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Epithelial-mesenchymal transition (EMT) is defined by the loss of epithelial characteristics and the acquisition of a mesenchymal phenotype. In carcinoma cells, EMT can be associated with increased aggressiveness, and invasive and metastatic potential. To assess the occurrence of EMT in human breast tumors, we conducted a tissue microarray–based immunohistochemical study in 479 invasive breast carcinomas and 12 carcinosarcomas using 28 different markers. Unsupervised hierarchical clustering of the tumors and statistical analysis showed that up-regulation of EMT markers (vimentin, smooth-muscle-actin, N-cadherin, and cadherin-11) and overexpression of proteins involved in extracellular matrix remodeling and invasion (SPARC, laminin, and fascin), together with reduction of characteristic epithelial markers (E-cadherin and cytokeratins), preferentially occur in breast tumors with the “basal-like phenotype.” Moreover, most breast carcinosarcomas also had a basal-like phenotype and showed expression of mesenchymal markers in their sarcomatous and epithelial components. To assess whether basal-like cells have intrinsic phenotypic plasticity for mesenchymal transition, we performed in vitro studies with the MCF10A cell line. In response to low cell density, MCF10A cells suffer spontaneous morphologic and phenotypic EMT-like changes, including cytoskeleton reorganization, vimentin and Slug up-regulation, cadherin switching, and diffuse cytosolic relocalization of the catenins. Moreover, these phenotypic changes are associated with modifications in the global genetic differentiation program characteristic of the EMT process. In summary, our data indicate that in breast tumors, EMT likely occurs within a specific genetic context, the basal phenotype, and suggests that this proclivity to mesenchymal transition may be related to the high aggressiveness and the characteristic metastatic spread of these tumors. [Cancer Res 2008;68(4):989–97]

Supplementary Tables 1-3 from Inactivation of the Candidate Tumor Suppressor Par-4 in Endometrial Cancer

Supplementary Figure 1 from SNAI1 Is Required for Tumor Growth and Lymph Node Metastasis of Human Breast Carcinoma MDA-MB-231 Cells

The transcription factors Snail, Slug, and bHLH E47 have been recently described as direct repressors of E-cadherin and inducers of epithelial-mesenchymal transition (EMT) and invasion when overexpressed in epithelial cells. Although a role of those factors in tumor progression and invasion has been proposed, whether the different repressors play distinct or redundant roles in the tumorigenic process has not been established. To further investigate this important issue, we have analyzed the gene expression profiling of Madin-Darby canine kidney (MDCK) epithelial cells expressing the different repressors (MDCK-Snail, MDCK-Slug, and MDCK-E47 cells) versus control MDCK cells by cDNA microarrays. A total of 243 clones (228 genes and 15 expressed sequence tags) were found to be differentially expressed between either of the three MDCK-derived cell lines and control MDCK cells. Twenty two of the candidate genes were validated by Northern blot, Western blot, immunofluorescence, and promoter analyses in cell lines and by immunohistochemistry in xenografted tumors. Gene clustering analysis indicated that about a third of the 243 candidate genes were common to MDCK cells expressing Snail, Slug, or E47 factors, whereas the rest of the genes were regulated in only one or two cell types. Differentially regulated genes include those related to EMT (45 genes), transcriptional regulation (18 genes), cell proliferation and signaling (54 genes), apoptosis (12 genes), and angiogenesis (9 genes). These results indicate that Snail, Slug, and E47 transcription factors induce common and specific genetic programs, supporting a differential role of the factors in tumor progression and invasion. (Cancer Res 2006; 66(19): 9543-56)

Supplementary Figure 3 from Lysyl Oxidase–Like 2 as a New Poor Prognosis Marker of Squamous Cell Carcinomas

Supplementary Figure 2 from SNAI1 Is Required for Tumor Growth and Lymph Node Metastasis of Human Breast Carcinoma MDA-MB-231 Cells

Supplementary Table 1 from Lysyl Oxidase–Like 2 as a New Poor Prognosis Marker of Squamous Cell Carcinomas

Supplementary Table 2 from Lysyl Oxidase–Like 2 as a New Poor Prognosis Marker of Squamous Cell Carcinomas

Supplementary Methods and Materials from SNAI1 Is Required for Tumor Growth and Lymph Node Metastasis of Human Breast Carcinoma MDA-MB-231 Cells

Supplementary Figure 5 from SNAI1 Is Required for Tumor Growth and Lymph Node Metastasis of Human Breast Carcinoma MDA-MB-231 Cells

Supplementary Table 4 from Epithelial-Mesenchymal Transition in Breast Cancer Relates to the Basal-like Phenotype

The transcription factor, SNAI1 (Snail), has recently been proposed as an important mediator of tumor invasion because of its role in E-cadherin down-regulation and induction of epithelial-mesenchymal transition. In human breast cancer, the expression of SNAI1 and/or the homologous SNAI2 (Slug) has been associated with E-cadherin repression, local or distant metastasis, tumor recurrence, or poor prognosis in different tumor series. However, the specific contribution of either factor to breast tumor progression is still unclear. We have analyzed the role of SNAI1 in human breast cancer by loss of function studies and provide evidence of a major role for SNAI1 in both primary tumor growth and metastasis of human breast carcinoma MDA-MB-231 cells. Specific silencing of SNAI1 by short hairpin RNA induces a decrease in mesenchymal and proinvasive markers (MMP9, ID1, SPARC) in MDA-MB-231 cells, concomitant with reduced in vitro invasive behavior. More importantly, stable SNAI1 silencing in MDA-MB-231 cells leads to a dramatic reduction of in vivo tumor incidence and growth rate. Tumors induced by MDA-MB-231-SNAI1–silenced cells show extensive necrotic regions and a significant decrease in invasive and angiogenic markers. Moreover, SNAI1 silencing increases the sensitivity of MDA-MB-231 cells to chemotherapeutics relevant in breast cancer treatments, gemcitabine and docetaxel. Remarkably, analysis of cell lines derived from lymph node metastasis indicates that SNAI1 expression is required for metastatic dissemination. [Cancer Res 2007;67(24):11721–31]

In this work we propose the use of putrescine, a precursor for higher polyamine biosynthesis for the preparation of cationic nanosystems for cancer gene therapy.

Correction for ‘Effectiveness of a novel gene nanotherapy based on putrescine for cancer treatment’ by Saínza Lores et al., Biomater. Sci., 2023, https://doi.org/10.1039/d2bm01456d.

Germline and tumor BRCA testing constitutes a valuable tool for clinical decision-making in the management of epithelial ovarian cancer (EOC) patients. Tissue testing is able to identify both germline (g) and somatic (s) BRCA variants, but tissue preservation methods and the widespread implementation of NGS represent pre-analytical and analytical challenges that need to be managed. This study was carried out on a multicenter prospective GEICO cohort of EOC patients with known gBRCA status in order to determine the inter-laboratory reproducibility of tissue sBRCA testing. The study consisted of two independent experimental approaches, a bilateral comparison between two reference laboratories (RLs) testing 82 formalin-paraffin-embedded (FFPE) EOC samples each, and a Ring Test Trial (RTT) with five participating clinical laboratories (CLs) evaluating the performance of tissue BRCA testing in a total of nine samples. Importantly, labs employed their own locally adopted next-generation sequencing (NGS) analytical approach. BRCA mutation frequency in the RL sub-study cohort was 23.17%: 12 (63.1%) germline and 6 (31.6%) somatic. Concordance between the two RLs with respect to BRCA status was 84.2% (gBRCA 100%). The RTT study distributed a total of nine samples (three commercial synthetic human FFPE references, three FFPE, and three OC DNA) among five CLs. The median concordance detection rate among them was 64.7% (range: 35.3–70.6%). Analytical discrepancies were mainly due to the minimum variant allele frequency thresholds, bioinformatic pipeline filters, and downstream variant interpretation, some of them with consequences of clinical relevance. Our study demonstrates a wide range of concordance in the identification and interpretation of BRCA sequencing data, highlighting the relevance of establishing standard criteria for detecting, interpreting, and reporting BRCA variants., This research was funded by Astra Zéneca Farmacéutica Spain SA (Grant Number GEICO60-0).

Germline and tumor

The formation of Gasdermin (GSDM) pores, leading to pyroptosis or other context-dependent consequences, is directly involved in multiple diseases. Gasdermin-B (GSDMB) plays complex and controversial roles in pathologies, with pyroptosis-dependent and independent functions. GSDMB is promising oncologic therapeutic target since it exhibits either antitumor function, when immunocyte-mediated Granzyme-A (GZMA) cleaves GSDMB releasing its cytotoxic N-terminal domain, or pro-tumoral activities (invasion, metastasis, and drug resistance). However, it is still unknown the precise regulatory mechanisms of GSDMB pyroptosis as well as the differential effects of the four translated GSDMB variants (GSDMB1-4, that differ in the alternative usage of exons 6-7) in this process. Here, we first prove that exon 6 translation (in the interdomain protein linker) is essential for pyroptosis, and therefore, GSDMB isoforms lacking this exon (GSDMB1-2) cannot provoke cancer cell death. Consistently, in large series of breast cancer samples GSDMB2 expression, and not of exon6-containing variants (GSDMB3-4), associates with unfavourable clinical-pathological parameters. Moreover, cellular, and biochemical analyses combined with confocal, live cell imaging, and electron microscopy studies, demonstrated that diverse GSDMB N-terminal constructs containing exon-6 induce mitochondrial damage (increased mitochondrial ROS, membrane potential loss and mitochondrial DNA release) together with pyroptotic membrane cell lysis. While exon-6 residues are not required for membrane or mitochondrial localization, we also identified other key residues for N-terminal domain cytotoxicity. Additionally, we demonstrated that all GSDMB variants share the cleavage sites for GZMA, Neutrophil Elastase (identified in this study) and caspases. Interestingly, whereas Neutrophil Elastase and caspases produce N-terminal fragments in all GSDMB isoforms with no pyroptotic activity, thus acting as a potential inhibitory mechanism, GZMA cleavage activates pyroptosis in an isoform-dependent way. Summarizing, our results have important implications for understanding the complex roles of GSDMB isoforms in cancer and other pathologies and for the future design of GSDMB-targeted therapies.

[Background and purpose]: Ovarian cancer (OC) is the deadliest gynaecologic cancer characterised by a high heterogeneity not only at the clinical point of view but also at the molecular level. This review focuses on the new insights about the OC molecular classification., [Materials and methods]: We performed a bibliographic search for different indexed articles focused on the new molecular classification of OC. All of them have been published in PubMed and included information about the most frequent molecular alterations in OC confirmed by omics approaches. In addition, we have extracted information about the role of liquid biopsy in the OC diagnosis and prognosis., [Results]: New molecular insights into OC have allowed novel clinical entities to be defined. Among OC, high-grade serous ovarian carcinoma (HGSOC) which is the most common OC is characterised by omics approaches, mutations in TP53 and in other genes involved in the homologous recombination repair, especially BRCA1/2. Recent studies in HGSOC have allowed a new molecular classification in subgroups according to their mutational, transcriptional, methylation and copy number variation signatures with a real impact in the characterisation of new therapeutic targets for OC to be defined. Furthermore, despite the intrinsic intra-tumour heterogeneity, the advances in next generation sequencing (NGS) analyses of ascetic liquid from OC have opened new ways for its characterisation and treatment., [Conclusions]: The advances in genomic approaches have been used for the identification of new molecular profiling techniques which define OC subgroups and has supposed advances in the diagnosis and in the personalised treatment of OC., 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, Grant AECC-2019) to GMB.

Leptin receptor (Ob-R)/leptin axis significantly modulates tumour-infiltrating lymphocytes (TILs) and PD-1 expression in early HER2+ breast cancer (BC) emerging as a new surrogate marker for immunotherapy. Background: There is strong pre-clinical evidence that obesity produces T-cell dysfunction and high PD-1 expression resulting in a paradoxical benefit from immunotherapy. This effect is driven, at least in part, by leptin that exerts its action through binding Ob-R, which is known to be highly expressed in HER2+ BC. TILs correlate with pathological response and long-term outcomes in BC; however the precise mechanism by which these T-cells are activated in and around tumour remains partially unknown. The primary aim of this study was to investigate the role of Ob-R/leptin axis in modulating TILs and PD-1 expression and its effect in pathological response in early HER2+ BC patients who have received neoadjuvant systemic treatment (NST). Methods: Women with HER2+ BC receiving anti-HER2-based NST followed by surgical resection were evaluated. Patient’s height and weight were measured before NST to calculate the body mass index (BMI). Based on the IHC results in diagnostic biopsy, tumors were categorized as HER2+/HR+ and HER2+/HR-. Ob-R expression was routinely measured in the diagnostic biopsy using the BOND RX Research Platform (Leica Biosystems). The Ob-R was classified as over-expressed if there were more than 50% positive cells with weak or strong staining. TILs and PD-1 expression were scored centrally in pre-treatment biopsy. TILs were considered as binary, < 30.0% versus ≥30.0% and PD-1 positive (>1%). Associations with pathological complete response (pCR; ypT0/isN0) were assessed using chi-squared or Wilcoxon test. Results: Of the 74 HER2+ BC patients included in the study, 47 (63.5%) had over-expression of Ob-R, 26 (35.1%) were overweight/obese (BMI ≥25kg/m2), and 42 (56.8%) had pCR status. Ob-R expression was similar regardless of menopausal status, age or HR expression. Patients with Ob-R overexpressed were 21 (80.8%) of 26 with BMI ≥25kg/m2 versus 26 (54.2%) of 48 with BMI < 25Kg/m2 (p=0.023). Tumors with Ob-R overexpressed had significantly higher mean levels of TILs than those with non-overexpressed Ob-R (21.4% [IQR, 7.5-30] vs 12.4% [IQR, 5-10]; p=0.009). Despite higher rates of TILs, the rate of pCR in Ob-R overexpressed tumours (57.4% [27 of 47 patients]) was not higher than in non-overexpressed tumours (55.6% [15 of 27 patients]; p=0.874). This could be due to the fact that Ob-R-overexpressed tumours had a significant higher median PD-1 expression than Ob-R-negative tumours (2% [IQR, 0.5-7.5] vs 0% [IQR, 0-1]; p< 0.001). Finally, no differences were found in terms of Ob-R expression and pathological response by hormone receptor expression. Conclusions: This multidisciplinary clinical study decodes for the first time how obesity, through the OB-R/leptin axis, might activate TILs but apparently dysfunctional as it is not translated into higher pCR; probably due to the presence of exhausted features such as high PD-1 expression. The role of Ob-R together with PD-1 as a potential biomarker for immunotherapy should be further explored. Citation Format: Laura García-Estévez, Adela Fernández, José Palacios, Miguel Sampayo, Isabel Calvo, Eva Díaz, Marta González, Belén Pérez-Míes, Silvia González, José Manuel Pérez-García, Javier Cortés, Gema Moreno-Bueno. Leptin receptor (Ob-R)/leptin axis significantly modulates tumour-infiltrating lymphocytes (TILs) and PD-1 expression in early HER2+ breast cancer (BC) emerging as a new surrogate marker for immunotherapy. [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 P5-02-38.

© 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-).

Gasdermins (

© 2021 The Authors., Cancer stem cells (CSC) are considered responsible for tumor initiation, therapeutic resistance, and metastasis. A comprehensive knowledge of the mechanisms governing the acquisition and maintenance of cancer stemness is crucial for the development of new therapeutic approaches in oncology. E2A basic helix–loop–helix (bHLH) transcription factors are associated with epithelial–mesenchymal transition (EMT) and tumor progression, but knowledge of their functional contributions to cancer biology is still limited. Using a combination of in vivo and in vitro analyses in a novel PyMT-E2A conditional knockout mouse model and derived primary tumor cell lines, we report here an essential role of E2A in stemness, metastasis, and therapeutic resistance in breast cancer. Targeted deletion of E2A in the mammary gland impaired tumor-initiating ability and dedifferentiation potential and severely compromised metastatic competence of PyMT-driven mammary tumors. Mechanistic studies in PyMT-derived cell lines indicated that E2A actions are mediated by the upregulation of Snai1 transcription. Importantly, high E2A and SNAIL1 expression occurred in aggressive human basal-like breast carcinomas, highlighting the relevance of the E2A–Snail1 axis in metastatic breast cancer. In addition, E2A factors contributed to the maintenance of genomic integrity and resistance to PARP inhibitors in PyMT and human triple-negative breast cancer cells. Collectively, these results support the potential for E2A transcription factors as novel targets worthy of translational consideration in breast cancer., This work has been supported by grants from the Spanish Ministry of Economy and Innovation (SAF2013–44739-R, SAF2016–76504-R to A. Cano and F. Portillo), PID2019–111052RB-I00 to F. Portillo, PID2019–104644RB-I00 to G. Moreno-Bueno; the Spanish Institute of Health Carlos III (CIBERONC-CB16/12/00295 to A. Cano, G. Moreno-Bueno; all of them partly supported by FEDER funds); the AECC Scientific Foundation 2019 (PROYE19036MOR to G. Moreno-Bueno); the Association International for Cancer Research (AICR; 12–1057 to A. Cano, P.G. Santamaría); and Worldwide Cancer Research (16–0295 to A. Cano, F. Portillo, and P.G. Santamaría). A. Vázquez-Naharro was funded by a PhD contract from SAF2016–76504-R grant; V. Santos by AICR (12–1057) and SAF2016–76504-R grants; P.G. Santamaría by a research contract of the AECC Scientific Foundation, and from Worldwide Cancer Research (16–0295) and SAF2016–76504-R grants.

[Objective]: The lysyl oxidase-like protein 2 (LOXL2) contributes to tumour progression and metastasis in different tumour entities, but its role in pancreatic ductal adenocarcinoma (PDAC) has not been evaluated in immunocompetent in vivo PDAC models., [Design]: Towards this end, we used PDAC patient data sets, patient-derived xenograft in vivo and in vitro models, and four conditional genetically-engineered mouse models (GEMMS) to dissect the role of LOXL2 in PDAC. For GEMM-based studies, K-Ras +/LSL-G12D;Trp53 LSL-R172H;Pdx1-Cre mice (KPC) and the K-Ras +/LSL-G12D;Pdx1-Cre mice (KC) were crossed with Loxl2 allele floxed mice (Loxl2Exon2 fl/fl) or conditional Loxl2 overexpressing mice (R26Loxl2 KI/KI) to generate KPCL2KO or KCL2KO and KPCL2KI or KCL2KI mice, which were used to study overall survival; tumour incidence, burden and differentiation; metastases; epithelial to mesenchymal transition (EMT); stemness and extracellular collagen matrix (ECM) organisation., [Results]: Using these PDAC mouse models, we show that while Loxl2 ablation had little effect on primary tumour development and growth, its loss significantly decreased metastasis and increased overall survival. We attribute this effect to non-cell autonomous factors, primarily ECM remodelling. Loxl2 overexpression, on the other hand, promoted primary and metastatic tumour growth and decreased overall survival, which could be linked to increased EMT and stemness. We also identified tumour-associated macrophage-secreted oncostatin M (OSM) as an inducer of LOXL2 expression, and show that targeting macrophages in vivo affects Osm and Loxl2 expression and collagen fibre alignment., [Conclusion]: Taken together, our findings establish novel pathophysiological roles and functions for LOXL2 in PDAC, which could be potentially exploited to treat metastatic disease., JCL-G received support from a 'la Caixa' Foundation (ID 100010434) fellowship (LCF/BQ/DR21/11880011). This study was supported by ISCIII FIS grants PI18/00757 and PI21/01110 (BSJ) and PI18/00267 (LG-B), and grants from the Spanish Ministry of Economy and Innovation SAF2016-76504-R (ACan and FP), PID2019-111052RB-I00 (FP), PID2019-104644RB-I00 (GM-B), a Ramón y Cajal Merit Award RYC-2012–12104 (BSJ) and ISCIII, CIBERONC, CB16/12/00446 (ACar) and CB16/12/00295 (ACan and GM-B), all of them co-financed through Fondo Europeo de Desarrollo Regional (FEDER) 'Una manera de hacer Europa'; a Fero Foundation Grant (BSJ); a Coordinated grant (GC16173694BARB) from the Fundación Científica Asociación Española Contra el Cáncer (FC-AECC) (BSJ); a Miguel Servet award (CP16/00121) (PS); a DFG, German Research Foundation Grant—Project no: 492 436 553 (KG); and a Max Eder Fellowship of the German Cancer Aid (111746) (PCH)

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.

BackgroundGasdermin-B gene (GSDMB) is frequently over-expressed in tumors, and its shortest translated variant (isoform 2; GSDMB2) increases aggressive behavior in breast cancer cells. Paradoxically, GSDMB could have either pro-tumor or tumor suppressor properties depending on the biological context. Since GSDMB gene is not present in the mouse genome, deciphering fully the functional roles of GSDMB in cancer requires novel in vivo models.MethodsWe first generated by gene targeting a conditional knock-in mouse model (R26-STOP-GB2) harboring human GSDMB2 transcript within the ROSA26 locus. We next derived the R26-GB2 model ubiquitously expressing GSDMB2 in multiple tissues (confirmed by western blot and immunohistochemistry) and performed a comprehensive histopathological analysis in multiple tissues from 75 male and female mice up to 18 months of age. Additionally, we produced the double transgenic model R26-GB2/MMTV-PyMT, co-expressing GSDMB2 and the Polyoma-Middle-T oncogene, and assessed breast cancer generation and progression in GSDMB2-homozygous (n=10) and control (n=17) female mice up to 15 weeks of age.ResultsIn the R26-GB2 model, which showed different GSDMB2 cytoplasmic and/or nuclear localization among tissues, we investigated if GSDMB2 expression had intrinsic tumorigenic activity. 41% of mice developed spontaneous lung tumors, but neither the frequency nor the histology of these neoplasias was significantly different from wildtype animals. Strikingly, while 17% control mice developed gastric carcinomas, no GSDMB2-positive mice did. No other tumor types or additional histological alterations were frequently seen in these mice. In the R26-GB2/MMTV-PyMT model, the strong nucleus-cytoplasmic GSDMB2 expression in breast cancer cells did not significantly affect cancer formation (number of tumors, latency, tumor weight, histology or proliferation) or lung metastasis potential compared to controls.ConclusionsGSDMB2 expression alone does not have an overall tumorigenic potential in mice, but it might reduce gastric carcinogenesis. Contrary to human cancers, GSDMB2 upregulation does not significantly affect breast cancer generation and progression in mouse models. However, to evidence the GSDMB functions in cancer and other pathologies in vivo may require the presence of specific stimulus or cellular contexts. Our novel mouse strains will serve as the basis for the future development of more precise tissue-specific and context-dependent cancer models.

The prognosis and quality of life of HER2 breast cancer patients have significantly improved due to the crucial clinical benefit of various anti-HER2 targeted therapies. However, HER2 tumors can possess or develop several resistance mechanisms to these treatments, thus leaving patients with a limited set of additional therapeutic options. Fortunately, to overcome this problem, in recent years, multiple different and complementary approaches have been developed (such as antibody–drug conjugates (ADCs)) that are in clinical or preclinical stages. In this review, we focus on emerging strategies other than on ADCs that are either aimed at directly target the HER2 receptor (i.e., novel tyrosine kinase inhibitors) or subsequent intracellular signaling (e.g., PI3K/AKT/mTOR, CDK4/6 inhibitors, etc.), as well as on innovative approaches designed to attack other potential tumor weaknesses (such as immunotherapy, autophagy blockade, or targeting of other genes within the HER2 amplicon). Moreover, relevant technical advances such as anti-HER2 nanotherapies and immunotoxins are also discussed. In brief, this review summarizes the impact of novel therapeutic approaches on current and future clinical management of aggressive HER2 breast tumors.

Endometrial cancer (EC) is the 4th most common neoplasm of the female genital tract, with 15–20% of patients being of high risk of recurrence which leads to a significant decrease in patient survival. Current therapeutic options for patients with EC are poor, being the combined therapy of carboplatin and paclitaxel the standard of care, with limited efficacy. Therefore, new therapeutic options and better monitoring tools are needed to improve the management of the disease. In the current case report, we showcase the value of liquid biopsy analyses in a microsatellite instability EC patient with initially good prognosis that however underwent rapid progression disease within 6 months post-surgery; through the study of plasma cfDNA/ctDNA dynamics to assess the tumour evolution during treatment, as well as the study of the uterine aspirate as a valuable sample that captures the intra-tumour heterogeneity that allows a comprehensive genomic profiling of the disease to identify potential therapeutic options. Furthermore, preclinical models were generated at the time of tumour progression to assess the efficacy of the identified targeted therapies.

© The Author(s) 2021., [Background]: The promoter hypermethylation of the methylguanine-DNA methyltransferase gene is a frequently used biomarker in daily clinical practice as it is associated with a favorable prognosis in glioblastoma patients treated with temozolamide. Due to the absence of adequately standardized techniques, international harmonization of the MGMT methylation biomarker is still an unmet clinical need for the diagnosis and treatment of glioblastoma patients. [Results]: In this study we carried out a clinical validation of a quantitative assay for MGMT methylation detection by comparing a novel quantitative MSP using double-probe (dp_qMSP) with the conventional MSP in 100 FFPE glioblastoma samples. We performed both technologies and established the best cutoff for the identification of positive-methylated samples using the quantitative data obtained from dp_qMSP. Kaplan–Meier curves and ROC time dependent curves were employed for the comparison of both methodologies. [Conclusions]: We obtained similar results using both assays in the same cohort of patients, in terms of progression free survival and overall survival according to Kaplan–Meier curves. In addition, the results of ROC(t) curves showed that dp_qMSP increases the area under curve time-dependent in comparison with MSP for predicting progression free survival and overall survival over time. We concluded that dp_qMSP is an alternative methodology compatible with the results obtained with the conventional MSP. Our assay will improve the therapeutic management of glioblastoma patients, being a more sensitive and competitive alternative methodology that ensures the standardization of the MGMT-biomarker making it reliable and suitable for clinical use., This study was supported by the “Fondo de Investigación Sanitaria-Instituto de Salud Carlos III” PI18/00050, DTS20/00029 and the European Regional Development Fund/European Social Fund FIS FEDER/FSE, Una Manera de Hacer Europa.

Introduction: mTOR inhibitors are anticancer agents affecting mTOR/AKT/PI3K pathway that is one of the most important in human cancer cells. Hyperactivation of mTOR/AKT/PI3K and overexpression of this pathway members are frequently reported in uterine sarcoma and carcinosarcoma. Present study is aimed to assess the activity of the two mTOR inhibitors (rapamycin - RAP and sapanisertib - MLN) as a single agent and combined with gemcitabine (GEM, one of substances commonly used in systemic anticancer treatment) in uterine sarcoma and carcinosarcoma in vitro models. Material and methods: SK-UT-1 and SK-UT1-B (uterine carcinosarcoma), MES-SA (leiomyosarcoma) and ESS-1 (endometrial stromal sarcoma) cell lines were used. An MTT assay was performed to examine the cytotoxicity of RAP, MLN and mixtures: RAP+MLN, RAP+GEM, MLN+GEM against these cells. The interactions between tested compounds were assessed in isobolographic analysis. Results and conclusions: Carcinosarcoma cell lines (both SK-UT-1 and SK-UT-1B) do not respond to RAP and respond relatively weakly to MLN treatment. Additive and supraadditive effects were noted for combined treatment with GEM and MLN. Endometrial stromal sarcoma cell line (ESS-1) occured to be sensitive to both RAP and MLN, but the response was stronger for MLN. Additive effect of all tested drug combinations was observed for ESS-1. Leiomyosarcoma cell line (MES-SA) was found sensitive to both mTOR inhibitors. Additive effects in combinations of GEM, RAP and MLN were observed, what makes them promising for future preclinical and clinical trials. Additivity with slight tendency towards antagonism between GEM and MLN observed in MES-SA cell line is unexpected finding and might prompt the mechanistic research aimed to explain this phenomenon.

Malignant melanoma is a highly aggressive tumor causing most skin cancer-related deaths. Understanding the fundamental mechanisms responsible for melanoma progression and therapeutic evasion is still an unmet need for melanoma patients. Progression of skin melanoma and its dissemination to local or distant organs relies on phenotypic plasticity of melanoma cells, orchestrated by EMT-TFs and microphthalmia-associated TF (MITF). Recently, melanoma phenotypic switching has been proposed to uphold context-dependent intermediate cell states benefitting malignancy. LOXL3 (lysyl oxidase-like 3) promotes EMT and has a key role in human melanoma cell survival and maintenance of genomic integrity. To further understand the role of Loxl3 in melanoma, we generated a conditional Loxl3-knockout (KO) melanoma mouse model in the context of BrafV600E-activating mutation and Pten loss. Melanocyte-Loxl3 deletion increased melanoma latency, decreased tumor growth, and reduced lymph node metastatic dissemination. Complementary in vitro and in vivo studies in mouse melanoma cells confirmed Loxl3’s contribution to melanoma progression and metastasis, in part by modulating phenotypic switching through Snail1 and Prrx1 EMT-TFs. Importantly, a novel LOXL3-SNAIL1-PRRX1 axis was identified in human melanoma, plausibly relevant to melanoma cellular plasticity. These data reinforced the value of LOXL3 as a therapeutic target in melanoma., This research was funded by grants from the Spanish Ministry of Science and Innovation MCIN (SAF2016-76504-R to A.C. and F.P., PID2019-111052RB-100 to F.P., and PID2019–104644RBI00 to G.M.-B.), Instituto de Salud Carlos III (CIBERONC-CB16/12/00295 to A.C. and G.M.-B.), all of them partly supported by EU-FEDER funds, Worldwide Cancer Research UK (grant ref. 16-0295 to A.C., F.P., and P.G.S.), and FC AECC (Grupos Estables de Investigación 2018-AECC and PROYE19036MOR to G.M.-B., and 2015-AECC to À.F., GCB15152978SOEN). S.S.O. was supported by a 2019 AECC postdoctoral contract. A.V.-N. was supported by a FPI predoctoral fellowship from MCIN (BES-2017-081082). J.B.-T. was supported by a FPI fellowship associated with SAF2013-44739-R (A.C. and F.P.).

Traditionally, studies to address the characterization of mechanisms promoting tumor aggressiveness and progression have been focused only on primary tumor analyses, which could provide relevant information but have limitations to really characterize the more aggressive tumor population. To overcome these limitations, circulating tumor cells (CTCs) represent a noninvasive and valuable tool for real-time profiling of disseminated tumor cells. Therefore, the aim of the present study was to explore the value of CTC enumeration and characterization to identify markers associated with the outcome and the aggressiveness of triple-negative breast cancer (TNBC). For that aim, the CTC population from 32 patients diagnosed with TNBC was isolated and characterized. This population showed important cell plasticity in terms of expression of epithelia/mesenchymal and stemness markers, suggesting the relevance of epithelial to mesenchymal transition (EMT) intermediate phenotypes for efficient tumor dissemination. Importantly, the CTC signature demonstrated prognostic value to predict the patients&rsquo, outcome and pointed to a relevant role of tissue inhibitor of metalloproteinases 1 (TIMP1) and androgen receptor (AR) for TNBC biology. Furthermore, we also analyzed the usefulness of the AR and TIMP1 blockade to target TNBC proliferation and dissemination using in vitro and in vivo zebra fish and mouse models. Overall, the molecular characterization of CTCs from advanced TNBC patients identifies highly specific biomarkers with potential applicability as noninvasive prognostic markers and reinforced the value of TIMP1 and AR as potential therapeutic targets to tackle the most aggressive breast cancer.

The incidence and mortality of endometrial cancer (EC) have risen in recent years, hence more precise management is needed. Therefore, wecombined di erent types of liquid biopsies to better characterize the genetic landscape of EC in a non-invasive and dynamic manner. Uterine aspirates (UAs) from 60 patients with EC were obtained during surgery and analyzed by next-generation sequencing (NGS). Blood samples, collected at surgery, were used for cell-free DNA (cfDNA) and circulating tumor cell (CTC) analyses. Finally, personalized therapies were tested in patient-derived xenografts (PDXs) generated from the UAs. NGS analyses revealed the presence of genetic alterations in 93% of the tumors. Circulating tumor DNA (ctDNA) was present in 41.2% of cases, mainly in patients with high-risk tumors, thus indicating a clear association with a more aggressive disease. Accordingly, the results obtained during the post-surgery follow-up indicated the presence of ctDNA in three patients with progressive disease. Moreover, 38.9% of patients were positive for CTCs at surgery. Finally, the e cacy of targeted therapies based on the UA-specific mutational landscape was demonstrated in PDX models. Our study indicates the potential clinical applicability of a personalized strategy based on a combination of different liquid biopsies to characterize and monitor tumor evolution, and to identify targeted therapies, This work was supported by grants and support from the Instituto de Salud Carlos III (ISCIII) and FEDER (PI17/01919, PI17/02071), CIBERONC (CB16/12/00328), and the AECC (Grupos Estables de Investigacion 2018-AECC) to A.G.-M. and M.A.; 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.M.-B.; and the AECC to L.M.-R.

[Objectives]: The function of NOTCH signaling (oncogenic or oncosuppressive) remains controversial in head and neck squamous cell carcinomas (HNSCC). The purpose of this work is to investigate the role of NOTCH pathway in HNSCC prognosis., [Methods]: Immunohistochemical NOTCH1 and HES1 expression was jointly evaluated and correlated with other NOTCH1 targets, p21 (WAF1/Cip1) and Cyclin D1, using an unbiased cohort of 372 surgically treated HPV-negative HNSCC patients., [Results]: Membranous NOTCH1 expression was detected in 197 (61%) out of 324 evaluable tumor samples, and nuclear NOTCH1 expression in 91 samples (28%). Nuclear HES1 expression was found in 224 (67%) cases. Membranous and nuclear NOTCH1 expression were consistently and significantly correlated with nuclear HES1 (P < 0.001) and p21 (P = 0.03) expression, but not with Cyclin D1. NOTCH1 expression was significantly associated to early stages (I-II), non-recurrent disease, and better disease-specific (DSS) and overall survival (OS) rates (P < 0.001). Moreover, triple-positive cases (NOTCH1+/HES1+/p21+) exhibited significantly improved DSS (P < 0.001) and OS (P = 0.004), thus reinforcing the association of NOTCH pathway activation with a better prognosis in HNSCC. Multivariate analysis further revealed membranous NOTCH1 expression as a robust independent predictor of better DSS (HR = 0.554; 95% IC 0.412–0.745; P < 0.001) and better OS (HR = 0.640; 95% CI 0.491–0.835; P = 0.001)., [Conclusion]: These findings show the association of NOTCH pathway activation with a better prognosis in HNSCC patients, also revealing membranous NOTCH1 expression as a robust independent predictor of improved survival. Accordingly, these results suggest a tumor suppressive rather than an oncogenic role for NOTCH pathway in HNSCC., This study was supported by grants from the Plan Nacional de I+D+I 2013–2016 [ISCIII (PI16/00280 and PI19/00560 to JMGP), CIBERONC (CB16/12/00390 to JPR and CB16/12/00295 to AC), Asociación Española contra el Cáncer (AECC; 2019/ INVES19001ALVA to MAF), the Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Ayudas a Grupos PCTI Principado de Asturias (IDI2018/155 to JPR), Fundación Bancaria Caja de Ahorros de Asturias-IUOPA and the FEDER Funding Program from the European Union.

© 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).

Therapy resistance is responsible for tumour recurrence and represents one of the major challenges in present oncology. Significant advances have been made in the understanding of the mechanisms underlying resistance to conventional and targeted therapies improving the clinical management of relapsed patients. Unfortunately, in too many cases, resistance reappears leading to a fatal outcome. The recent introduction of immunotherapy regimes has provided an unprecedented success in the treatment of specific cancer types; however, a good percentage of patients do not respond to immune-based treatments or ultimately become resistant. Cellular plasticity, cancer cell stemness and tumour heterogeneity have emerged as important determinants of treatment resistance. Epithelial-to-mesenchymal transition (EMT) is associated with resistance in many di erent cellular and preclinical models, although little evidence derives directly from clinical samples. The recognition of the presence in tumours of intermediate hybrid epithelial/mesenchymal states as the most likely manifestation of epithelial plasticity and their potential link to stemness and tumour heterogeneity, provide new clues to understanding resistance and could be exploited in the search for anti-resistance strategies. Here, recent evidence linking EMT/epithelial plasticity to resistance against conventional, targeted and immune therapy are summarized. In addition, future perspectives for related clinical approaches are also discussed, This work was supported by grants from the Spanish Ministerio de Economía y Competividad (SAF2013-44739-R, SAF2016-76504-R) and Instituto de Salud Carlos III (CIBERONC 16/12/00295; PI16/00134), all of them partly supported by EU-FEDER fund, FC AECC (Grupos Estables de Investigación 2018-AECC) and Worldwide Cancer Research UK (formerly AICR, 12-1057 and 16-0295)

© 2021 by the authors., The primary aim of this retrospective study was to investigate the correlation between the immunohistochemical expression of Ob-R (leptin receptor) with pCR (pathological complete response) in early breast cancer patients receiving neoadjuvant systemic treatment (NST). A total of 100 women with breast cancer receiving NST (2017–2020) followed by surgical resection were retrospectively obtained. Demographic parameters and clinicopathological factors (e.g., treatment modalities, immunohistochemistry (IHC), and cancer subtype) were obtained from the patient’s clinical records. In the analyzed breast cancer cohort, high expression of Ob-R was found in 52% of tumors and there was a significantly higher incidence in the HER2+ and TNBC subgroups. Overall, a significantly greater percentage of patients with Ob-R positive tumors achieved pCR compared with Ob-R negative patients (57.7% vs. 27.1%; p = 0.002). This result was observed in most breast cancer subtypes. In patients with HER2+ breast cancer, there was no difference in Ob-R expression in relation to the HR status. Ob-R cell positivity was significantly higher in younger breast cancer patients (p = 0.008), those who were premenopausal (p = 0.011), and in those with a BMI > 25 kg/m2 (p = 0.019). A significantly greater percentage of early breast cancer patients with Ob-R positive tumors achieved pCR compared with Ob-R negative patients. Furthermore, breast cancer patients with positive Ob-R expression were significantly younger than those with negative Ob-R expression. This association was not explained by differences in BMI between young and old patients., This study has been supported by the Spanish Ministry of Economy and Innovation (PID2019- 104644RB-I00 -GMB-, the Instituto de Salud Carlos III (CIBERONC, CB16/12/00295 -GMBl partly supported by FEDER funds]) and by the AECC Scientific Foundation (FC_AECC PROYE19036MOR -GMB-). Sara S Oltra work has been funded by the AECC (Ayudas para Investigadores en Oncología 2020).

In developed countries, endometrial carcinoma is the most common cancer that affects the female genital tract. Endometrial carcinoma is divided into two main histological types, type I or endometrioid and type II or non-endometrioid, each of which have characteristic, although not exclusive, molecular alterations and mutational profiles. Nevertheless, information about the implication and relevance of some of these genes in this disease is lacking. We sought here to identify new recurrently mutated genes in endometrioid cancers that play a role in tumourigenesis and that influence the clinical outcome. We focused on low-grade, non-ultramutated tumours as these tumours have a worse prognosis than the ultramutated POLE-positive endometrioid endometrial carcinomas (EECs). We performed exome-sequencing of 11 EECs with matched normal tissue and subsequently validated 15 candidate genes in 76 samples. For the first time, we show that mutations in chromatin remodelling-related genes (KMT2D, KMT2C, SETD1B and BCOR) and in DNA-repair-related genes (BRCA1, BRCA2, RAD50 and CHD4) are frequent in this subtype of endometrial cancer. The alterations to these genes occurred with frequencies ranging from 35.5% for KMT2D to 10.5% for BRCA1 and BCOR, with some showing a tendency toward co-occurrence (RAD50-KMT2D and RAD50-SETD1B). All these genes harboured specific mutational hotspots. In addition, the mutational status of KMT2C, KMT2D and SETD1B helps to predict the degree of myometrial invasion, a critical prognostic feature. These results highlight the possible implication of these genes in this disease, creating opportunities for new therapeutic approaches.

Endometrial cancer is the most common gynaecological cancer in western countries, being the most common subtype of endometrioid tumours. Most patients are diagnosed at an early stage and present an excellent prognosis. However, a number of those continue to suffer recurrence, without means of identification by risk classification systems. Thus, finding a reliable marker to predict recurrence becomes an important unmet clinical issue. ALCAM is a cell-cell adhesion molecule and member of the immunoglobulin superfamily that has been associated with the genesis of many cancers. Here, we first determined the value of ALCAM as a marker of recurrence in endometrioid endometrial cancer by conducting a retrospective multicentre study of 174 primary tumours. In early-stage patients (N = 134), recurrence-free survival was poorer in patients with ALCAM-positive compared to ALCAM-negative tumours (HR 4.237; 95% CI 1.01-17.76). This difference was more significant in patients with early-stage moderately-poorly differentiated tumours (HR 9.259; 95% CI 2.12-53.47). In multivariate analysis, ALCAM positivity was an independent prognostic factor in early-stage disease (HR 6.027; 95% CI 1.41-25.74). Then we demonstrated in vitro a role for ALCAM in cell migration and invasion by using a loss-of-function model in two endometrial cancer cell lines. ALCAM depletion resulted in a reduced primary tumour size and reduced metastatic local spread in an orthotopic murine model. Gene expression analysis of ALCAM-depleted cell lines pointed to motility, invasiveness, cellular assembly, and organization as the most deregulated functions. Finally, we assessed some of the downstream effector genes that are involved in ALCAM-mediated cell migration; specifically FLNB, TXNRD1, and LAMC2 were validated at the mRNA and protein level. In conclusion, our results highlight the potential of ALCAM as a recurrent biomarker in early-stage endometrioid endometrial cancer and point to ALCAM as an important molecule in endometrial cancer dissemination by regulating cell migration, invasion, and metastasis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

PHARMACOLOGY Correction for "Therapeutic targeting of HER2-CB2R heteromers in HER2-positive breast cancer," by Sandra Blasco-Benito, Estefania Moreno, Marta Seijo-Vila, Isabel Tundidor, Clara Andradas, Maria M. Caffarel, Miriam Caro-Villalobos, Leyre Uriguen, Rebeca Diez-Alarcia, Gema Moreno-Bueno, Lucia Hernandez, Luis Manso, Patricia Homar-Ruano, Peter J. McCormick, Lucka Bibic, Cristina Bernado-Morales, Joaquin Arribas, Meritxell Canals, Vicent Casado, Enric I. Canela, Manuel Guzman, Eduardo Perez-Gomez, and Cristina Sanchez, which was first published February 7, 2019; 10.1073/pnas.1815034116 (Proc Natl Acad Sci USA 116:3863-3872). The authors note that, due to a printer's error, the affiliation for Lucka Bibic appeared incorrectly. It should instead appear as School of Pharmacy, University of East Anglia, Norwich, Norfolk NR4 7TJ, United Kingdom. The corrected author and affiliation lines appear below. The online version has been corrected.

Erratum in: Correction for Blasco-Benito et al., Therapeutic targeting of HER2-CB2R heteromers in HER2-positive breast cancer., Although human epidermal growth factor receptor 2 (HER2)-targeted therapies have dramatically improved the clinical outcome of HER2-positive breast cancer patients, innate and acquired resistance remains an important clinical challenge. New therapeutic approaches and diagnostic tools for identification, stratification, and treatment of patients at higher risk of resistance and recurrence are therefore warranted. Here, we unveil a mechanism controlling the oncogenic activity of HER2: heteromerization with the cannabinoid receptor CB2R. We show that HER2 physically interacts with CB2R in breast cancer cells, and that the expression of these heteromers correlates with poor patient prognosis. The cannabinoid Δ9-tetrahydrocannabinol (THC) disrupts HER2-CB2R complexes by selectively binding to CB2R, which leads to (i) the inactivation of HER2 through disruption of HER2-HER2 homodimers, and (ii) the subsequent degradation of HER2 by the proteasome via the E3 ligase c-CBL. This in turn triggers antitumor responses in vitro and in vivo. Selective targeting of CB2R transmembrane region 5 mimicked THC effects. Together, these findings define HER2-CB2R heteromers as new potential targets for antitumor therapies and biomarkers with prognostic value in HER2-positive breast cancer., This work was supported by grants from the Spanish Ministry of Economy and Competitiveness [supported with European Regional Development funds: PI14/01101 and PI17/00041 (to C.S. and E.P.-G.), and PI16/00134 (to G.M.-B.); CIBERONC (CB16/12/00295, to G.M.-B.); Fundació La Marató TV3 (20140610, to E.I.C.); Fundación Asociación Española Contra el Cáncer (to E.P.-G.); Zelda Therapeutics (to C.S. and M.G.); and Fundación Mutua Madrileña (2013/0072, to L.M.)]. S.B.-B. and I.T. are recipients of a Formación de Profesorado Universitario and pFIS fellowship (from the Spanish Ministry of Economy and Competitiveness). P.J.M. would like to acknowledge funding from the Biotechnology and Biological Sciences Research Council (BBSRC) Doctoral Training Programme.

Ovarian cancer is the most lethal gynecological malignancy due to the silent nature on its early onset and the rapid acquisition of drug resistance. Histologically heterogeneous, it includes several subtypes with different mutational landscapes, hampering the development of effective targeted therapies. Non-coding RNAs are emerging as potential new therapeutic targets in cancer. To search for a microRNA signature related to ovarian carcinomas and study its potential as effective targeted therapy, we examined the expression of 768 miRNA in a large collection of tumor samples and found miR-654-5p to be infraexpressed in ovarian serous carcinomas, the most common and aggressive type. Restoration of miR-654-5p levels reduced tumor cell viability in vitro and in vivo and impaired sphere formation capacity and viability of ovarian cancer patient-derived ascitic cells ex vivo. CDCP1 and PLAGL2 oncogenes were found to be the most relevant direct miR-654-5p targets and both genes convey in a molecular signature associated with key cancer pathways relevant to ovarian tumorigenesis, such as MYC, WNT and AKT pathways. Together, we unveiled the tumor suppressor function of miR-654-5p, suggesting that its restoration or co-targeting of CDCP1 and PLAGL2 may be an effective therapeutic approach for ovarian cancer., This work was supported in part by grants from Instituto de la Mujer Dexeus (DEXEUS-B29/012), CIBER (CB16/12/00328), SGR (2017 SGR 1661), the Ministerio de Economia y Competitividad and Fondos FEDER (RTC-2015-3821-1), Instituto Carlos III (PI15/00238 to A.S. and PI17/00564 to M.F.S) and the Miguel Servet Program (CP13/00158 and CPII18/00027 to AS. and CPII16/00006 to MFS). AP and LS were supported by predoctoral VHIR fellowships and CJ by an AGAUR predoctoral fellowship (VHIR: PRED-VHIR-2014-11 and PRED-VHIR-2017; AGAUR: 2017FI_B_00095, respectively).

[Background]: Breast cancer is the second most common cause of cancer-related death among women. Advances in our understanding of the disease have translated into better diagnostics and more effective therapeutics, leading to earlier detection and improved outcomes. Several studies have pointed at lifestyle and environmental factors as contributory for the onset and progression of the disease. Obesity and cholesterol stand out for their potential causal relationship with breast cancer and ease of modification., [Main text]: Obesity and cholesterol represent risk factors for breast cancer, but their impact is largely affected by cofounding variables including menopausal status, disease subtype, and inflammation. Establishing a causal relationship between lifestyle factors and clinical outcomes may be challenging. Epidemiological studies and meta-analyses have rendered conflicting or sometimes contradictory results, possibly owing to the multifactorial nature of the disease. We discuss the supporting evidence and limitations in our understanding of obesity and cholesterol as risk factors for breast cancer., [Conclusions]: There is sufficient evidence that obesity and cholesterol impact clinical outcomes. Physicians are advised to take steps to help patients with their weight, such as recommending dietary and lifestyle interventions., This work was supported by grants from the Instituto de Salud Carlos III (ISCIII) and FEDER (PI16/00134 and CIBERONC CB16/12/00295).

© 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).

Prostaglandin (PG) F2αhas been scarcely studied in cancer. We have identified a new role for PGF2αin ovarian cancer, stimulating the production of TGFβ and the consequent induction of PMEPA1. We show that this induction increases cell plasticity and proliferation, enhancing tumor growth through PMEPA1. Thus, PMEPA1 overexpression in ovarian carcinoma cells, significantly increased cell proliferation rates, whereas PMEPA1 silencing decreased proliferation. In addition, PMEPA1 overexpression buffered TGFβ signaling, via reduction of SMAD-dependent signaling. PMEPA1 overexpressing cells acquired an epithelial morphology, associated to higher E-cadherin expression levels while β-catenin nuclear translocation was inhibited. Interestingly, in mouse xenografts, PMEPA1 overexpressing ovarian cells had a clear survival and proliferative advantage, resulting in higher metastatic capacity, while PMEPA1 silencing had the opposite effect. Furthermore, high PMEPA1 expression in a cohort of advanced ovarian cancer patients was observed, correlating with E-cadherin expression. Most importantly, high PMEPA1 mRNA levels were associated with lower patient survival.SignificanceThis work identifies Prostaglandin F2αas an inducer, through NFAT, of TGFβ and together with this up-regulate PMEPA1, which is identified both as a drug target and as a prognostic biomarker in ovarian tumors, potentially useful in clinical practice.

Although Ras genes are frequently mutated in human tumors, these mutations are uncommon in breast cancer. However, many breast tumors show evidences of Ras pathway activation. In this manuscript, we have analyzed and characterized mouse mammary tumors generated by random Sleeping Beauty transposon mutagenesis and identify ERAS -a member of the RAS family silenced in adult tissues- as a new gene involved in progression and malignancy of breast cancer. Forced expression of ERAS in human non-transformed mammary gland cells induces a process of epithelial-to-mesenchymal transition and an increase in stem cells markers; these changes are mediated by miR-200c downregulation. ERAS expression in human tumorigenic mammary cells leads to the generation of larger and less differentiated tumors in xenotransplant experiments. Immunohistochemical, RT-qPCR and bioinformatics analysis of human samples show that ERAS is aberrantly expressed in 8–10% of breast tumors and this expression is associated with distant metastasis and reduced metastasis-free survival. In summary, our results reveal that inappropriate activation of ERAS may be important in the development of a subset of breast tumors. These findings open the possibility of new specific treatments for this subset of ERAS-expressing tumors., This research was supported partially by funds from Fondo Europeo de Desarrollo Regional (FEDER) and by grants from the Spanish Government PI16/00161, PI16/00134, PIE15/00076, PI17/00578, CB/16/00228, CB16/12/00295 and RD12/0036/0009 from Instituto de Salud Carlos III (Ministerio de Economía y Competitividad) and SAF-2015-66015-R from the Ministerio de Economía y Competitividad. Biobanco 1 + 12 is supported by Instituto de Salud Carlos III.

The hair follicle is a biological oscillator that alternates growth, regression, and rest phases driven by the sequential activation of the proliferation/differentiation programs of resident stem cell populations. The activation of hair follicle stem cell niches and subsequent entry into the growing phase is mainly regulated by Wnt/β-catenin signalling, while regression and resting phases are mainly regulated by Tgf-β/Bmp/Smad activity. A major question still unresolved is the nature of the molecular switch that dictates the coordinated transition between both signalling pathways. Here we have focused on the role of Endoglin (Eng), a key co-receptor for members of the Tgf-β/Bmp family of growth factors. Using an Eng haploinsufficient mouse model, we report that Eng is required to maintain a correct follicle cycling pattern and for an adequate stimulation of hair follicle stem cell niches. We further report that β-catenin binds to the Eng promoter depending on Bmp signalling. Moreover, we show that β-catenin interacts with Smad4 in a Bmp/Eng-dependent context and both proteins act synergistically to activate Eng promoter transcription. These observations point to the existence of a growth/rest switching mechanism in the hair follicle that is based on an Eng-dependent feedback cross-talk between Wnt/β-catenin and Bmp/Smad signals., This work was supported by grants from Ministerio de Economía y Competitividad of Spain (RTC-2014-2626-1 to J.E., SAF2013-46183-R to M.Q., and SAF2013-43421-R to C.B.), Comunidad de Madrid (S2010/BMD-2359, SkinModel to J.E. and M.Q.), Instituto de Salud Carlos III (PI15/01458 to J.E.), and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER; ISCIII-CB06/07/0038 to C.B.) financed jointly by the European Regional Development Funds (FEDER). E.C. and M.I.C. were supported by Spanish MECD-FPU and UAM-FPI fellowships, respectively.

Epithelial ovarian cancer is characterized by a low recovery rate because the disease is typically diagnosed at an advanced stage, by which time most patients (80%) already exhibit disseminated neoplasia. The cytokine receptor CXCR4 has been implicated in the development of metastasis in various tumor types. Using a patient-derived tissue macroarray and mRNA expression analysis, we observed high CXCR4 levels in high-grade serous epithelial ovarian carcinomas, the most metastatic tumor, compared with those in endometrioid carcinomas. CXCR4 inhibition by treatment with the CXCR4 antagonist AMD3100 or by expression of shRNA anti-CXCR4 similarly inhibited angiogenesis in several models of ovarian carcinomas orthotopically grown in nude mice, but the effect on tumor growth was correlated with the levels of CXCR4 expression. Moreover, CXCR4 inhibition completely blocked dissemination and metastasis. This effect was associated with reduced levels of active Src, active ERKs, the inhibition of EMT transition, and block of hematogenous ovarian cancer dissemination decreasing circulating human tumoral cells (CTC). In tumors, CXCR4-expressing cells also had more mesenchymal characteristics. In conclusion, our results indicate that CXCR4 expression confers a proinvasive phenotype to ovarian carcinoma cells. Thus, anti-CXCR4 therapy is a possible agent for a complementary treatment of advanced disseminated epithelial high-grade serous ovarian cancer patients., This study was supported by research grants from the Spanish Ministerio de Economía y Competitividad (SAF2013-46063R), The Spanish Institute of Health Carlos III (ISCIII) and the European Regional Development Fund (ERDF) under the Integrated Project of Excellence no. PIE13/00022 (ONCOPROFILE), and the Generalitat de Catalunya (2014SGR364) to F. Vinals. Work supported by the Xarxa de Bancs de Tumors de Catalunya sponsored by Pla Director d'Oncología de Catalunya (XBTC), IDIBELL and PLATAFORMA BIOBANCOSPT13/0010/0013 and for the MD Anderson Foundation Biobank (B.0000745, ISCIII National Biobank Record). Grants from the AECC (Grupos Estables de Investigacion 2011-AECC-GCB 110333 REVE), the Instituto de Salud Carlos III (ISCIII), FEDER (PI16/00134), and CIBERONC (CB16/12/00295) to G. Moreno-Bueno. E. Alsina-Sanchís is a recipient of a predoctoral fellowship from the Ministerio de Economía y Competitividad.

OXFORD UNIVERSITY: This is a pre-copyedited, author-produced version of an article accepted for publication in Carcinogenesis following peer review. The version of record David Rincón-Fernández, Michael D Culler, Natia Tsomaia, Gema Moreno-Bueno, Raúl M Luque, Manuel D Gahete, Justo P Castaño; In1-ghrelin splicing variant is associated with reduced disease-free survival of breast cancer patients and increases malignancy of breast cancer cells lines, Carcinogenesis, Volume 39, Issue 3, 8 March 2018, Pages 447–457, https://doi.org/10.1093/carcin/bgx146, Ghrelin gene generates several variants that regulate multiple pathophysiological functions, including tumor-related processes. In1-ghrelin is a splicing variant that was previously shown to be overexpressed in breast cancer (BCa), where it correlated with proliferation markers; however, its possible association with clinical outcome of BCa patients and underlying mechanisms are still unknown. To address this issue, expression levels and clinical associations of In1-ghrelin were analyzed in a cohort of 117 BCa samples. Additionally, a battery of cellular and molecular assays was implemented using two BCa cell lines (MCF-7 and MDA-MB-231), wherein the role of In1-ghrelin on proliferation, migration, dedifferentiation and signaling pathways was explored. The results generated revealed that high expression of In1-ghrelin in BCa samples was associated with lymph node metastasis and reduced disease-free survival. Indeed, In1-ghrelin overexpression stimulated proliferation and migration in MCF-7 and MDA-MB-231 cells. Similar results were found by treating MDA-MB-231 and MCF-7 with In1- ghrelin-derived peptides. Conversely, In1-ghrelin silencing decreased proliferation and migration capacities of MDA-MB-231. Furthermore, In1-ghrelin (but not ghrelin) overexpression increased the capacity to form mammospheres in both cell lines. These effects could be associated with activation of MAPK-ERK, Jag1/Notch, Wnt/β-catenin and/or TGF-β1 pathways. Altogether, our data indicate that In1-ghrelin could play relevant functional roles in the regulation of BCa development and progression and may provide insights to identify novel biomarkers and new therapeutic approaches for this pathology., BIO-0139, CTS-1406, PI-639-2012, PI-0541-2013 (Junta de Andalucia), BFU2013-43282-R, BFU2016-80360-R (MINECO), PI13-00651, PI16/00264 (Proyectos de Investigación en Salud FIS, funded by Instituto de Salud Carlos III), GETNE Grant 2014, Merck Serono Grant 2013 and CIBERobn (to RML and JPC); PI13/00132, RETICC RD12/0036/0007, CIBERonc and S2010/BMD-2303 (to GMB)