Role of ADAM17 in the non-cell autonomous effects of oncogene-induced senescence

Proteomic analysis of the secretome of p95HER2-induced senescence. a MCF7 Tet-Off p95HER2 cells were cultured with or without doxycycline for 1 week and stained for senescence-associated β-galactosidase. Representative images of the stained cultures are shown. b The secretomes of the same cells as in a were analyzed by label-free quantitative proteomics. The results are shown as unsupervised hierarchical clustering analysis corresponding to three technical replicas (a-c) of two independent experiments (1 and 2). c The proteins identified in b were classified according to the presence of transmembrane or glycophosphatidylinositol domains (cell membrane), signal peptide but not transmembrane domain (secreted, canonical), or the lack of these domains (secreted, unknown). See also Additional file 2: Table S1. Doxy doxycycline. (JPEG 585 kb

HER2 and p95HER2 differentially regulate miRNA expression in MCF-7 breast cancer cells and downregulate MYB proteins through miR-221/222 and miR-503

Mecanismes de la malaltia; Càncer de mamaMecanismos de la enfermedad; Cáncer de mamaDisease Mechanisms; Breast CancerThe HER2 oncogene and its truncated form p95HER2 play central roles in breast cancer. Here, we show that although HER2 and p95HER2 generally elicit qualitatively similar changes in miRNA profile in MCF-7 breast cancer cells, a subset of changes are distinct and p95HER2 shifts the miRNA profile towards the basal breast cancer subtype. High-throughput miRNA profiling was carried out 15, 36 and 60 h after HER2 or p95HER2 expression and central hits validated by RT-qPCR. miRNAs strongly regulated by p95HER2 yet not by HER2, included miR-221, miR-222, miR-503, miR-29a, miR-149, miR-196 and miR-361. Estrogen receptor-α (ESR1) expression was essentially ablated by p95HER2 expression, in a manner recapitulated by miR-221/-222 mimics. c-Myb family transcription factors MYB and MYBL1, but not MYBL2, were downregulated by p95HER2 and by miR-503 or miR-221/-222 mimics. MYBL1 3′UTR inhibition by miR-221/222 was lost by deletion of a single putative miR-221/222 binding sites. p95HER2 expression, or knockdown of either MYB protein, elicited upregulation of tissue inhibitor of matrix metalloprotease-2 (TIMP2). miR-221/222 and -503 mimics increased, and TIMP2 knockdown decreased, cell migration and invasion. A similar pathway was operational in T47D- and SKBr-3 cells. This work reveals important differences between HER2- and p95HER2- mediated miRNA changes in breast cancer cells, provides novel mechanistic insight into regulation of MYB family transcription factors by p95HER2, and points to a role for a miR-221/222– MYB family–TIMP2 axis in regulation of motility in breast cancer cells.This work was supported by the Danish Council for Independent Research (grants no. 12-126942 and 12-127290 to SFP), by the Hartmann foundation (SFP), Fondation Juchum (SFP), Kirsten og Freddy Johansens Fond (SFP), the Breast Cancer Research Foundation (BCRF-17-008) (JA), Instituto de Salud Carlos III (PI16/00253) (JA) and the Harboe foundation (SFP). Katrine Franklin Mark is gratefully acknowledged for excellent technical assistance. We are grateful to Pascal Pineau from Institut Pasteur, France for the MYBL1 3′UTR/psiCHECK2 construct

Modeling anti-IL-6 therapy using breast cancer patient-derived xenografts

The pleiotropic cytokine IL-6 accelerates the progression of breast cancer in a variety of preclinical models through the activation of the STAT3 (signal transducer and activator of transcription 3) signaling pathway. However, the proportion of breast cancers sensitive to anti-IL-6 therapies is not known. This study evaluates the efficacy of anti-IL-6 therapies using breast cancer patient derived xenografts (PDXs). During the generation of our collection of PDXs, we showed that the successful engraftment of tumor tissue in immunodeficient mice correlates with bad prognosis. Four PDXs out of six were resistant to anti-IL-6 therapies and the expression of IL-6, its receptor or the levels of phospho-STAT3 (the active form of the signal transducer) did not correlate with sensitivity. Using cell cultures established from the PDXs as well as samples from in vivo treatments, we showed that only tumors in which the activation of STAT3 depends on IL-6 respond to the blocking antibodies. Our results indicate that only a fraction of breast tumors are responsive to anti-IL-6 therapies. In order to identify responsive tumors, a functional assay to determine the dependence of STAT3 activation on IL-6 should be performed

The target antigen determines the mechanism of acquired resistance to T cell-based therapies

Cancer; Antigen; ResistanceCáncer; Antígeno; ResistenciaCàncer; Antigen; ResistènciaDespite the revolution of immunotherapy in cancer treatment, patients eventually progress due to the emergence of resistance. In this scenario, the selection of the tumor antigen can be decisive in the success of the clinical response. T cell bispecific antibodies (TCBs) are engineered molecules that include binding sites to the T cell receptor and to a tumor antigen. Using gastric CEA+/HER2+ MKN45 cells and TCBs directed against CEA or HER2, we show that the mechanism of resistance to a TCB is dependent on the tumor antigen. Acquired resistant models to a high-affinity-CEA-targeted TCB exhibit a reduction of CEA levels due to transcriptional silencing, which is reversible upon 5-AZA treatment. In contrast, a HER2-TCB resistant model maintains HER2 levels and exhibit a disruption of the interferon-gamma signaling. These results will help in the design of combinatorial strategies to increase the efficacy of cancer immunotherapies and to anticipate and overcome resistances.This work was supported by Asociación Española Contra el Cancer (AECC), Breast Cancer Research Foundation (BCRF-21-008), and Instituto de Salud Carlos III (PI19/01181). A.M.S. was funded by the Spanish Government (PFIS FI20/00188). B.M. was funded by a fellowship from PERIS (Departament de Salut, Generalitat de Catalunya). M.R.A. was funded by Agency for Management of University and Research Grants (AGAUR, 2022 FI_B2 00080). P.O.R. was funded by the BBVA. E.J.A. was funded by the AECC (POSTD211413AREN). VHIO acknowledges the Cellex Foundation for providing research facilities and equipment, the Centro de Investigación Biomédica en Red de Cáncer (CIBERONC) from the Institute of Health Carlos III (ISCIII), and the Department of Health (Generalitat de Catalunya, SLT008/18/00198 SLT008/18/00205) for their support on this research. The authors acknowledge financial support from the State Agency for Research (Agencia Estatal de Investigación) (CEX2020-001024-S/AEI/10.13039/501100011033) and for the Cancer Immunology and Immunotherapy (CAIMI-2) program funded by BBVA Foundation. We would like to remark the funding from B.M PERIS (Spain). The authors thank Dr. Anne Freimoser-Grundschober and Roche for helping provide the TCBs. The graphical abstract was created with BioRender.com

Therapy-Induced Senescence Enhances the Efficacy of HER2-Targeted Antibody–Drug Conjugates in Breast Cancer

Eficàcia; Conjugats de fàrmacs; Càncer de mamaEficacia; Conjugados de medicamentos; Cáncer de mamaEfficacy; Drug conjugates; Breast cancerAntibody–drug conjugates (ADC) are antineoplastic agents recently introduced into the antitumor arsenal. T-DM1, a trastuzumab-based ADC that relies on lysosomal processing to release the payload, is approved for HER2-positive breast cancer. Next-generation ADCs targeting HER2, such as [vic-]trastuzumab duocarmazine (SYD985), bear linkers cleavable by lysosomal proteases and membrane-permeable drugs, mediating a bystander effect by which neighboring antigen-negative cells are eliminated. Many antitumor therapies, like DNA-damaging agents or CDK4/6 inhibitors, can induce senescence, a cellular state characterized by stable cell-cycle arrest. Another hallmark of cellular senescence is the enlargement of the lysosomal compartment. Given the relevance of the lysosome to the mechanism of action of ADCs, we hypothesized that therapies that induce senescence would potentiate the efficacy of HER2-targeting ADCs. Treatment with the DNA-damaging agent doxorubicin and CDK4/6 inhibitor induced lysosomal enlargement and senescence in several breast cancer cell lines. While senescence-inducing drugs did not increase the cytotoxic effect of ADCs on target cells, the bystander effect was enhanced when HER2-negative cells were cocultured with HER2-low cells. Knockdown experiments demonstrated the importance of cathepsin B in the enhanced bystander effect, suggesting that cathepsin B mediates linker cleavage. In breast cancer patient-derived xenografts, a combination treatment of CDK4/6 inhibitor and SYD985 showed improved antitumor effects over either treatment alone. These data support the strategy of combining next-generation ADCs targeting HER2 with senescence-inducing therapies for tumors with heterogenous and low HER2 expression. Significance: Combining ADCs against HER2-positive breast cancers with therapies that induce cellular senescence may improve their therapeutic efficacy by facilitating a bystander effect against antigen-negative tumor cells.This work was supported by Breast Cancer Research Foundation (BCRF-20-008), Instituto de Salud Carlos III (project reference numbers AC15/00062, CB16/12/00449 and PI19/01181), the EC under the framework of the ERA-NET TRANSCAN-2 initiative co-financed by FEDER, Fundación Mutua Madrileña and Asociación Española Contra el Cáncer. S. Duro-Sánchez is supported by the Spanish Ministerio de Universidades by the grant Formación de Profesorado Universitario (FPU20/05388). A. Esteve-Codina is funded by ISCIII /MINECO (PT17/0009/0019) and co-funded by FEDER. The authors acknowledge Alyson MacInnes for reviewing and editing the article

RANK signaling increases after anti-HER2 therapy contributing to the emergence of resistance in HER2-positive breast cancer

Background: Around 15-20% of primary breast cancers are characterized by HER2 protein overexpression and/or HER2 gene amplification. Despite the successful development of anti-HER2 drugs, intrinsic and acquired resistance represents a major hurdle. This study was performed to analyze the RANK pathway contribution in HER2-positive breast cancer and anti-HER2 therapy resistance. Methods: RANK and RANKL protein expression was assessed in samples from HER2-positive breast cancer patients resistant to anti-HER2 therapy and treatment-naive patients. RANK and RANKL gene expression was analyzed in paired samples from patients treated with neoadjuvant dual HER2-blockade (lapatinib and trastuzumab) from the SOLTI-1114 PAMELA trial. Additionally, HER2-positive breast cancer cell lines were used to modulate RANK expression and analyze in vitro the contribution of RANK signaling to anti-HER2 resistance and downstream signaling. Results: RANK and RANKL proteins are more frequently detected in HER2-positive tumors that have acquired resistance to anti-HER2 therapies than in treatment-naive ones. RANK (but not RANKL) gene expression increased after dual anti-HER2 neoadjuvant therapy in the cohort from the SOLTI-1114 PAMELA trial. Results in HER2-positive breast cancer cell lines recapitulate the clinical observations, with increased RANK expression observed after short-term treatment with the HER2 inhibitor lapatinib or dual anti-HER2 therapy and in lapatinib-resistant cells. After RANKL stimulation, lapatinib-resistant cells show increased NF-κB activation compared to their sensitive counterparts, confirming the enhanced functionality of the RANK pathway in anti-HER2-resistant breast cancer. Overactivation of the RANK signaling pathway enhances ERK and NF-κB signaling and increases lapatinib resistance in different HER2-positive breast cancer cell lines, whereas RANK loss sensitizes lapatinib-resistant cells to the drug. Our results indicate that ErbB signaling is required for RANK/RANKL-driven activation of ERK in several HER2-positive cell lines. In contrast, lapatinib is not able to counteract the NF-κB activation elicited after RANKL treatment in RANK-overexpressing cells. Finally, we show that RANK binds to HER2 in breast cancer cells and that enhanced RANK pathway activation alters HER2 phosphorylation status. Conclusions: Our data support a physical and functional link between RANK and HER2 signaling in breast cancer and demonstrate that increased RANK signaling may contribute to the development of lapatinib resistance through NF-κB activation. Whether HER2-positive breast cancer patients with tumoral RANK expression might benefit from dual HER2 and RANK inhibition therapy remains to be elucidated

Effect of p95HER2/611CTF on the Response to Trastuzumab and Chemotherapy

Human epidermal growth factor receptor 2 (HER2)–positive breast cancers are currently treated with trastuzumab, an anti-HER2 antibody. About 30% of these tumors express a group of HER2 fragments collectively known as p95HER2. Our previous work indicated that p95HER2-positive tumors are resistant to trastuzumab monotherapy. However, recent results showed that tumors expressing the most active of these fragments, p95HER2/611CTF, respond to trastuzumab plus chemotherapy. To clarify this discrepancy, we analyzed the response to chemotherapy of cell lines transfected with p95HER2/611CTF and patient-derived xenografts (n = 7 mice per group) with different levels of the fragment. All statistical tests were two-sided. p95HER2/611CTF-negative and positive tumors showed different responses to various chemotherapeutic agents, which are particularly effective on p95HER2/611CTF-positive cells. Furthermore, chemotherapy sensitizes p95HER2/611CTF-positive patient-derived xenograft tumors to trastuzumab (mean tumor volume, trastuzumab alone: 906mm3, 95% confidence interval = 1274 to 538 mm3; trastuzumab+doxorubicin: 259mm3, 95% confidence interval = 387 to 131 mm3; P < .001). This sensitization may be related to HER2 stabilization induced by chemotherapy in p95HER2/611CTF-positive cells

New adipokines vaspin and omentin. Circulating levels and gene expression in adipose tissue from morbidly obese women

<p>Abstract</p> <p>Background</p> <p>Vaspin and omentin are recently described molecules that belong to the adipokine family and seem to be related to metabolic risk factors. The objectives of this study were twofold: to evaluate vaspin and omentin circulating levels and mRNA expression in subcutaneous and visceral adipose tissues in non-diabetic morbidly obese women; and to assess the relationship of vaspin and omentin with anthropometric and metabolic parameters, and other adipo/cytokines.</p> <p>Design</p> <p>We analysed vaspin and omentin circulating levels in 71 women of European descent (40 morbidly obese [BMI ≥ 40 kg/m²] and 31 lean [BMI ≤ 25]). We assessed vaspin and omentin gene expression in paired samples of visceral and subcutaneous abdominal adipose tissue from 46 women: 40 morbidly obese and 6 lean. We determined serum vaspin and plasma omentin levels with an Enzyme-Linked Immunosorbent Assay and adipose tissue mRNA expression by real time RT-PCR.</p> <p>Results</p> <p>Serum vaspin levels in the morbidly obese were not significantly different from those in controls. They correlated inversely with levels of lipocalin 2 and interleukin 6. Vaspin mRNA expression was significantly higher in the morbidly obese, in both subcutaneous and visceral adipose tissue.</p> <p>Plasma omentin levels were significantly lower in the morbidly obese and they correlated inversely with glucidic metabolism parameters. Omentin circulating levels, then, correlated inversely with the metabolic syndrome (MS). Omentin expression in visceral adipose tissue was significantly lower in morbidly obese women than in controls.</p> <p>Conclusions</p> <p>The present study indicates that vaspin may have a compensatory role in the underlying inflammation of obesity. Decreased omentin circulating levels have a close association with MS in morbidly obese women.</p

Analysis of the transcriptional activity of endogenous NFAT5 in primary cells using transgenic NFAT-luciferase reporter mice

<p>Abstract</p> <p>Background</p> <p>The transcription factor NFAT5/TonEBP regulates the response of mammalian cells to hypertonicity. However, little is known about the physiopathologic tonicity thresholds that trigger its transcriptional activity in primary cells. Wilkins et al. recently developed a transgenic mouse carrying a luciferase reporter (9xNFAT-Luc) driven by a cluster of NFAT sites, that was activated by calcineurin-dependent NFATc proteins. Since the NFAT site of this reporter was very similar to an optimal NFAT5 site, we tested whether this reporter could detect the activation of NFAT5 in transgenic cells.</p> <p>Results</p> <p>The 9xNFAT-Luc reporter was activated by hypertonicity in an NFAT5-dependent manner in different types of non-transformed transgenic cells: lymphocytes, macrophages and fibroblasts. Activation of this reporter by the phorbol ester PMA plus ionomycin was independent of NFAT5 and mediated by NFATc proteins. Transcriptional activation of NFAT5 in T lymphocytes was detected at hypertonic conditions of 360–380 mOsm/kg (isotonic conditions being 300 mOsm/kg) and strongly induced at 400 mOsm/kg. Such levels have been recorded in plasma in patients with osmoregulatory disorders and in mice deficient in aquaporins and vasopressin receptor. The hypertonicity threshold required to activate NFAT5 was higher in bone marrow-derived macrophages (430 mOsm/kg) and embryonic fibroblasts (480 mOsm/kg). Activation of the 9xNFAT-Luc reporter by hypertonicity in lymphocytes was insensitive to the ERK inhibitor PD98059, partially inhibited by the PI3-kinase inhibitor wortmannin (0.5 μM) and the PKA inhibitor H89, and substantially downregulated by p38 inhibitors (SB203580 and SB202190) and by inhibition of PI3-kinase-related kinases with 25 μM LY294002. Sensitivity of the reporter to FK506 varied among cell types and was greater in primary T cells than in fibroblasts and macrophages.</p> <p>Conclusion</p> <p>Our results indicate that NFAT5 is a sensitive responder to pathologic increases in extracellular tonicity in T lymphocytes. Activation of NFAT5 by hypertonicity in lymphocytes was mediated by a combination of signaling pathways that differed from those required in other cell types. We propose that the 9xNFAT-Luc transgenic mouse model might be useful to study the physiopathological regulation of both NFAT5 and NFATc factors in primary cells.</p

Regulation of NFAT5 by signaling pathways involved in osmotic stress responses and cell growth

NFAT5 is the main regulator of an osmoprotective gene program that is switched on by osmotic stress. Hypertonicity causes a harmful increase in the intracellular concentration of inorganic ions and NFAT5-regulated genes allow the accumulation of organic osmolytes and chaperones in order to protect the cells. However, little is known about the physiopathologic tonicity thresholds that trigger NFAT5 transcriptional activity, and which signaling pathways are involved, in primary cells. We have studied the regulation of NFAT5 transcriptional activity in several types of primary cells obtained from transgenic mice carrying the 9xNFAT-Luc reporter developed by the Molkentin laboratory. Our results indicate that NFAT5 is able to respond to pathological tonicity levels in primary cells and it requires a combination of signaling mediators, some of which are more relevant in specific cell types