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Purpose:Gasdermin B (GSDMB) overexpression/amplification occurs in about 60% of HER2 breast cancers, where it promotes cell migration, resistance to anti-HER2 therapies, and poor clinical outcome. Thus, we tackle GSDMB cytoplasmic overexpression as a new therapeutic target in HER2 breast cancers.Experimental Design:We have developed a new targeted nanomedicine based on hyaluronic acid–biocompatible nanocapsules, which allow the intracellular delivery of a specific anti-GSDMB antibody into HER2 breast cancer cells both in vitro and in vivo.Results:Using different models of HER2 breast cancer cells, we show that anti-GSDMB antibody loaded to nanocapsules has significant and specific effects on GSDMB-overexpressing cancer cells' behavior in ways such as (i) lowering the in vitro cell migration induced by GSDMB; (ii) enhancing the sensitivity to trastuzumab; (iii) reducing tumor growth by increasing apoptotic rate in orthotopic breast cancer xenografts; and (iv) diminishing lung metastasis in MDA-MB-231-HER2 cells in vivo. Moreover, at a mechanistic level, we have shown that AbGB increases GSDMB binding to sulfatides and consequently decreases migratory cell behavior and may upregulate the potential intrinsic procell death activity of GSDMB.Conclusions:Our findings portray the first evidence of the effectiveness and specificity of an antibody-based nanomedicine that targets an intracellular oncoprotein. We have proved that intracellular-delivered anti-GSDMB reduces diverse protumor GSDMB functions (migration, metastasis, and resistance to therapy) in an efficient and specific way, thus providing a new targeted therapeutic strategy in aggressive HER2 cancers with poor prognosis.

Supplementary Methods, References and Table 1

Supplementary Figures S1-S10

© 2021 The Authors., The involvement of the Gasdermin (GSDM) protein family in cancer and other pathologies is one of the hottest topics in biomedical research. There are six GSDMs in humans (GSDMA, B, C, D, GSDME/DFNA5 and PJVK/DFNB59) and, except PJVK, they can trigger cell death mostly by pyroptosis (a form of lytic and pro-inflammatory cell death) but also other mechanisms. The exact role of GSDMs in cancer is intricate, since depending on the biological context, these proteins have diverse cell-death dependent and independent functions, exhibit either pro-tumor or anti-tumor functions, and promote either sensitization or resistance to oncologic treatments. In this review we provide a comprehensive overview on the multifaceted roles of the GSDMs in cancer, and we critically discuss the possibilities of exploiting GSDM functions as determinants of anti-cancer treatment and as novel therapeutic targets, with special emphasis on innovative GSDM-directed nano-therapies. Finally, we discuss the issues to be resolved before GSDM-mediated oncologic therapies became a reality at the clinical level., This study was supported by the Ministerio de Ciencia e Innovación (PID2019-104644RB-I00 -GMB-), the Instituto de Salud Carlos III (CIBERONC, CB16/12/00295 –GMB-; partly supported by FEDER funds) and by the Fundación Científica de la AECC (FC_AECC PROYE19036MOR -GMB-).

PurposeGasdermin B (GSDMB) over-expression promotes poor prognosis and aggressive behavior in HER2 breast cancer by increasing cell invasion, metastasis and resistance to therapy. Decoding the molecular mechanism of GSDMB-mediated drug resistance is crucial to identify novel effective targeted treatments for HER2/GSDMB aggressive tumors.Experiment designTo decipher the functional relevance of GSDMB in promoting resistance to HER2-targeted therapies we performed several molecular approaches (immunoblot, qRT-PCR, flow cytometry, immunoprecipitation and confocal microscopy) in different breast and gastric carcinoma cell models. The results were confirmed in Patient Derived Xenografts (PDX) by qRT-PCR and in clinical human cancer samples by immunohistochemistry. Finally, we validated the efficacy of the identified targeted treatment in HER2/GSDMB cancers using two complementary in vivo preclinical models (tumor xenografts in mice and zebrafish).ResultsWe discovered that GSDMB up-regulation renders HER2 breast and gastric cancer cells more resistant to anti-HER2 agents by promoting protective autophagy. Consistent with this, we proved that the combination of lapatinib with the autophagy inhibitor chloroquine increases the therapeutic response specifically in GSDMB-positive tumors in vitro and in vivo using zebrafish and mice preclinical cancer models. Mechanistically, we confirmed that the GSDMB N-terminal domain interacts with the autophagy protein LC3B. Finally, we validated these results in clinical samples of breast and gastric cancers, where GSDMB/LC3B co-expression associates significantly with relapse.ConclusionOur findings uncovered a novel functional link between GSDMB over-expression and LC3B-mediated protective autophagy in response to HER2-targeted therapies and provide a new and accessible therapeutic approach for HER2/GSDMB+ cancers with adverse clinical outcome.TRANSLATIONAL RELEVANCEIdentifying the biomarkers and mechanisms of therapy resistance is a main challenge in current oncology. In this regard, Gasdermin-B (GSDMB) over-expression, which was initially found in >60% HER2 breast cancers, promotes resistance to therapy through an unknown molecular mechanism. In the present work, we revealed for the first time that in HER2 gastric and breast cancers GSDMB mediates innate and acquired resistance to HER2-targeted drugs through the promotion of a pro-survival autophagy mechanism that requires the interaction of GSDMB with LC3B. Accordingly, GSDMB/LC3B co-expression in human breast and gastric cancer clinical samples associates with relapse. To reverse this anti-drug effect, we developed a therapeutic approach based on the combination of the autophagy inhibitor chloroquine with lapatinib that showed significant efficacy both in vitro and in vivo on GSDMB-positive tumors. Our findings provide an accessible (FDA-approved drugs) therapeutic combination to treat effectively HER2/GSDMB over-expressing tumors with poor clinical outcome.

© 2019 American Association for Cancer Research., [Purpose]: Gasdermin B (GSDMB) overexpression/amplification occurs in about 60% of HER2 breast cancers, where it promotes cell migration, resistance to anti-HER2 therapies, and poor clinical outcome. Thus, we tackle GSDMB cytoplasmic overexpression as a new therapeutic target in HER2 breast cancers. Experimental Design: We have developed a new targeted nanomedicine based on hyaluronic acid–biocompatible nanocapsules, which allow the intracellular delivery of a specific anti-GSDMB antibody into HER2 breast cancer cells both in vitro and in vivo., [Results]: Using different models of HER2 breast cancer cells, we show that anti-GSDMB antibody loaded to nanocapsules has significant and specific effects on GSDMB-overexpressing cancer cells' behavior in ways such as (i) lowering the in vitro cell migration induced by GSDMB; (ii) enhancing the sensitivity to trastuzumab; (iii) reducing tumor growth by increasing apoptotic rate in orthotopic breast cancer xenografts; and (iv) diminishing lung metastasis in MDA-MB-231-HER2 cells in vivo. Moreover, at a mechanistic level, we have shown that AbGB increases GSDMB binding to sulfatides and consequently decreases migratory cell behavior and may upregulate the potential intrinsic procell death activity of GSDMB., [Conclusions]: Our findings portray the first evidence of the effectiveness and specificity of an antibody-based nanomedicine that targets an intracellular oncoprotein. We have proved that intracellular-delivered anti-GSDMB reduces diverse protumor GSDMB functions (migration, metastasis, and resistance to therapy) in an efficient and specific way, thus providing a new targeted therapeutic strategy in aggressive HER2 cancers with poor prognosis., This work was supported by grants from the Instituto de Salud Carlos III (ISCIII) and FEDER (PI16/00134), CIBERONC (CB16/12/00295), and the AECC (Grupos Estables de Investigacion 2018-AECC) to G. Moreno-Bueno. D. Torres and M.J. Alonso received support from the Xunta de Galicia (Competitive Reference Groups: ED431C 2017/09). J. Palacios received support from CIBERONC (CB16/12/00316). A. Molina-Crespo and D. Sarrio are funded by CIBERONC (CB16/12/00295). A. Cadete was funded in the Nanofar program by the European Commission (EACEA), an Erasmus Mundus Joint Doctorate (EMJD). A. Olivera was funded by grant BES-2015-071236 (Spanish Ministry of Science, Innovation and Universities). A. Gonella was funded by Marie Sklodowska - Curie Grant 642028 (NABBA).

© The Author(s) 2019., [Background]: Lack of effective tumor-specific delivery systems remains an unmet clinical challenge for successful translation of innovative therapies, such as, therapeutic oligonucleotides. In the past decade, exosomes have been suggested to be ideal drug delivery systems with application in a broad range of pathologies including cancer, due to their organotropic properties. Tumor-derived exosomes, having tumor-homing properties, can efficiently reach cancer cells and therefore behave as carriers for improved drug delivery to the primary tumor and metastases. However, due to their complex composition, and still undefined biological functions, safety concerns arise hampering their translation to the clinics., [Results]: We propose here the development of exosome-mimetic nanosystems (EMNs) that simulate natural tumor-derived exosomes with respect to their structure and functionality, but with a controlled composition, for the targeted delivery of therapeutic oligonucleotides to lung adenocarcinoma cells (microRNA-145 mimics). Making use of the well-known liposome technology, EMNs can be engineered, loaded with the therapeutic compounds, and tailored with specific proteins (integrin α6β4) providing them organotropic properties. EMNs show great similarities to natural exosomes with respect to their physicochemical properties, drug loading capacity, and ability to interact with the cancer target cells in vitro and in vivo, but are easier to manufacture, can be produced at high yields, and are safer by definition., [Conclusions]: We have designed a multifunctional nanoplatform mimicking exosomes, EMNs, and proved their potential to reach cancer cells with a similar efficient that tumor-derived exosomes but providing important advantages in terms of production methodology and regulations. Additionally, EMNs are highly versatile systems that can be tunable for a broader range of applications., This work was in part supported by grants from Instituto de Salud Carlos III (ISCIII) and European Regional Development Fund (FEDER) (CP12/03150, PI15/00828, and CB16/12/00328 from M.F. and PI16/0014, CIBERONC CB16/12/00295 from G.M.B). A.V.R. also acknowledges the fnancial support from the Spanish Ministry of Education, Culture, and Sport (FPU15/06595).

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al., Gasdermin B (GSDMB) belongs to the Gasdermin protein family that comprises four members (GSDMA-D). Gasdermin B expression has been detected in some tumor types such as hepatocarcinomas, gastric and cervix cancers; and its overexpression has been related to tumor progression. At least four splicing isoforms of GSDMB have been identified, which may play differential roles in cancer. However, the implication of GSDMB in carcinogenesis and tumor progression is not well understood. Here, we uncover for the first time the functional implication of GSDMB in breast cancer. Our data shows that high levels of GSDMB expression is correlated with reduced survival and increased metastasis in breast cancer patients included in an expression dataset (>1,000 cases). We demonstrate that GSDMB is upregulated in breast carcinomas compared to normal breast tissue, being the isoform 2 (GSDMB-2) the most differentially expressed. In order to evaluate the functional role of GSDMB in breast cancer two GSDMB isoforms were studied (GSDMB-1 and GSDMB-2). The overexpression of both isoforms in the MCF7 breast carcinoma cell line promotes cell motility and invasion, while its silencing in HCC1954 breast carcinoma cells decreases the migratory and invasive phenotype. Importantly, we demonstrate that both isoforms have a differential role on the activation of Rac-1 and Cdc-42 Rho-GTPases. Moreover, our data support that GSMDB-2 induces a pro-tumorigenic and pro-metastatic behavior in mouse xenograft models as compared to GSDMB-1. Finally, we observed that although both GSDMB isoforms interact in vitro with the chaperone Hsp90, only the GSDMB-2 isoform relies on this chaperone for its stability. Taken together, our results provide for the first time evidences that GSDMB-2 induces invasion, tumor progression and metastasis in MCF7 cells and that GSDMB can be considered as a new potential prognostic marker in breast cancer., This work was supported by grants from the Spanish Ministry of Science and Innovation, MICINN (SAF2007-63075 and SAF2010-20175), AVON Foundation 2012, Comunidad de Madrid (S2010/BMD-2302), AECC network 2011, Instituto de Salud Carlos III (ISCIII) (PI13_00132) to GMB and Breast Network from ISCIII RD12036/0007 to AC. MHR has been funded by a predoctoral contract associated to SAF2007-63075 and now has a postdoc contract from S2010/BMD-23. DS and PGS are funded by postdoc contracts from the AECC Scientific Foundation, AM is funded by a predoctoral fellowship from MECD; ACM is funded by ISCIII RD12036/0007. Dr HP’s work is supported by the Melanoma Research Alliance, Pediatric Oncology Experimental Therapeutics Investigators Consortium, The Nancy C. and Daniel P. Paduano Foundation, The Manning Foundation, NCI (U01 CA169538, RO1 CA169416-01) and the DoD (BC123187, BC121988).