. The aim of this work is to evaluate the participation of sTNF and transmembrane TNF (tmTNF) in LAP resistance in vivo and in the anti-tumor innate immune response. We used the LAP-resistant human BC cell line JIMT-1 and compared etanercept (E), a fusion protein that non-selectively blocks both sTNF and tmTNF, with the dominant negative-TNF protein INB03 (DN), that neutralizes sTNF without affecting tmTNF. Nude mice bearing JIMT-1 tumors (~50 mm3), received LAP (100 mg/kg) daily by oral gavage and IgG (5 mg/kg), E (5 mg/kg), DN (10 mg/kg), LAP+E or LAP+DN, twice a week i.p. Tumor volume was monitored routinely. At the end of the experiment, tumor-infiltrating immune cells were evaluated by immunofluorescence and analyzed by flow cytometry. DN or E treatments did not exhibit any anti-tumor effect alone, but in combination with LAP (LAP+DN and LAP+E) tumor growth decreased in a 54% and 34% vs. IgG, respectively (p Citation Format: Sofia Bruni, Florencia L. Mauro, Mara De Martino, Agustina Roldán Deamicis, María F. Mercogliano, Patricia V. Elizalde, Roxana Schillaci. Soluble TNFα blockade overcomes lapatinib resistance and induces an innate immune response in HER2-positive breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1913.

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. 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: Béguelin, W.. 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: 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: 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: Galigniana, Natalia Maricel. 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: Tkach, Mercedes. 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, P.. Universidad de La Frontera; Chile Fil: Roa, J.C.. Universidad de La Frontera; Chile Fil: O'Brien, N.A.. David Geffen School of Medicine at UCLA; Estados Unidos 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

Stat3 is a signaling node for multiple oncogenic pathways and is therefore frequently active in breast cancer. As experimental and clinical evidence reveals that progestins are key players in controlling mammary gland tumorigenesis, we studied Stat3 participation in this event. We have previously shown that progestins induce Stat3Tyr705 phosphorylation and its transcriptional activation in breast cancer cells. In this study, we demonstrate that progestins also induce Stat3 phosphorylation at Ser727 residue, which occurs via activation of c-Src/p42/p44 MAPK pathways in murine progestin-dependent C4HD cells and in T-47D cells. Expression of a Stat3S727A vector, which carries a serine-to-alanine substitution at codon 727, shows that Stat3Ser727 phosphorylation is required for full transcriptional activation of cyclin D1 gene expression by progestins and for in vivo Stat3 recruitment on cyclin D1 promoter. Transfection of Stat3S727A in murine and human breast cancer cells abolished progestin-induced in vitro and in vivo growth. Moreover, we found a positive correlation between progesterone receptor expression and nuclear localization of Stat3Ser727 phosphorylation in breast cancer biopsies. These data highlight Stat3 phosphorylation in Ser727 residue as a nongenomic action by progestins, necessary to promote breast cancer growth. Fil: Tkach, Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Rosemblit, Cinthia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Rivas, Martin Alfredo. Vall d; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Proietti Anastasi, Cecilia Jazmín. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Díaz Flaqué, María Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Mercogliano, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Beguelin, Wendy. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Maronna, Esteban. Sanatorio Mater Dei; Argentina Fil: Guzman, Pablo. Universidad de la Frontera. Facultad de Medicina; Chile Fil: Gercovich, Felipe G.. Instituto Oncológico Henry Moore; Argentina Fil: Gil Deza, Ernesto. Instituto Oncológico Henry Moore; Argentina Fil: Elizalde, Patricia Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Schillaci, Roxana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina

Approximately 15-20% of breast cancers (BC) show either membrane overexpression of ErbB-2 (MErbB-2), a member of the ErbBs family of receptor tyrosine kinases, or ERBB2 gene amplification. Until the development of MErbB-2-targeted therapies, this BC subtype, called ErbB-2-positive, was associated with increased metastatic potential and poor prognosis. Although these therapies have significantly improved overall survival and cure rates, resistance to available drugs is still a major clinical issue. In its classical mechanism, MErbB-2 activates downstream signaling cascades, which transduce its effects in BC. The fact that ErbB-2 is also present in the nucleus of BC cells was discovered over twenty years ago. Also, compelling evidence revealed a non-canonical function of nuclear ErbB-2 as a transcriptional regulator. As a deeper understanding of nuclear ErbB-2 actions would be crucial to the disclosure of its role as a biomarker and a target of therapy in BC, we will here review its function in BC, in particular, its role in growth, metastatic spreading and response to currently available MErbB-2-positive BC therapies. 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: 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: 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: 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

The incidence of metabolic disorders including obesity, type 2 diabetes and metabolic syndrome have seriously increased in the last decades. These diseases - with growing impact in modern societies - constitute major risk factors for neurodegenerative disorders such as Alzheimer's disease (AD), sharing insulin resistance, inflammation and associated cognitive impairment. However, cerebral cellular and molecular pathways involved are not yet clearly understood. Thus, our aim was to study the impact of a non-severe high fat diet (HFD) that resembles western-like alimentary habits, particularly involving juvenile stages where the brain physiology and connectivity are in plain maturation. To this end, one-month-old C57BL/6J male mice were given either a control diet or HFD during 4 months. Exposure to HFD produced metabolic alterations along with changes in behavioral and central parameters, in the absence of obesity. Two-month-old HFD mice showed increased glycemia and plasmatic IL1β but these values normalized at the end of the HFD protocol at 5 months of age, probably representing an acute response that is compensated at later stages. After four months of HFD exposure, mice presented dyslipidemia, increased Lipoprotein-associated phospholipase A2 (Lp-PLA2) activity, hepatic insulin resistance and inflammation. Alterations in the behavioral profile of the HFD group were shown by the impediment in nest building behavior, deficiencies in short and mid-term spatial memories, anxious and depressive- like behavior. Regarding the latter disruptions in emotional processing, we found an increased neural activity in the amygdala, shown by a greater number of c-Fos+ nuclei. We found that hippocampal adult neurogenesis was decreased in HFD mice, showing diminished cell proliferation measured as Ki67+ cells and neuronal differentiation in SGZ by doublecortin labeling. These phenomena were accompanied by a neuroinflammatory and insulin-resistant state in the hippocampus, depicted by a reactive phenotype in Iba1+ microglia cells (increased in number and soma size) and an impaired response to insulin given by decreased phosphorylated Akt levels and increased levels of inhibitory phosphorylation of IRS1. Our data portray a set of alterations in behavioral and neural parameters as a consequence of an early-life exposure to a quite moderate high fat diet, many of which can resemble AD-related features. These results highly emphasize the need to study how metabolic and neurodegenerative disorders are interrelated in deep, thus allowing the finding of successful preventive and therapeutic approaches. Fil: Vinuesa, María Angeles. 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. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; Argentina Fil: Pomilio, Carlos Javier. 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. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: Menafra, Martín. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina Fil: Bonaventura, Maria Marta. 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: Garay, Laura 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: 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: Lux, Victoria Adela R.. 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: Brites, Fernando Daniel. Universidad Nacional de Rosario. Facultad de Cs.bioquímicas y Farmaceuticas. Departamento de Bioquímica Clinica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Beauquis, Juan. 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. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: Saravia, Flavia Eugenia. 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. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina