RESUMO
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 receptor tyrosine kinase ErbB-2/HER2. Due to TNBC heterogeneity, clinical biomarkers and targeted therapies for this disease remain elusive. We demonstrated that ErbB-2 is 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). Here, we revealed that the inhibitors of the retrograde transport Retro-2 and its cyclic derivative Retro-2.1 evict both WTErbB-2 and ErbB-2c from the nucleus of BC cells and tumors. Using BC cells from several molecular subtypes, as well as normal breast cells, we demonstrated that Retro-2 specifically blocks proliferation of BC cells expressing NErbB-2. Importantly, Retro-2 eviction of both ErbB-2 isoforms from the nucleus resulted in a striking growth abrogation in multiple TNBC preclinical models, including tumor explants and xenografts. Our mechanistic studies in TNBC cells revealed that Retro-2 induces a differential accumulation of WTErbB-2 at the early endosomes and the plasma membrane, and of ErbB-2c at the Golgi, shedding new light both on Retro-2 action on endogenous protein cargoes undergoing retrograde transport, and on the biology of ErbB-2 splicing variants. In addition, we revealed that the presence of a functional signal peptide and a nuclear export signal (NES), both located at the N-terminus of WTErbB-2, and absent in ErbB-2c, accounts for the differential subcellular distribution of ErbB-2 isoforms upon Retro-2 treatment. Our present discoveries provide evidence for the rational repurposing of Retro-2 as a novel therapeutic agent for TNBC.
Assuntos
Neoplasias de Mama Triplo Negativas , Núcleo Celular/metabolismo , Humanos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptor ErbB-2/metabolismo , Receptores de Progesterona/metabolismo , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
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.
Assuntos
Receptor ErbB-2/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/enzimologia , Núcleo Celular/metabolismo , Proliferação de Células/fisiologia , Feminino , Humanos , Proteína Quinase 7 Ativada por Mitógeno/biossíntese , Proteína Quinase 7 Ativada por Mitógeno/genética , Inclusão em Parafina , Isoformas de Proteínas , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/biossíntese , Receptor ErbB-2/genética , Neoplasias de Mama Triplo Negativas/enzimologia , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Stat3 is constitutively activated in several tumor types and plays an essential role in maintaining their malignant phenotype and immunosupression. To take advantage of the promising antitumor activity of Stat3 targeting, it is vital to understand the mechanism by which Stat3 regulates both cell autonomous and non-autonomous processes. Here, we demonstrated that turning off Stat3 constitutive activation in different cancer cell types induces senescence, thus revealing their Stat3 addiction. Taking advantage of the senescence-associated secretory phenotype (SASP) induced by Stat3 silencing (SASP-siStat3), we designed an immunotherapy. The administration of SASP-siStat3 immunotherapy induced a strong inhibition of triple-negative breast cancer and melanoma growth associated with activation of CD4 + T and NK cells. Combining this immunotherapy with anti-PD-1 antibody resulted in survival improvement in mice bearing melanoma. The characterization of the SASP components revealed that type I IFN-related mediators, triggered by the activation of the cyclic GMP-AMP synthase DNA sensing pathway, are important for its immunosurveillance activity. Overall, our findings provided evidence that administration of SASP-siStat3 or low dose of Stat3-blocking agents would benefit patients with Stat3-addicted tumors to unleash an antitumor immune response and to improve the effectiveness of immune checkpoint inhibitors.
Assuntos
Melanoma , Neoplasias de Mama Triplo Negativas , Animais , Senescência Celular , Humanos , Imunoterapia , Camundongos , Vício Oncogênico , Fator de Transcrição STAT3/genéticaRESUMO
BACKGROUND: 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. METHODS: We retrospectively studied 86 HER2-positive breast cancer patients treated with trastuzumab and chemotherapy in the adjuvant setting. We explored the association of the IMPC component with clinicopathological parameters at diagnosis and its prognostic value. We compared MUC4 expression in IMPC with respect to other histological breast cancer subtypes by immunohistochemistry. RESULTS: IMPC, either as a pure entity or associated with invasive ductal carcinoma (IDC), was present in 18.6% of HER2-positive cases. It was positively correlated with estrogen receptor expression and tumor size and inversely correlated with patient's age. Disease-free survival was significantly lower in patients with IMPC (hazard ratio = 2.6; 95%, confidence interval 1.1-6.1, P = 0.0340). MUC4, a glycoprotein associated with metastasis, was strongly expressed in all IMPC cases tested. IMPC appeared as the histological breast cancer subtype with the highest MUC4 expression compared to IDC, lobular and mucinous carcinoma. CONCLUSION: In HER2-positive breast cancer, the presence of IMPC should be carefully examined. As it is often not informed, because it is relatively difficult to identify or altogether overlooked, we propose MUC4 expression as a useful biomarker to highlight IMPC presence. Patients with MUC4-positive tumors with IMPC component should be more frequently monitored and/or receive additional therapies.
Assuntos
Neoplasias da Mama/mortalidade , Carcinoma Ductal de Mama/mortalidade , Carcinoma Papilar/mortalidade , Mucina-4/metabolismo , Receptor ErbB-2/metabolismo , Trastuzumab/farmacologia , Adulto , Idoso , Antineoplásicos Imunológicos , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/tratamento farmacológico , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patologia , Carcinoma Papilar/tratamento farmacológico , Carcinoma Papilar/metabolismo , Carcinoma Papilar/patologia , Estudos de Casos e Controles , Quimioterapia Adjuvante , Feminino , Seguimentos , Humanos , Pessoa de Meia-Idade , Invasividade Neoplásica , Prognóstico , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/imunologia , Estudos Retrospectivos , Taxa de SobrevidaRESUMO
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.