Gámez‑Chiachio et al. J Exp Clin Cancer Res (2022) 41:285 https://doi.org/10.1186/s13046-022-02497-w RESEARCH Open Access Gasdermin B over‑expression modulates HER2‑targeted therapy resistance by inducing protective autophagy through Rab7 activation Manuel Gámez‑Chiachio1,2†, Ángela Molina‑Crespo1†, Carmen Ramos‑Nebot1,2, Jeannette Martinez‑Val3, Lidia Martinez1, Katja Gassner4, Francisco J. Llobet4, Mario Soriano5, Alberto Hernandez6, Marco Cordani1, Cristina Bernadó‑Morales2,7,8, Eva Diaz9, Alejandro Rojo‑Sebastian9, Juan Carlos Triviño10, Laura Sanchez4, Ruth Rodríguez‑Barrueco11, Joaquín Arribas2,7,12,13, David Llobet‑Navás4, David Sarrió1,2* and Gema Moreno‑Bueno1,2,9* Abstract Background: Gasdermin B (GSDMB) over-expression promotes poor prognosis and aggressive behavior in HER2 breast cancer by increasing 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. Methods: Different in vitro approaches (immunoblot, qRT-PCR, flow cytometry, proteomic analysis, immunopre‑ cipitation, and confocal/electron microscopy) were performed in HER2 breast and gastroesophageal carcinoma cell models. Results were then validated using in vivo preclinical animal models and analyzing human breast and gastric cancer samples. Results: GSDMB up-regulation renders HER2 cancer cells more resistant to anti-HER2 agents by promoting protec‑ tive autophagy. Accordingly, the combination of lapatinib with the autophagy inhibitor chloroquine increases the therapeutic response of GSDMB-positive cancers in vitro and in zebrafish and mice tumor xenograft in vivo models. Mechanistically, GSDMB N-terminal domain interacts with the key components of the autophagy machinery LC3B and Rab7, facilitating the Rab7 activation during pro-survival autophagy in response to anti-HER2 therapies. Finally, we validated these results in clinical samples where GSDMB/Rab7/LC3B co-expression associates significantly with relapse in HER2 breast and gastric cancers. Conclusion: Our findings uncover for the first time a functional link between GSDMB over-expression and protective autophagy in response to HER2-targeted therapies. GSDMB behaves like an autophagy adaptor and plays a pivotal role in modulating autophagosome maturation through Rab7 activation. Finally, our results provide a new and acces‑ sible therapeutic approach for HER2/GSDMB + cancers with adverse clinical outcome. † Manuel Gámez-Chiachio and Ángela Molina-Crespo equally contributed to this work. *Correspondence: dsarrio@iib.uam.es; gmoreno@iib.uam.es 1 Departamento de Bioquímica, Universidad Autónoma de Madrid (UAM) Instituto de Investigaciones Biomédicas ‘Alberto Sols’ (CSIC-UAM), IdiPAZ, C/ Arturo Duperier 4, 28029 Madrid, Spain Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Gámez‑Chiachio et al. J Exp Clin Cancer Res (2022) 41:285 Page 2 of 19 Keywords: Gasdermin B, Protective autophagy, Anti-HER2 therapy, Drug resistance, HER2 breast cancer, Gastroesophageal tumors, LC3B Background inducing cancer cell death in vivo [14]. Given that HER2 Gasdermin B (GSDMB) is one of the six Gasder- amplification/overexpression also occurs frequently min (GSDM) genes in the human genome (along with in gastroesophageal cancers [15], and considering that GSDMA, C, D, GSDME/DFNA5, and DFNB59/PJVK) some cancer patients can experience drug resistance to [1]. Recent data suggest that all GSDMs, under spe- diverse available anti-HER2-targeted therapies (anti- cific circumstances, can trigger a lytic and pro-inflam- bodies like trastuzumab, pertuzumab, T-DM1; tyros- matory cell death mechanism, known as pyroptosis [2, ine kinase inhibitors (TKI) such as lapatinib neratinib 3]. This pro-cell death function is usually auto-inhib- or tucatinib) [16], we proposed that identifying the ited through the intramolecular interaction of GSDM functional mechanisms by which GSDMB over-expres- N-terminal (NT) and C-terminal (CT) domains, but sion modulates the therapeutic response of different upon cleavage by specific caspases and other pro- HER2 tumors could have novel clinical utility. Indeed, teases, the released N-terminal domain forms mem- there is a growing necessity to discover the resistance brane pores that subsequently lead to cell lysis [2, 3]. In mechanisms of the FDA-approved anti-HER2 TKIs and the case of GSDMB, the pro-cell death function under therapeutic strategies to overcome it, since they have physiological conditions is a matter of intense debate advantageous characteristics (such as oral administra- [4–7]. Recently, it has been revealed that lymphocyte- tion, capability to reach CNS metastasis, multiple HER derived Granzyme-A can cleave GSDMB within tumor family targets, lower cardiotoxicity), and importantly cells, thus provoking a pyroptotic cancer cell death [8]. they are the therapeutic alternatives to the treatment of In this sense, an activated anti-tumor immune response anti-HER2 antibodies-refractory HER2 + breast cancer mediated by NK and T-cytotoxic cells could reduce patients [17]. Furthermore, diverse evidence showed tumor growth of murine colon and melanoma cells that there is some cross-resistance to these three TKIs, exogenously overexpressing GSDMB [8]. Moreover, thus being lapatinib resistant cells less sensitive to the GSDMB pore-forming activity can also have microbi- others and the other way round [17–19]. ocidal function during enterobacteria infection, point- To this end, here we demonstrate that GSDMB over- ing out a new role of GSDMB in host immunity [9]. expression mediates pro-survival autophagy after anti- However, GSDMB plays other diverse non-pyroptotic HER2 TKI treatment. While protective autophagy functions in inflammatory pathologies [10, 11] and can- has been linked to cancer resistance to diverse HER2- cer. In tumors, GSDMB is frequently over-expressed in targeted drugs [20] and autophagy inhibition can par- breast, liver, colon, cervical and gastric carcinomas, and tially abrogate this effect [21, 22], the precise molecular GSDMB upregulation can promote multiple pro-tumor mechanisms involved in this process are still unclear. functions [3]. Our data prove that the combination of anti-HER2 TKI In particular, GSDMB is frequently (> 60%) over- treatment with the autophagy inhibitor chloroquine expressed in HER2 breast cancer, mostly due to (CQ) increases the therapeutic response specially in GSDMB-HER2 co-amplification [12]. In these tumors, GSDMB-positive tumors in vitro and in vivo. In addi- GSDMB over-expression and/or amplification, asso- tion, we show that GSDMB increases pro-survival ciates significantly with poor prognosis and reduced autophagy through a cooperation of its N-terminal response to anti-HER2 standard therapy (trastuzumab) with the microtubule‑associated protein light chain independently of the hormone receptor status or his- 3B (LC3B), an essential modulator of the autophagic tological grade [12]. Moreover, GSDMB overexpres- machinery [23, 24] and Rab7, a small GTPase involved sion promotes cell motility, invasion, and metastasis in the autophagosome maturation [25, 26]. Overall, in breast cancer cell lines [13, 14], but it does not we have uncovered a novel functional link between affect cell proliferation [13]. Interestingly, our recent GSDMB over-expression, Rab7 and LC3B, where data showed that GSDMB over-expression is a novel GSDMB acts like an autophagy adaptor by inducing therapeutic target in HER2 breast tumors [14] since Rab7 activation and subsequently enhancing protec- the intracellular delivery of a GSDMB antibody using tive autophagy in response to HER2-targeted therapies. nanoparticles significantly reduces the tumor growth Besides, this work provides a new therapeutic approach and metastasis development in HER2 breast tumors by for HER2/GSDMB + cancers, characterized by poor clinical outcome. Gámez‑Chiachio et al. J Exp Clin Cancer Res (2022) 41:285 Page 3 of 19 Methods techniques were performed as described in Supplemen- Human tumor samples tary Material and Methods. The present study was carried out using the 31 availa- ble HER2 breast carcinoma series treated with adjuvant Animal in vivo studies regimen that was previously reported [12] and a novel All the experimental procedures with mice were intestinal gastric tumor cohort (n = 59). Gastric tumors approved by the internal ethical research and animal were acquired from the Biobank of the Anatomy Pathol- welfare committee (IDIBELL and IIB, UAM), and by the ogy Department (record number B.0000745, ISCIII Local Authorities (Generalitat Catalana, B-9900010 and National Biobank network) of the MD Anderson Cancer Comunidad de Madrid, PROEX 235.6/20, respectively). Center Madrid, Madrid, Spain. The mean patient age at They complied with the European Union (Directive diagnosis was 59.8 ± 12.7 years (range, 29 to 85 years) 2010/63/UE) and Spanish Government guidelines (Real and 24.2% of tumors were diagnosed in women. All Decreto 53/20133). Furthermore, zebrafish studies (AB tumors were high grade and 5 were stage I, 20 were stage strain, Danio rerio) were performed with the agreement II, and 34 stage III-IV (among HER2 gastric tumors, 11 of the Bioethics Committee for animal experimentation were stage II and 21 stage III-IV) according to the TNM of the University of Santiago of Compostela (CEEA-LU), staging system. HER2 staining was performed at the REGA code: ES270280346401. The detailed description diagnosis following the established protocols [27] and of the different in vivo experiments is provided in Sup- 31 were considered as HER2-positive. Immunohisto- plementary Material and Methods. chemical and clinical data of HER2 breast and gastric tumors are provided in Supplementary Table 1. This Statistics and reproducibility study was performed following standard ethical proce- The Chi-square contingency test with Yates’s correction, dures of the Spanish regulation (Ley de Investigación or Fisher’s exact test, was used to determine the statisti- Orgánica Biomédica, 14 July 2007) and was approved cal significance of the relationships between immunohis- by the ethic committees of the MD Anderson Cancer tochemical and clinico-pathological features. GraphPad Center Madrid, Madrid, Spain. Prism software was used for graphic representation and statistical analysis. Error bars represent the mean ± s.e.m of at least three independent experiments. Data were Cell culture and in vitro assays tested for normality, and paired sets of data were com- HCC1954 (derived from a human invasive ductal breast pared using unpaired Student’s t-test (two-tailed). carcinoma), NCI-N87 (human gastric adenocarci- noma) and HEK293T cell lines were obtained from the Results American Type Cell Culture (ATCC) and OE19 (human GSDMB upregulation in response to anti‑HER2 therapies esophageal adenocarcinoma) cell line from the Deutsche associates with drug resistance Sammlung von Mikroorganismen und Zellkulturen Our previous work proved that GSDMB over-expres- (DSMZ). Cells were cultured following the supplier sion is a maker of poor prognosis associated with tras- conditions. Cells were authenticated by STR-profiling tuzumab resistance in HER2 breast carcinoma patients according to ATCC or DSMZ guidelines. To generate in both neoadjuvant and adjuvant treatment settings lapatinib resistant (named LR) cell lines, both HCC1954 [12, 14]. Moreover, high levels of GSDMB decrease sen- and OE19 parental cells were cultured for up to 6 months sitivity to trastuzumab in vitro in HCC1954 and SKBR3 in the presence of increasing concentrations of lapatinib cells, and its expression increases during the acquisition from 0.2 μM up to 2 μM (HCC1954 cells, which corre- of trastuzumab resistance in HER2 + breast cancer PDX sponds to IC50) or 1.5 μM (OE19, IC70). Resistant sta- models [12]. Here, to address whether GSDMB also plays tus was analyzed by cell viability assays using AlamarBlue a role in the clinical behavior of gastric tumors, we first (Bio-Rad), according to the manufacturer’s protocol. observed that strong cytoplasmic and nuclear GSDMB Then, HCC1954 LR and OE19 LR cells were cultured staining (Fig. 1A and Supplementary Table 1) associ- in the continuous presence of 2 μM and 1.5 μM lapat- ated statistically with HER2 positive status (GSDMB inib, respectively. In parallel, HCC1954 and OE19 con- high expression in 18/31 (58.1%) of HER2-positive and trol cells (C) were generated by chronic treatment with 8/28 (29%) of HER2-negative tumors; p = 0.023). In the vehicle DMSO (same amount as the correspond- HER2 gastric carcinomas, alike HER2 breast tumors ing lapatinib-resistant cells). For the in vivo studies with [12], GSDMB over-expression associates with relapse mice, mCherry-luc transduced HCC1954 cells previously (p = 0.060, Supplementary Table 2), thus supporting the generated [14] were used. Cell viability assays, biochemi- relationship between high levels of GSDMB and poor cal studies, immunofluorescence, and confocal imaging prognosis in gastric tumors. Next, to test if in HER2 Gámez‑Chiachio et al. J Exp Clin Cancer Res (2022) 41:285 Page 4 of 19 breast and gastroesophageal cancers GSDMB is function- cells (chronically grown with high doses of the vehicle ally involved in regulating drug response/resistance also DMSO) (Fig. 1D). Interestingly, GSDMB upregulation to the HER2 tyrosine kinase inhibitor lapatinib, we used was dependent on lapatinib presence since drug removal three HER2 + cancer cell lines that endogenously express for 10 days restored GSDMB protein levels (Fig. 1D). GSDMB, HCC1954 (breast cancer), OE19 (esophageal) Moreover, as a complementary model, we used primary and NCI-N87 (gastric). First, we treated these cells for cultures from previously generated HER2 + breast cancer different time points (up to 72 h) with their correspond- PDXs [31]. Thereby, compared to the parental cell line ing IC50 of lapatinib (Fig. 1B-C and Supplementary (C), which is sensitive to trastuzumab, the in vitro resist- Fig. 1A). Additionally, for comparison, trastuzumab ant clones to trastuzumab (TRC1 and TRC2) or lapatinib treatment was carried out only in OE19 and NCI-N87 (LRC1 and LRC2) showed a significant GSDMB upreg- cells (Supplementary Fig. 1B-C) since HCC1954 cells are ulation (Fig. 1E). These in vitro results in various cell intrinsically highly resistant to this drug [14, 28]. Both models (breast and gastric cancer cell lines, breast PDX lapatinib (Fig. 1B-C and Supplementary Fig. 1A) and primary culture), together with our published observa- trastuzumab (Supplementary Fig. 1B-C) provoke a sharp tions in vivo with PDXs and HER2 breast carcinoma induction of GSDMB mRNA, which peaked at 24–48 h, patients [12], suggest that the GSDMB over-expression in all tested models. HER2 upregulation was also would induce resistance to trastuzumab as well as lapa- detected as previously reported [29]. At the protein level, tinib. Since TKIs are mainly utilized in trastuzumab while GSDMB was strongly upregulated by lapatinib in pretreated patients [17] (indeed HCC1954 cells are HCC1954 cells, the total amount of GSDMB protein was intrinsically resistant to trastuzumab [14, 28]) and there not clearly increased in gastroesophageal cancer cells is a need to understand the molecular mechanisms that (OE19 and N87) neither after lapatinib nor trastuzumab underlies the TKI derived resistance, we selected lapa- treatment. This is due to the appearance of a processed tinib as the suitable (second line) anti-HER2 therapy to form of GSDMB protein (p37) (Fig. 1B-C and Supple- further decipher the connection between GSDMB and mentary Fig. 1A-C) at the latest treatment time points, drug resistance. that corresponds to the previously identified C-terminal To study if GSDMB is functionally involved in the cleavage product generated by apoptotic caspases-3/6/7 early response and long-term resistance to lapatinib, we [30] (Supplementary Fig. 1D). GSDMB processing by silenced GSDMB in OE19 and HCC1954 cells by shRNA caspases-3/6/7 generates N- (p10) and C-terminal (p37) (two different sequences shGB1 and shGB2), as previ- fragments [30] that do not have an effect on cell death ously reported [13, 14]. These shRNAs robustly decrease induction [6]. GSDMB expression at the protein (Supplementary Next, to assess further if GSDMB induction correlates Fig. 1E) and mRNA levels (all four GSDMB isoforms that with acquired resistance to HER2-targeted therapies, we translate to protein) in OE19 (Supplementary Fig. 1F) and generated HCC1954 and OE19 cells long-term resistant HCC1954 cells [14], and do not target other GSDM genes (LR) to high doses (> IC50) of lapatinib. In both mod- (Supplementary Fig. 1G). Furthermore, in the resistant els, again, we observed an upregulation of GSDMB and HCC1954 LR and OE19 LR cells, GSDMB expression HER2, and as expected, a strong decrease in HER2 phos- was transiently reduced by two different GSDMB-specific phorylation [29], compared to their respective control siRNAs (siGB1/2, Fig. 1F). Stable GSDMB-silencing by (See figure on next page.) Fig. 1 GSDMB is induced in response to anti-HER2 therapies, and its silencing increases the sensitive to lapatinib treatment. A Representative immunofluorescence images of GSDMB and HER2 expression in GSDMB/HER2-positive gastric carcinomas. Scale bar, 20 µm. Nuclei were counterstained with DAPI. Representative pie chart of GSDMB expression statistics in HER2 + gastric carcinomas (see Supplementary Table 1 for extra clinical features and statistics). B-C Relative mRNA (left) and protein levels (right) of GSDMB and HER2 in HCC1954 (B) and OE19 (C) cells treated with IC50 of lapatinib (2 µM and 0.7 µM, respectively) at indicated time points. D GSDMB and HER2 protein levels in lapatinib resistant HCC1954 and OE19 cells (LR) and their corresponding control cells (C) treated chronically with lapatinib and DMSO, respectively, and after ten days of drug removal. Study of the different cytotoxic effect of the chronic lapatinib treatment in these cells was analyzed by cell viability assays. E Relative mRNA levels of GSDMB and HER2 in trastuzumab (TRC1 and TRC2) and lapatinib (LRC1 and LRC1) resistant tumors, compared to the parental tumor (C) derived from a HER2 breast cancer PDXs. F GSDMB expression was reduced in HCC1954 LR (left) and OE19 LR cells (right) by two specific siRNAs (siGB1 and siGB2), in comparison with the control (siNTC). Cytotoxic effect of the presence of lapatinib (2 µM and 1.5 µM, respectively) in GSDMB-siRNAs-silenced cells HCC1954 LR (left) and OE19 LR (right) cells was assessed by cell viability assays. G Cytotoxic effect after 72 h treatment with IC50 of lapatinib (2 µM and 0.7 µM, respectively) in GSDMB-shRNAs-silenced cells HCC1954 and OE19 (right) cells was assessed by cell growth (viability assays, left panel) and death (Annexin V FITC and PI, right panel). Annexin V-FITC positive cells alone (A + /PI-) and Annexin V-FITC and PI doubled stained (A + /PI +) were defined as apoptotic cells. The number over the bars indicate the ratio of cell death relative to the shNTC condition. Statistical significance was determined by two-tailed unpaired t-test (*P < 0.05; **P < 0.01). Data are shown as the mean ± s.e.m. Three independent experiments with similar results were performed. In (B, C, E), gene expression was normalized to the mRNA levels of GAPDH. A, Annexin V; PI, propidium iodide. NTC, non-targeting control. DMSO, dimethyl sulfoxide. Lap, lapatinib Gámez‑Chiachio et al. J Exp Clin Cancer Res (2022) 41:285 Page 5 of 19 Fig. 1 (See legend on previous page.) Gámez‑Chiachio et al. J Exp Clin Cancer Res (2022) 41:285 Page 6 of 19 lentiviral shRNA transduction could not be obtained in treatment, the enhanced cell death resulted in an overall these models because they exhibited intrinsic resistance degradation of proteins, including GSDMB and GAPDH to the selection antibiotic puromycin. (Fig. 2A-B). Besides, a significant increase in the relative GSDMB-shRNA-silenced HCC1954 and OE19 cells volume density of autophagic vacuoles was observed by were significantly more sensitive to lapatinib treatment, transmission electron microscopy in GSDMB-expressing as they exhibited an important reduction in cell viabil- HCC1954 cells, compared to GSDMB-silenced cells, after ity and an increase in cell death compared to shNTC the treatment with lapatinib and its combination with control cells (Fig. 1G). Likewise, in the LR models with CQ (Fig. 2C). In the same way, HCC1954 LR and OE19 siGSDMB, we found a slight but statistically significant LR cells, which express high levels of GSDMB, exhibit decrease (around 20%) in cell viability in the presence of increased LC3B-II accumulation by western blot and lapatinib (Fig. 1F). LC3B-II puncta by confocal imaging (Fig. 2D-E) com- pared to their respective control cells. Thus, these results GSDMB increases pro‑survival autophagy in response suggest that high GSDMB expression somehow increases to lapatinib treatment the intrinsic autophagic response to cell stress. Accord- GSDMB silencing did not affect the levels of HER2 recep- ingly, we confirmed that the autophagic flux induced by tor in any of our cell models (Supplementary Fig. 2A), lapatinib (Fig. 2A-C), or by serum starvation (Supple- suggesting that GSDMB does not promote response/ mentary Fig. 3A-B) is reduced in GSDMB-silenced cells resistance to lapatinib through direct modulation of compared to control lines. Moreover, in HCC1954 LR HER2 quantity. Therefore, to decipher the molecu- cells grown in the presence of high doses of lapatinib, the lar mechanism by which GSDMB modulates lapatinib reduction of GSDMB expression by siRNAs also dimin- response, we focused on autophagy, as this process has ishes the autophagic flux (Fig. 2F). Furthermore, we dis- been demonstrated to act as a resistance mechanism carded that this observed autophagic response involved to anti-HER2 therapies both in vivo and in vitro [22]. selective autophagy subtypes such as mitophagy (Supple- Autophagy induction during tumor progression can lead mentary Fig. 4A), aggrephagy (Supplementary Fig. 4B) or to either survival (pro-tumor) or cell death (anti-tumor) lipophagy (Supplementary Fig. 4C). depending on the stimulus and the cellular context [21]. Then, given that GSDMB-high cells exhibit increased Hence, we first tested in HCC1954 and OE19 parental autophagic lapatinib response and that this process pro- cells if lapatinib treatment induced autophagy with sur- motes survival in our cell models (Supplementary Fig. 2), vival or death consequences (Supplementary Fig. 2B-E). we postulated that GSDMB-high cells could be par- In both cell lines, lapatinib treatment induced autophagic ticularly sensitive to the combination of lapatinib with response at 24–48 h, measured by the increase in the lev- autophagy inhibitors. Indeed, the autophagy blockage els of LC3B-II (Supplementary Fig. 2B). Importantly, this with CQ in GSDMB-high (shNTC) HCC1954 and OE19 autophagy is protective since blocking autophagic flux cells treated with lapatinib produces a significant reduc- with chloroquine (CQ), which affects the completion of tion in the cell viability (Fig. 3A-C) as well as an increase the latter stages of autophagy [32], significantly increased in cell death rates (Fig. 3B-D) compared to lapatinib the cytotoxicity of lapatinib (Supplementary Fig. 2C). alone. In contrast, no such dramatic effect was observed Similarly, blocking the formation of autophagosomes in the GSDMB-silenced cells. Importantly, in HCC1954 [33] by an ATG5-specific siRNA (Supplementary Fig. 2D) LR and OE19 LR cells, which over-express GSDMB, the enhanced the effect on cell viability of lapatinib (Supple- addition of CQ strongly increases the cytotoxic effect mentary Fig. 2E). of lapatinib, and thus revert their resistance to this anti- Next, we assessed whether cells with high or low HER2 drug (Fig. 3E-H). Consistent with these results, the GSDMB expression could have different endogenous autophagy blockage by siATG5 confirmed the increased autophagic responses by measuring autophagic flux as sensitivity to lapatinib in GSDMB-high cells in all cell the accumulation of the lipidated LC3B (LC3B-II) form in models (Supplementary Fig. S5a-d). It should be noted western blots [34, 35]. In this regard, we analyzed LC3B- that particularly in OE19 LR cells, which show the high- II turnover in the presence and absence of lysosomal deg- est GSDMB levels, the autophagy inhibition alone (either radation using CQ. Therefore, higher LC3-II levels were by CQ or siATG5) has a significant effect on cell viabil- observed in GSDMB-expressing (shNTC) HCC1954 and ity (Fig. 3G and Supplementary Fig. 5D), supporting that OE19 cells in comparison with GSDMB-silenced cells in after long-term challenge with this anti-HER2 therapy basal autophagy, (Fig. 2A-B, see grey bars) and this effect the subsistence of these cells mostly relies on the endog- was significantly exacerbated upon autophagy activa- enous pro-survival autophagic process. tion with lapatinib (Fig. 2A-B, see blue bars). It should Taken together, these results confirm that GSDMB be noted that due to the high efficacy of the combined enhances the pro-survival autophagy in response to Gámez‑Chiachio et al. J Exp Clin Cancer Res (2022) 41:285 Page 7 of 19 Fig. 2 GSDMB-high cells show an increased autophagic flux in response to lapatinib. A-B GSDMB and LC3B protein levels in shNTC, shGB1 and shGB2 HCC1954 (A) and OE19 (B) cells treated with lapatinib (Lap, 2 µM and 0.7 µM, respectively) and/or CQ (10 µM and 50 µM, respectively) for 72 h. Quantification of the relative LC3B-II expression was conducted as described before [34, 35]. C Representative transmission electron microscopy images of shNTC and shGB2 HCC1954 cells treated with the treatment regimens indicated in (A). Quantification of the relative volume density of autophagic vacuoles is shown on the right. At least 25 cells were analyzed per experimental condition. D-E Western blot analysis of GSDMB and LC3B (left panels) in HCC1954 LR (D) and OE19 LR (E) cells and their respective controls (C) treated with or without CQ (10 µM and 50 µM, respectively) for 72 h. LC3B expression (green) analysis by confocal microscopy (right panels) in HCC1954 LR (D) and OE19 LR (E) cells and their controls (C) treated with or without CQ at the concentrations indicated in (A-B). Representative confocal microscopy images were shown, scale bar, 10 µm. Nuclei were counterstained with DAPI. F GSDMB and LC3B protein levels in GSDMB-siRNA-silenced HCC1954 LR cells treated with or without 10 µM CQ for 72 h. Quantification of LC3B-II expression (showed on the right of panels, A-B, D-F) was carried out by densitometric scanning and normalized to GAPDH expression following previous methods [34, 35]. Statistical significance was determined by two-tailed unpaired t-test (*P < 0.05; **P < 0.01). Data are shown as the mean ± s.e.m. Three independent experiments with similar results were performed. NTC, non-targeting control. LR, Lapatinib resistant cells. CQ, chloroquine. Lap, lapatinib Gámez‑Chiachio et al. J Exp Clin Cancer Res (2022) 41:285 Page 8 of 19 lapatinib in HER2 + breast and gastric cancer cells, and addition of CQ enhanced the effect of lapatinib on the thus GSDMB-over-expressing cells are more sensitive reduction of tumor growth exclusively in GSDMB- to the addition of autophagy inhibitors both in the early expressing (shNTC) tumors (Fig. 4D-E). The therapeutic response to HER2-targeted treatment and in drug resist- response was independent of cell proliferation since no ant cells. differences in PCNA immunohistochemical expression were detected in any of the experimental conditions (Sup- Autophagy inhibition enhances lapatinib efficacy in vivo plementary Fig. 7B). As observed in vitro, GSDMB was specifically in GSDMB‑expressing breast cancer cells also up-regulated in vivo in GSDMB-expressing tumors Next, to validate if the combination of lapatinib and CQ (shNTC) by lapatinib, noting an increase in the necrotic on HER2/GSDMB positive tumors would be an effective areas in the case of the combined treatment (Fig. 4H therapeutic approach, we assayed its functional effect and Supplementary Fig. 7B), and again it was associated on tumor growth in vivo using two different preclinical with a reduced response to the treatment compared to models, zebrafish, and mice (Fig. 4). In zebrafish, which GSDMB-silenced tumors (Fig. 4D,F,G and Supplemen- allows testing the drug response in a large number of bio- tary Fig. 7C). These in vivo data prove that the autophagy logical replicates [36], we first calculated the acute dose- blockage is essential to improve the anti-HER2 therapy dependent toxicity of both lapatinib and CQ by analyzing response in HER2/GSDMB tumors and reinforce the role the embryo mortality (Supplementary Table 3). Next, the of GSDMB in the promotion of pro-survival autophagy different HCC1954 models mentioned above (shNTC, as a resistance mechanism to these therapies. shGB1/2 as well as LR and the corresponding control cells) that stably express GFP were inoculated into the GSDMB acts as an autophagy adaptor inducing Rab7 yolk sac of zebrafish embryos and treated with either activation with a predictive value to anti‑HER2 therapies lapatinib, CQ or the combination of both (Fig. 4A-B and To unravel further the role of GSDMB in autophagy, Supplementary Fig. 6A-B). As expected, lapatinib treat- HCC1954 cells exogenously over-expressing myc-tagged ment produced a significant reduction in cancer cell full length GSDMB (GB) after treatment with lapatinib, growth of GSDMB-silenced tumors (shGB1 and shGB2), CQ, or both drugs were immunoprecipitated with a but not in HCC1954 shNTC (Fig. 4A). Remarkably, Myc-tag antibody followed by mass-spectrometry stud- autophagy blockage by CQ provoked an increase in the ies. Among potential GSDMB interacting cancer and effect of lapatinib on tumor growth reduction (p < 0.001) autophagy proteins, some Rab GTPases were found only in GSDMB-expressing (shNTC) tumors, compared (Rab5C, Rab7A, Rab9A and Rab15, Fig. 5A and Supple- to lapatinib alone, while no such effect was observed in mentary Table 4). These have been implicated in differ- GSDMB-silenced (shGB1, shGB2) tumors. Furthermore, ent steps of the autophagy process [37]; especially, Rab7 similar results were found on lapatinib resistant cells which participates in the autophagosome-lysosome (LR), where the combined treatment (CQ plus lapat- fusion, the autolysosome maturation and transport [25, inib) practically abolished the resistant phenotype of 38] and has shown a pivotal role in resistance to chemo- HCC1954 LR cells (Fig. 4B and Supplementary Fig. 6B). therapeutic agents [39]. Rab7a was found with a higher These findings support that the combination therapy was score both in CQ and combined treatment in the mass- effective specifically in high GSDMB-expressing tumors. spectrometry assay compared to untreated cells (Fig. 5A). Subsequently, to validate these results, we performed To identify the interaction region between GSDMB and similar treatment experiments in mice bearing ortho- Rab7a, an in silico prediction assay was carried out, topically injected HCC1954 (shNTC, shGB1 and shGB2) revealing an energetically feasible GSDMB/Rab7a com- breast cancer xenografts (Fig. 4C-H and Supplemen- plex (ΔG = -2.73 kJ/mol), in which the most probable tary Fig. 7). Once again, while lapatinib alone mainly GSDMB residues involved in this interaction are distrib- decreased tumor weight and volume in GSDMB-silenced uted through its N-terminal region (amino acids V7, D30, tumors (Fig. 4D,F,G and Supplementary Fig. 7A), the F46, Q60 and E185 from GSDMB, Fig. 5B). Interestingly, (See figure on next page.) Fig. 3 GSDMB-high cells are significantly more sensitive to the combination of lapatinib plus chloroquine. A-D The cytotoxic effect of the treatment with lapatinib and/or chloroquine in shNTC, shGB1 and shGB2 HCC1954 (A, B) and OE19 (C, D) cells was evaluated by cell viability assays and Annexin V-FITC plus PI. E–H The outcome in terms of cell viability and apoptosis of the different treatment regimens was analyzed in HCC1954 LR (E–F) and OE19 LR (G-H) cells compared to their corresponding control cells C. Statistical significance was determined by two-tailed unpaired t-test (*P < 0.05; **P < 0.01; ***P < 0.001). Data are shown as the mean ± s.e.m. Annexin V-FITC positive cells alone (A + /PI-) and Annexin V-FITC and PI doubled stained (A + /PI +) were defined as apoptotic cells. The number over the bars indicate the fold increase in cell death between the indicated conditions (B, D, F, H). Three independent experiments with similar results were performed. A, Annexin V; PI, propidium iodide; CQ, chloroquine. NTC, non-targeting control. LR, Lapatinib resistant cells. Lap, lapatinib, CQ, chloroquine Gámez‑Chiachio et al. J Exp Clin Cancer Res (2022) 41:285 Page 9 of 19 Fig. 3 (See legend on previous page.) Gámez‑Chiachio et al. J Exp Clin Cancer Res (2022) 41:285 Page 10 of 19 we also found in the GSDMB N-terminal domain two (Fig. 5F and Supplementary Fig. 8F). Besides, to prove putative LC3B-interacting region (LIR) motifs (“LIR1” the involvement of Rab7 in GSDMB-mediated resistance corresponding to 3SVFEEI8 sequence and “LIR2” cover- to anti-HER2 therapies, we silenced Rab7a (siRab7a) and ing 82AEFQIL87 amino acids, Supplementary Fig. 8A). confirmed the increased sensitivity to lapatinib especially Despite LC3B was not detected in the proteomic analy- in GSDMB-expressing HCC1954 cells (Fig. 5G and Sup- sis, we hypothesized that a GSDMB-Rab7a-LC3B multi- plementary Fig. 8G) and in HCC1954 LR cells (Fig. 5H). protein complex could occur. In fact, the HawkDock web Together, these data indicate that the GSDMB N-termi- server [40], predicted a highly possible GSDMB/Rab7a/ nal region comprising the amino acids 1–91 is neces- LC3B complex (ΔG = -2.17 kJ/mol, Supplementary sary for the interaction with Rab7 and LC3B and for the Fig. 8B). By contrast, a similar complex was not feasible subsequent autophagy induction mediated by GSDMB when the rest of human GSDMs were analyzed (data not over-expression during lapatinib treatment. Supporting shown), supporting the idea that although other GSDMs this idea, a significant cell growth increase was observed have been previously implicated in autophagic pro- in HCC1954 GB cells compared to HCC1954 control cesses [4, 41, 42], only GSDMB might regulate autophagy or ­GB92−416 cells in response to lapatinib (Fig. 5I). Like- through Rab7a/LC3B interaction. Thus, to validate these wise, the cell death rate after the combination of lapat- results experimentally, we utilized HCC1954 paren- inib plus CQ was higher in HCC1954 GB cells than in the tal cells with endogenous GSDMB levels (Control, C), other conditions (Fig. 5J). Moreover, although the basal cells with exogenous GSDMB overexpression (GB) and autophagy, measuring LC3B-II levels, induced by CQ a model exogenously over-expressing the C-terminal treatment in HCC1954 GB, ­GB92−416 and control cells ­(GB92−416) fragment produced by caspase 3/6/7 process- was similar (Supplementary Fig. 8C, see grey bars), the ing [30], which lacks both LIR motifs. increase in autophagic flux by lapatinib plus CQ treat- First, we noticed that neither GB nor G ­ B92−416 over- ment was significantly stronger in HCC1954 GB cells, expression significantly modifies Rab7 and LC3B compared to control or G ­ B92−416 cells (Supplemen- endogenous levels (Supplementary Fig. 8C). In the co- tary Fig. 8C, compare blue and grey bars). Together, the immunoprecipitation assays, the interaction between results support the key role of the GSDMB N-terminal GSDMB and Rab7 was detected only in HCC1954 region, particularly the first 91 residues, in the control of GB cells but not in G ­ B92−416 or control cells (Fig. 5C the autophagy response after anti-HER2 treatment. and Supplementary Fig. S8D). As expected, LC3B and In parallel, to validate our results in a GSDMB null Rab7 co-immunoprecipitation was mainly found after biological context, thus avoiding the effects of low autophagy inhibition by CQ treatment in HCC1954 endogenous GSDMB expression in shGB models, we GB cells (Fig. 5C). Similar results were observed in the generated complete GSDMB-knockout cells by CRISPR/ binding assays between GSDMB and LC3B (Fig. 5D Cas9 (sgGB1). As expected, the autophagic flux induc- and Supplementary Fig. 8D). Furthermore, by confo- tion in sgGB1 cells was indeed lower than in control cells cal microscopy, GSDMB colocalization with L3CB or (Supplementary Fig. 9A) and consequently the cyto- Rab7 was significantly higher in GB than in G ­ B92−416 toxic effect of lapatinib treatment was much higher in expressing cells treated with lapatinib plus CQ (Fig. 5E GSDMB-null cells (Supplementary Fig. 9B). In agreement and Supplementary Fig. 8E). Consistently, Rab7-LC3B with the results in the other cell models, after lapatinib colocalization was also significantly higher in GSDMB- plus CQ treatment the colocalization between LC3B and expressing cells (shNTC) compared to shGB1/2 cells Rab7 was lower in GSDMB-knockout cells, confirming (See figure on next page.) Fig. 4 Autophagy blockade with chloroquine improves lapatinib efficacy in vivo in zebrafish and mouse xenografts of GSDMB-expressing tumors. A-B Representative fluorescence stereomicroscope images (left panel) of GFP expressing control (shNTC), and GSDMB-silenced (shGB1) HCC1954 xenografts (A) or HCC1954 LR and control (C) tumors (B) treated with the LC50 of lapatinib and chloroquine (35,1 mM and 116,4 mM, respectively). Insets represent an augmented image of GFP-positive tumor. Tumor proliferation rate (right panel) of the different HCC1954 xenografts was analyzed by measuring the fluorescence intensity ratio (48 hpt/0 hpt). Statistical significance was determined by two-tailed unpaired t-test. At least, n = 20 per each indicated condition. C Experimental design of the mouse xenograft model (n = 5 per condition). Mice were inoculated with either HCC1954-mCherry-luc control (shNTC), or GSDMB-silenced cells (shGB1 and shGB2) and treated with lapatinib (100 mg/kg, orally, once daily), CQ (50 mg/kg, intraperitoneally, once daily), or a combination of both (lapatinib + CQ). An aqueous solution containing 0.1% Tween 80 and 0.5% Hypromellose was used as vehicle. The experiment was performed for 30 days, according to the approved protocol and conditions of animal research (detailed in Supplementary Methods). D-G Quantification of the tumor weight (D) tumor volume evolution of shNTC (E), shGB1 (F) and shGB2 (G) HCC1954 xenografts, treated with the indicated regimens. Statistical significance was determined by multiple unpaired t-test – comparing vehicle with each of the other conditions at every time point. H Representative images of GSDMB immunohistochemical analysis and hematoxylin and eosin staining in shNTC tumors, treated with the different therapeutic strategies indicated in (C). Immunohistochemical images were taken on 10X and 40X (insets) magnification. (*P < 0.05; **P < 0.01; ***P < 0.001; ns, nonsignificant). Data are shown as the mean ± s.e.m. LC50, 50% lethal concentration; hpt, hours post-treatment. NTC, non-targeting control. LR, Lapatinib resistant cells, CQ, chloroquine. Lap, lapatinib Gámez‑Chiachio et al. J Exp Clin Cancer Res (2022) 41:285 Page 11 of 19 Fig. 4 (See legend on previous page.) GSDMB involvement in the formation of an autophagic patterns, being LC3B very diffuse and Rab7 rarely local- multiprotein complex (Supplementary Fig. 9C). Indeed, ized into vesicles (Supplementary Fig. 9C). This suggest compared to shGB1/2 cells (Supplementary Fig. 8F), that complete lack of GSDMB strongly decreases Rab7- sgGB1 cells exhibited distinct Rab7 and LC3B staining mediated maturation of autophagosomes. Gámez‑Chiachio et al. J Exp Clin Cancer Res (2022) 41:285 Page 12 of 19 Therefore, to demonstrate the functional connection Table 2). Third, although GSDMB localization is usu- between GSDMB and Rab7 and the subsequent modu- ally diffuse cytoplasmic or nuclear [12, 14, 44], a con- lation of pro-survival autophagy we measured Rab7 current dotted staining was observed for GSDMB, Rab7 activity (by co-immunoprecipitation assays with an anti- and LC3B puncta within tumor cells (Fig. 6C-D). Four, Rab7GTP antibody) in cells treated with lapatinib and and most notably, the GSDMB/Rab7 and GSDMB/LC3B the combined treatment (Fig. 6A). GSDMB-expressing puncta positive co-expression significantly correlated cells showed higher Rab7-GTP levels after lapatinib or with relapse in HER2/GSDMB + breast and gastric car- the combination treatment (P = 0.2195) compared to cinomas (Fig. 6C-D and Supplementary Table 5). Given shGSDMB cells (Fig. 6A), thus demonstrating an impor- that these tumors were treated, the association observed tant effect of GSDMB on Rab7 activity. Then, we hypoth- between GSDMB/LC3B/Rab7 staining patterns and clini- esized that GSDMB could act like an autophagy receptor cal behavior support the idea that GSDMB over-expres- or adaptor [43]. Both types of molecules bind Atg8 fam- sion could promote protective autophagic response to ily proteins, like LC3B, but autophagy receptors, such as therapy in HER2 positive cancer patients. p62, control cargo selection and therefore, are degraded Taken together, our results reveal that the autophagy by autophagy [43]; thus, their levels increase when the inhibition reverses, at least in part, the resistance to lapa- autophagic flux is blocked. In contrast to p62 or LC3B, tinib mediated by GSDMB over-expression in HER2 can- GSDMB does not accumulate upon autophagy blockage cer cells. Moreover, the multiprotein complex formed by by CQ (Fig. 6B). In consequence, our data indicate that GSDMB, Rab7 and LC3B plays a key role during the pro- GSDMB would act as an autophagy adaptor in response survival autophagy process in vitro and in vivo. Therefore, to anti-HER2 therapies due to its ability to interact with the combination of autophagy inhibitors with anti-HER2 LC3B located in the outer autophagosome membrane agents could provide new therapeutic options for HER2/ and regulates the autophagic machinery through Rab7 GSDMB-overexpressing breast and gastroesophageal activation. tumors (Fig. 7). Finally, we aimed to validate our in vitro and in vivo results on clinical specimens of HER2 breast and gastric Discussion carcinomas. The immunohistochemical study of GSDMB, The multifunctional protein GSDMB plays complex roles LC3B and Rab7 provided different pieces of important in inflammatory pathologies and cancer. On the one information. First, about 50% of HER2 breast and gastric hand, similar to other GSDM family members [2, 3], the tumors showed intense LC3B dotted (puncta) expres- released/activated GSDMB NT domain has pore-forming sion while Rab7 puncta was more frequent in gastric activity on biological membranes that can provoke lytic than breast carcinomas (Fig. 6C-D and Supplementary cell death of normal [5, 7], cancer cells [8] and intracel- Table 1). Second, strong Rab7 or LC3B puncta was signif- lular bacteria [9]. On the other hand, GSDMB full-length icantly more frequent in GSDMB over-expressing breast protein (GSDMB-FL) exhibits additional functions in and in gastric tumors (Fig. 6C-D and Supplementary pathogenesis, including cell motility and epithelial barrier (See figure on next page.) Fig. 5 GSDMB modulates autophagic response through Rab7 and LC3B interaction. A Heatmap representation of the mass spectrometry analysis performed on HCC1954 GB cells treated with the different treatment regimens, after co-immunoprecipitation assay using a Myc-tag antibody. Normalization to untreated cells was performed. Color key indicates protein expression score: dark blue: highest; dark orange: lowest. B In silico protein interaction prediction using the InterEvDock2 and PPCheck web servers identifies a potential interaction between Rab7a (yellow) and the N-terminal domain of GSDMB (green). Inset magnifies the predicting interacting region (pink). 3D structures were obtained from Uniprot data bases (Q8TAX9: GSDMB and P51149: Rab7a). C-D Co-immunoprecipitation assay of Rab7-GSDMB (C) and LC3B-GSDMB (D) interaction after CQ and lapatinib plus CQ treatment in HCC1954 cells exogenously expressing full length GSDMB (GB), a construct lacking the N-terminal domain ­(GB(92–416)) or control (C; empty vector) cells. E Representative images of the colocalization between Rab7 (red), LC3B (cyan) and GSDMB-Myc-tag (green) by confocal microscopy in HCC1954 GB and ­GB(92–416) cells after lapatinib (2 µM) plus CQ (10 µM) treatment. Nuclei were counterstained with DAPI. Quantification of the Manders’ Overlap Coefficient (Rab7 overlapping Myc-tag and LC3B overlapping Myc-tag) is shown on the right. Five independent experiments were performed obtaining at least 60 cells, per experimental condition. Scale bar, 10 µm. F Representative images of the colocalization between Rab7 (red) and LC3B (green) by confocal microscopy in shNTC, shGB1 and shGB2 HCC1954 cells after lapatinib (2 µM) plus CQ (10 µM) treatment. Nuclei were counterstained with DAPI. Quantification of the Manders’ Overlap Coefficient (LC3B overlapping Rab7) is shown on the right. Five independent experiments were performed obtaining at least 60 cells, per experimental condition. Scale bar, 10 µm. G-H The cytotoxic effect of the treatment with lapatinib plus/or siRab7a-silencing was evaluated by cell viability assays in shNTC, shGB1 and shGB2 HCC1954 (G) and HCC1954 LR (H) cells. I-J Comparative cytotoxic effect by cell viability assays (I) and Annexin V-FITC plus PI (J) in HCC1954 C, GB and ­GB(92–416) cells after 72 h treatment with lapatinib (2 µM) and/or CQ (10 µM). Annexin V-FITC positive cells alone (A + /PI-) and Annexin V-FITC and PI doubled stained (A + /PI +) were defined as apoptotic cells. Statistical significance was determined by two-tailed unpaired t-test (*P < 0.05; **P < 0.01; ***P < 0.001; ns, nonsignificant). Data are shown as the mean ± s.e.m. In (E-J), three independent experiments with similar results were performed. Lap, lapatinib, CQ, chloroquine Gámez‑Chiachio et al. J Exp Clin Cancer Res (2022) 41:285 Page 13 of 19 Fig. 5 (See legend on previous page.) Gámez‑Chiachio et al. J Exp Clin Cancer Res (2022) 41:285 Page 14 of 19 Fig. 6 GSDMB expression promotes Rab7 activation in response to anti-HER2 drugs. A Co-immunoprecipitation assay of Rab7-GTP levels in shNTC and shGB2 HCC1954 cells after the treatment with lapatinib (2 µM) and the combined treatment. Statistical significance was determined by two-tailed unpaired t-test (**P < 0.01). Data are shown as the mean ± s.e.m. Three independent experiments with similar results were performed. B GSDMB, p62 and LC3B protein levels in HCC1954 cells treated for 72 h with or without chloroquine (10 µM). C-D Representative multiplex immunofluorescence images of GSDMB (green), Rab7 (red) and LC3B (deep red) co-expression in adjuvant breast (C) and gastric carcinomas (D). Scale bar, 20 µm. Nuclei were counterstained with DAPI. Representative pie charts of Rab7 and GSDMB co-expression statistics and its relationship with relapse in adjuvant breast (C) and gastric carcinomas (D) (see Supplementary Tables 1, 2 and 5 for extra clinical features and statistics). CQ, chloroquine. Lap, lapatinib repair during inflammatory bowel disease [11], transcrip- over-expressed in diverse cancer types [3], where usu- tional control of disease-associated genes in asthma [10], ally associates with poor prognosis [3], the activation promoting invasion and/or metastasis of cancer cells [13, of GSDMB pro-cell death function has been previously 14, 45], metabolism regulation in tumors [45], and cancer explored as a novel therapeutic approach using two dif- therapy resistance [12, 14]. Since GSDMB is frequently ferent methods: a) an antibody-based nanomedicine that Gámez‑Chiachio et al. J Exp Clin Cancer Res (2022) 41:285 Page 15 of 19 Fig. 7 GSDMB acts as an autophagy adaptor in response to anti-HER2 therapies. Representative scheme of the proposed mechanism of action of GSDMB as mediator of protective autophagy response. It is highlighted the dualistic model on HER2/GSDMB over-expressing tumors behavior after the treatment anti-HER2 therapy (resistance phenotype) or its combination with autophagy inhibitors (sensible phenotype) seemed to activate the protein cytotoxic activity in breast in cancer models [8], and methotrexate in gut epithelium cancer xenografts in vivo [14]; b) triggering the proteo- [11], but the effects of this upregulation depends on the lytic cleavage of GSDMB NT pore-forming domain by cellular and pathological settings. Thus, GSDMB can lymphocyte Granzyme-A [8] via pharmacological acti- promote migration, adhesion and proliferation, through vation (anti-PD-1) of the antitumor immunity response FAK phosphorylation changes mediated by PDGF-A, [8]. Whereas both approaches yielded promising results, during gut epithelial barrier repair/restitution [11], while only partial tumor regression was achieved, and so far, it GSDMB effects on breast cancer invasion/metastasis and is still unproven if GSDMB-mediated pyroptosis actually tumor growth depend on the cellular and in vivo contexts occurs in clinical tumor samples. Therefore, to expand [12–14, 46]. Generally, GSDMB does not significantly GSDMB potential as therapy target, in the present work, affect breast tumor proliferation (as shown in this work we aimed at identifying other druggable pathways, con- and previous literature [12–14, 46]) but can mediate can- trolling GSDMB pro-tumor functions, that could be cer survival upon therapy challenge. Here we prove that readily translated into the clinic. Specifically, we focused high GSDMB levels are required for anti-HER2 therapy on tackling the mechanisms by which GSDMB mediates resistance in breast and gastroesophageal cancers since limited response to anti-HER2 therapy [12, 14] in tumors. GSDMB downregulation by si-/shRNAs or CRISPR/Cas9 While GSDMB over-expression in breast and likely gas- significantly increases sensitivity and partially reverts the tric [44] cancer is mostly due to GSDMB-HER2 gene co- resistant phenotype in these tumors. amplification, our results together with our published Importantly, we demonstrate for the first time that this data [12], prove that GSDMB transcription and trans- GSDMB function requires the activation of protective lation are further upregulated specifically in response autophagy. Autophagy, a catabolic mechanism where mol- (early response and during acquired resistance) to differ- ecules and damaged cellular organelles are degraded, has ent anti-HER2 drugs (trastuzumab, lapatinib) in HER2 a complex role in cancer development and clinical behav- breast cancer (cell lines and PDXs) and gastroesophageal ior, acting as either a pro-death or a pro-survival factor tumor cells. GSDMB can also be upregulated by different depending on the cancer type and stage as well as treat- chemokines (IFN-α, -β, and -γ, or a lesser extent TNF–α) ment [47]. In HER2 tumors, the anti-HER2 targeted drugs Gámez‑Chiachio et al. J Exp Clin Cancer Res (2022) 41:285 Page 16 of 19 lapatinib [22] and trastuzumab [48] trigger protective to autophagosomes, and its downstream effects on Rab7 autophagy to maintain the resistant phenotype [20], and activity, like other Rab7 regulators [26]. For instance, accordingly, abrogation of autophagy by specific inhibitors PI3P, essential in the initial steps of autophagy, is required (like chloroquine, CQ) can re-sensitize cancer cells to these by the Rab7 guanine nucleotide exchange factor (GEF), drugs and enhance tumor cell death in preclinical models Mon1-Ccz1, for binding to early autophagosomes and [20]. Nevertheless, the biological determinants affecting the following activation of Rab7 [26]. autophagic response to these drugs and the sensitivity to It is worth noting that our results, together with previous autophagy inhibitors are not well understood so far. Here, studies based on other GSDMs, indicate that these proteins we discovered that GSDMB-high cells exhibit upregulated can regulate diverse autophagic processes that result in autophagic flux, and the inhibition of this process by CQ cell- and context-dependent biological consequences. For significantly enhances the response to lapatinib in vitro, instance, GSDMA3 mutant proteins (with activated NT) inducing a shift from cell viability towards toxicity both provoke autophagy leading to cell death [4], whereas cleaved in early response and in lapatinib-resistant cells. Interest- GSDMD NT localizes to autophagosomes in neutrophils, ingly, lapatinib plus CQ nearly abrogates tumor growth inducing autophagy-mediated cell-death-independent in two preclinical in vivo models (zebrafish, mouse), thus IL-1β secretion [41], and full length PJVK/DFNB59 recruits proving the therapeutic utility of this combination. LC3-II to produce pexophagy, a type of selective autophagy Mechanistically, our data indicate that GSDMB, Rab7 for degrading damaged peroxisomes [42]. and LC3B form a complex after lapatinib and/or CQ While further research is required to fully decipher the treatment. The GSDMB-Rab7 functional axis is impor- importance of the autophagy-related functions of GSDMs tant for mediating survival autophagy, since Rab7a in disease conditions, our data in patient cohorts with silencing increases the sensitivity to lapatinib of GSDMB- HER2 gastric or breast carcinomas support that GSDMB/ expressing and lapatinib resistant cells, and higher Rab7/LC3B-autophagy axis has an impact on clinical GSDMB levels upregulate Rab7 activation (measured by behavior. Indeed, treated gastric and breast cancer patients the co-immunoprecipitated levels of Rab7-GTP). Rab7 with higher puncta tumor expression of both GSDMB/ performs multiple functions that depend on the interac- Rab7 and GSDMB/LC3B are more prone to relapse. Our tion with diverse downstream effectors [26]. In particu- results are in line with previous studies describing GSDMB lar, Rab7 activity during autophagosome-lysosome fusion [12, 14], Rab7 [51] and LC3 over-expression [52, 53] as is regulated by its effector, FYCO1 (FYVE and coiled- poor prognosis cancer factors and reinforce the connec- coil domain-containing autophagy adaptor 1), which tion between GSDMB/Rab7/LC3B co-expression, pro- promotes microtubule plus end–directed transport of tective autophagy, resistance to anti-HER2 therapies and autophagic vesicles [49], and other partners, such as Nlp adverse prognosis in clinical samples. (ninein-like protein) which enhances autophagosome- Finally, and most notably, our findings have two impor- lysosome fusion via promoting Rab7 and FYCO1 interac- tant therapeutic implications for the future treatment of tion [50]. Given that GSDMB is not an autophagic cargo HER2/GSDMB tumors. First, they open a new therapeu- (not degraded during autophagy), interacts with LC3B tic opportunity for lapatinib, whose use in current clini- and induces Rab7 activation, we propose that GSDMB cal practice is mostly restricted to the advanced stages acts, like FYCO1, as an autophagy adaptor that specifi- in combination with chemotherapy and/or other HER2- cally facilitates autophagosome maturation and the autol- directed targets [17]. Second, provide evidence that the ysosome formation in response to anti-HER2 therapies. FDA-approved chloroquine in combination with lapat- Further supporting this idea, we prove that a region of inib can be a novel effective therapy for anti-HER2-drug GSDMB NT domain (1–91 residues) harboring two LIR resistant HER2/GSDMB positive tumors. motifs is necessary for Rab7/LC3B interaction and the subsequent autophagy induction, since deletion of this Conclusions region (construct G ­ SDMB92−416) abrogates the co-immu- Our study reveals for the first time that GSDMB acts noprecipitation and colocalization of Rab7/GSDMB and like a pro-survival autophagy adaptor in response LC3B/GSDMB in HCC1954 GB cells. Moreover, similar to anti-HER2 drugs, binding autophagosome sur- to FYCO1, which function also depends on its binding to face-lipidated LC3B and promoting Rab7 activation. LC3 and phosphatidylinositol-3-phosphate (PI3P) [49], Moreover, we demonstrated the preclinical utility of GSDMB NT domain has affinity to specific lipids such combining the autophagy inhibitor CQ with the HER2- as mono-, bis- and tri-phosphoinositides and, weakly to targeted drug lapatinib; thus, contributing to the sulfatides and cardiolipin [14, 30]. Thus, it is reasonable evolving treatment paradigm in HER2 tumors over- to speculate that GSDMB NT, at least the first 91 resi- expressing GSDMB, which are largely associated with dues, might be important for the anchorage of GSDMB poor outcomes. Gámez‑Chiachio et al. J Exp Clin Cancer Res (2022) 41:285 Page 17 of 19 Abbreviations Funding ATG5: Autophagy related 5; CQ: Chloroquine; CNS: Central nervous system; This study has been supported by the Ministerio de Ciencia, Innovación DMSO: Dimethyl sulfoxide; FAK: Focal Adhesion Kinase; FDA: U.S. Food y Universidades, Agencia Estatal de Investigación (PID2019-104644RB- and drug administration; FYCO1: FYVE and coiled-coil domain-containing I00) -GMB-, the Instituto de Salud Carlos III (CIBERONC, CB16/12/00449 autophagy adaptor 1; GSDM: Gasdermin; GSDMB: Gasdermin B; HER2: -JA-, CB16/12/00231 -DLN- and CB16/12/00295 -GMB-, PI19/01181 -JA-, Human epidermal growth factor receptor 2; IF: Immunofluorescence; IHC: PI18/00795, CP17/00063 and RTI2018-095611-A-I00 -DLN- and ERA-NET TRAN‑ Immunohistochemistry; IP: Immunoprecipitation; Lap: Lapatinib; LC3B: SCAN-2 -JA- [all partly supported by FEDER funds]) and by the AECC Scientific Microtubule-associated proteins 1A/1B light chain 3B; LIR: LC3-interacting Foundation (FC_AECC PROYE19036MOR -GMB- and LABAE19004LLOB -DLN-). region; LR: Lapatinib resistant cells; NTC: Non-targeting control; PDGF- Furthermore, this work was supported by Breast Cancer Research Foundation A: Platelet-Derived Growth Factor-A; PDX: Patients-derived xenografts; (BCRF-19–08) -JA-. We are also grateful to the CERCA Programme (Generalitat PI: Propidium iodide; Rab7: Ras-related protein Rab-7; RT-qPCR: Reverse de Catalunya) for institutional support. MGC and DS contracts are funded by transcription-quantitative polymerase chain reaction; TKI: Tyrosine kinase CIBERONC, KG is a recipient of a PFIS fellowship (FI19/00188), RRB is recipient inhibitor; WB: Western blot. of a Ramón y Cajal grant (RyC-2016–19671) and DLN is recipient of a Miguel Servet grant (MS17/00063) (all partly supported by FEDER funds). We are also grateful to MD Anderson BIOBANK for providing tumor samples. The bank (ref‑ Supplementary Information erence # B.0000745) belongs to the National Registry of Biobanks coordinated The online version contains supplementary material available at https://​doi.​ by the Carlos III Health Institute. org/​10.​1186/​s13046-​022-​02497-w. Availability of data and materials Additional file 1. Supplementary Figure 1. Upregulation of GSDMB All data generated or analyzed during this study are included in this published in response to anti-HER2 therapies and expression of different GSDMs article and its supplementary information files. in GSDMB-silenced cells after treatment with lapatinib. Supplementary Figure 2. Lapatinib induces pro-survival autophagy in HCC1954 and OE19 Declarations cells. Supplementary Figure 3. GSDMB-high cells show an increased autophagic flux in response to starvation. Supplementary Figure 4. Ethics approval and consent to participate GSDMB-mediated autophagic response is not correlated with mitophagy, All mouse studies were performed in agreement with the procedures and aggrephagy or lipophagy. Supplementary Figure 5. ATG5-silencing protocols that have been approved by the internal committees of ethical renders GSDMB-high cells more sensitive to lapatinib. Supplementary and animal welfare of the Institutions (Autonomous University of Madrid Figure 6. The combination of lapatinib plus chloroquine increases the and Institute of Biomedical Sciences Alberto Sols-CSIC and IDIBELL) and therapeutic response in vivo in zebrafish xenografts of GSDMB-expressing the local authorities (Comunidad de Madrid, PROEX424/15 and Gener‑ tumors. Supplementary Figure 7. Immunohistochemical and histologi‑ alitat Catalana, B-9900010). The procedures comply with the European cal analysis in orthotopic tumor xenografts. Supplementary Figure 8. Union (Directive 2010/63/UE) and Spanish Government guidelines (Real GSDMB potentially forms a multiprotein complex with Rab7 and LC3B. Decreto 53/20133). All mice were housed within the same room under Supplementary Figure 9. GSDMB knockout cells show a decreased standard conditions, with a maximum of 4 animals per cage. No animals autophagic flux, correlated with higher sensitivity to lapatinib treatment, were caged individually. Moreover, zebrafish studies (AB strain, Danio rerio) compared to control cells. Supplementary Table 1. Immunohistochemi‑ were performed with the agreement of the Bioethics Committee for animal cal and clinical data of the HER2+ gastric carcinoma cohort and the experimentation of the University of Santiago of Compostela (CEEA-LU), HER2+ breast carcinoma series*. Supplementary Table 2. Relation‑ REGA code: ES270280346401. ship between GSDMB expression and clinical or immunohistochemical features in the adjuvant treated HER2-positive gastric carcinoma and Consent for publication breast carcinoma* cohorts. Supplementary Table 3. In vivo acute toxic‑ Not applicable. This work does not involve human studies. ity results in zebrafish. Supplementary Table 4. Summary of potential cancer and autophagy GSDMB interactors proteins obtained from immu‑ Competing interests noprecipitation and mass spectrometry. Supplementary Table 5. Co- The authors have no conflicts of interest to declare. expression of GSDMB and autophagy markers (LC3B and Rab7) and their associations with relapse in the adjuvant treated HER2-positive gastric Author details carcinoma and breast carcinoma* cohorts. Supplementary Table 6. List 1 Departamento de Bioquímica, Universidad Autónoma de Madrid (UAM) Insti‑ of primary antibodies used for western blot (WB), immunofluorescence tuto de Investigaciones Biomédicas ‘Alberto Sols’ (CSIC-UAM), IdiPAZ, C/Arturo (IF), immunoprecipitation (IP), and immunohistochemistry (IHC). Duperier 4, 28029 Madrid, Spain. 2 Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Instituto de Salud Carlos III, Madrid, Spain. 3 Departa‑ mento de Zoología Genética Antropología Física, Universidad Santiago de Acknowledgements Compostela, Lugo, Spain. 4 Mecanismos Moleculares Y Terapia Experimental We are grateful to members of Amparo Cano’s and Gema Moreno-Bueno’s en Oncologia-Programa OncobellIdibell, L’Hospitalet de Llobregat, Spain. laboratories for constructive suggestions, as well as Mónica Martín-Belinchon 5 Servicio de Microscopía Electrónica, Centro de Investigación Príncipe Felipe (Confocal Microscopy core facilities at IIBM, CSIC-UAM). We also thank to the (CIPF), Valencia, Spain. 6 Servicio de Microscopía Óptica Avanzada, Centro de Proteomics Unit-UCM a member of ProteoRed supported ISCIII and ERDF. Investigación Príncipe Felipe (CIPF), Valencia, Spain. 7 Programa de Investi‑ gación Preclínica, Vall d’Hebron Institute of Oncology (VHIO), Barcelona, Spain. Authors’ contributions 8 Leitat Medical Department, Leitat Technological Center, Barcelona, Spain. MGC, AMC, DS and GMB conceived and designed the study. MGC, AMC, 9 Fundación MD Anderson Internacional, Madrid, Spain. 10 Sistemas Genómi‑ CR, LM, and CGC performed most of the experiments, analyzed results cos, Paterna, Valencia, Spain. 11 Departamento de Patologia Y Terapèutica and interpreted data. JMV and LS performed experiments with zebra fish. Experimental Facultad de Medicina, Unidad de Anatomia Universidad de Bar‑ MGC, LM, KG, FJL, RR and DLN made mice experiments. ARS, ED and GMB celona (UB), L’Hospitalet de Llobregat, Spain. 12 Programa de Investigación en performed histological analyses and interpreted data from tumor samples. Cáncer, IMIM (Hospital del Mar Medical Research Institute), Barcelona, Spain. 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