RESUMO
Epidermal growth factor receptor (EGFR) is a well-exploited therapeutic target in metastatic colorectal cancer (mCRC). Unfortunately, not all patients benefit from current EGFR inhibitors. Mass spectrometry-based proteomics and phosphoproteomics were performed on 30 genomically and pharmacologically characterized mCRC patient-derived xenografts (PDXs) to investigate the molecular basis of response to EGFR blockade and identify alternative drug targets to overcome resistance. Both the tyrosine and global phosphoproteome as well as the proteome harbored distinctive response signatures. We found that increased pathway activity related to mitogen-activated protein kinase (MAPK) inhibition and abundant tyrosine phosphorylation of cell junction proteins, such as CXADR and CLDN1/3, in sensitive tumors, whereas epithelial-mesenchymal transition and increased MAPK and AKT signaling were more prevalent in resistant tumors. Furthermore, the ranking of kinase activities in single samples confirmed the driver activity of ERBB2, EGFR, and MET in cetuximab-resistant tumors. This analysis also revealed high kinase activity of several members of the Src and ephrin kinase family in 2 CRC PDX models with genomically unexplained resistance. Inhibition of these hyperactive kinases, alone or in combination with cetuximab, resulted in growth inhibition of ex vivo PDX-derived organoids and in vivo PDXs. Together, these findings highlight the potential value of phosphoproteomics to improve our understanding of anti-EGFR treatment and response prediction in mCRC and bring to the forefront alternative drug targets in cetuximab-resistant tumors.
Assuntos
Antineoplásicos , Neoplasias do Colo , Neoplasias Colorretais , Humanos , Anticorpos Monoclonais Humanizados/uso terapêutico , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Cetuximab/uso terapêutico , Neoplasias Colorretais/metabolismo , Resistencia a Medicamentos Antineoplásicos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Transdução de Sinais , Fosfoproteínas , ProteomaRESUMO
High-grade mucinous colorectal cancer (HGM CRC) is particularly aggressive, prone to metastasis and treatment resistance, frequently accompanied by "signet ring" cancer cells. A sizeable fraction of HGM CRCs (20-40%) arises in the context of the Lynch Syndrome, an autosomal hereditary syndrome that predisposes to microsatellite instable (MSI) CRC. Development of patient-derived preclinical models for this challenging subtype of colorectal cancer represents an unmet need in oncology. We describe here successful propagation of preclinical models from a case of early-onset, MSI-positive metastatic colorectal cancer in a male Lynch syndrome patient, refractory to standard care (FOLFOX6, FOLFIRI-Panitumumab) and, surprisingly, also to immunotherapy. Surgical material from a debulking operation was implanted in NOD/SCID mice, successfully yielding one patient-derived xenograft (PDX). PDX explants were subsequently used to generate 2D and 3D cell cultures. Histologically, all models resembled the tumor of origin, displaying a high-grade mucinous phenotype with signet ring cells. For preclinical exploration of alternative treatments, in light of recent findings, we considered inhibition of the proteasome by bortezomib and of the related NEDD8 pathway by pevonedistat. Indeed, sensitivity to bortezomib was observed in mucinous adenocarcinoma of the lung, and we previously found that HGM CRC is preferentially sensitive to pevonedistat in models with low or absent expression of cadherin 17 (CDH17), a differentiation marker. We therefore performed IHC on the tumor and models, and observed no CDH17 expression, suggesting sensitivity to pevonedistat. Both bortezomib and pevonedistat showed strong activity on 2D cells at 72 hours and on 3D organoids at 7 days, thus providing valid options for in vivo testing. Accordingly, three PDX cohorts were treated for four weeks, respectively with vehicle, bortezomib and pevonedistat. Both drugs significantly reduced tumor growth, as compared to the vehicle group. Interestingly, while bortezomib was more effective in vitro, pevonedistat was more effective in vivo. Drug efficacy was further substantiated by a reduction of cellularity and of Ki67-positive cells in the treated tumors. These results highlight proteasome and NEDD8 inhibition as potentially effective therapeutic approaches against Lynch syndrome-associated HGM CRC, also when the disease is refractory to all available treatment options.
RESUMO
PURPOSE: Regorafenib (REG) is approved for the treatment of metastatic colorectal cancer, but has modest survival benefit and associated toxicities. Robust predictive/early response biomarkers to aid patient stratification are outstanding. We have exploited biological pathway analyses in a patient-derived xenograft (PDX) trial to study REG response mechanisms and elucidate putative biomarkers. EXPERIMENTAL DESIGN: Molecularly subtyped PDXs were annotated for REG response. Subtyping was based on gene expression (CMS, consensus molecular subtype) and copy-number alteration (CNA). Baseline tumor vascularization, apoptosis, and proliferation signatures were studied to identify predictive biomarkers within subtypes. Phospho-proteomic analysis was used to identify novel classifiers. Supervised RNA sequencing analysis was performed on PDXs that progressed, or did not progress, following REG treatment. RESULTS: Improved REG response was observed in CMS4, although intra-subtype response was variable. Tumor vascularity did not correlate with outcome. In CMS4 tumors, reduced proliferation and higher sensitivity to apoptosis at baseline correlated with response. Reverse phase protein array (RPPA) analysis revealed 4 phospho-proteomic clusters, one of which was enriched with non-progressor models. A classification decision tree trained on RPPA- and CMS-based assignments discriminated non-progressors from progressors with 92% overall accuracy (97% sensitivity, 67% specificity). Supervised RNA sequencing revealed that higher basal EPHA2 expression is associated with REG resistance. CONCLUSIONS: Subtype classification systems represent canonical "termini a quo" (starting points) to support REG biomarker identification, and provide a platform to identify resistance mechanisms and novel contexts of vulnerability. Incorporating functional characterization of biological systems may optimize the biomarker identification process for multitargeted kinase inhibitors.
Assuntos
Neoplasias Colorretais/tratamento farmacológico , Compostos de Fenilureia/uso terapêutico , Piridinas/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Biomarcadores Tumorais , Neoplasias Colorretais/classificação , Neoplasias Colorretais/genética , Modelos Animais de Doenças , Camundongos , Resultado do TratamentoRESUMO
Blockade of epidermal growth factor receptor (EGFR) causes tumor regression in some patients with metastatic colorectal cancer (mCRC). However, residual disease reservoirs typically remain even after maximal response to therapy, leading to relapse. Using patient-derived xenografts (PDXs), we observed that mCRC cells surviving EGFR inhibition exhibited gene expression patterns similar to those of a quiescent subpopulation of normal intestinal secretory precursors with Paneth cell characteristics. Compared with untreated tumors, these pseudodifferentiated tumor remnants had reduced expression of genes encoding EGFR-activating ligands, enhanced activity of human epidermal growth factor receptor 2 (HER2) and HER3, and persistent signaling along the phosphatidylinositol 3-kinase (PI3K) pathway. Clinically, properties of residual disease cells from the PDX models were detected in lingering tumors of responsive patients and in tumors of individuals who had experienced early recurrence. Mechanistically, residual tumor reprogramming after EGFR neutralization was mediated by inactivation of Yes-associated protein (YAP), a master regulator of intestinal epithelium recovery from injury. In preclinical trials, Pan-HER antibodies minimized residual disease, blunted PI3K signaling, and induced long-term tumor control after treatment discontinuation. We found that tolerance to EGFR inhibition is characterized by inactivation of an intrinsic lineage program that drives both regenerative signaling during intestinal repair and EGFR-dependent tumorigenesis. Thus, our results shed light on CRC lineage plasticity as an adaptive escape mechanism from EGFR-targeted therapy and suggest opportunities to preemptively target residual disease.
Assuntos
Neoplasias Colorretais , Fosfatidilinositol 3-Quinases , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Receptores ErbB , Humanos , Recidiva Local de Neoplasia , Neoplasia Residual , Celulas de Paneth , FenótipoRESUMO
BACKGROUND: Targeting the epidermal growth factor receptor (EGFR) either alone or in combination with chemotherapy is an effective treatment for patients with RAS wild-type metastatic colorectal cancer (mCRC). However, only a small percentage of mCRC patients receive clinical benefits from anti-EGFR therapies, due to the development of resistance mechanisms. In this regard, HER2 has emerged as an actionable target in the treatment of mCRC patients with resistance to anti-EGFR therapy. METHODS: We have used SW48 and LIM1215 human colon cancer cell lines, quadruple wild-type for KRAS, NRAS, BRAF and PI3KCA genes, and their HER2-amplified (LIM1215-HER2 and SW48-HER2) derived cells to perform in vitro and in vivo studies in order to identify novel therapeutic strategies in HER2 gene amplified human colorectal cancer. RESULTS: LIM1215-HER2 and SW48-HER2 cells showed over-expression and activation of the HER family receptors and concomitant intracellular downstream signaling including the pro-survival PI3KCA/AKT and the mitogenic RAS/RAF/MEK/MAPK pathways. HER2-amplified cells were treated with several agents including anti-EGFR antibodies (cetuximab, SYM004 and MM151); anti-HER2 (trastuzumab, pertuzumab and lapatinib) inhibitors; anti-HER3 (duligotuzumab) inhibitors; and MEK and PI3KCA inhibitors, such as refametinib and pictilisib, as single agents and in combination. Subsequently, different in vivo experiments have been performed. MEK plus PI3KCA inhibitors treatment determined the best antitumor activity. These results were validated in vivo in HER2-amplified patient derived tumor xenografts from three metastatic colorectal cancer patients. CONCLUSIONS: These results suggest that combined therapy with MEK and PI3KCA inhibitors could represent a novel and effective treatment option for HER2-amplified colorectal cancer.
Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Amplificação de Genes , MAP Quinase Quinase Quinases/antagonistas & inibidores , Proteínas Nucleares/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Receptor ErbB-2/genética , Fatores de Transcrição/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Biomarcadores Tumorais , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , MAP Quinase Quinase Quinases/metabolismo , Camundongos , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Functional genomics approaches can overcome limitations-such as the lack of identification of robust targets and poor clinical efficacy-that hamper cancer drug development. Here we performed genome-scale CRISPR-Cas9 screens in 324 human cancer cell lines from 30 cancer types and developed a data-driven framework to prioritize candidates for cancer therapeutics. We integrated cell fitness effects with genomic biomarkers and target tractability for drug development to systematically prioritize new targets in defined tissues and genotypes. We verified one of our most promising dependencies, the Werner syndrome ATP-dependent helicase, as a synthetic lethal target in tumours from multiple cancer types with microsatellite instability. Our analysis provides a resource of cancer dependencies, generates a framework to prioritize cancer drug targets and suggests specific new targets. The principles described in this study can inform the initial stages of drug development by contributing to a new, diverse and more effective portfolio of cancer drug targets.
Assuntos
Sistemas CRISPR-Cas/genética , Descoberta de Drogas/métodos , Edição de Genes , Terapia de Alvo Molecular/métodos , Neoplasias/genética , Neoplasias/terapia , Animais , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Feminino , Genoma Humano/genética , Humanos , Camundongos , Instabilidade de Microssatélites , Transplante de Neoplasias , Neoplasias/classificação , Neoplasias/patologia , Especificidade de Órgãos , Reprodutibilidade dos Testes , Mutações Sintéticas Letais/genética , Síndrome de Werner/genética , Helicase da Síndrome de Werner/genéticaRESUMO
Background: The NEDD8 conjugation pathway modulates the ubiquitination and activity of a wide range of intracellular proteins, and its blockade by pevonedistat is emerging as a promising therapeutic approach in various cancer settings. However, systematic characterization of pevonedistat efficacy in specific tumor types and definition of response predictors are still missing. Methods: We investigated in vitro sensitivity to pevonedistat in 122 colorectal cancer (CRC) cell lines by an ATP-based proliferation assay and evaluated apoptosis and DNA content by flow cytometry. Associations between pevonedistat sensitivity and CRC molecular features were assessed by Student's t test. A 184-gene transcriptional predictor was generated in cell lines and applied to 87 metastatic CRC samples for which patient-derived xenografts (PDXs) were available. In vivo reponse to pevonedistat was assessed in PDX models (≥5 mice per group). All statistical tests were two-sided. Results: Sixteen (13.1%) cell lines displayed a marked response to pevonedistat, featuring DNA re-replication, proliferative block, and increased apoptosis. Pevonedistat sensitivity did not statistically significantly correlate with microsatellite instability or mutations in KRAS or BRAF and was functionally associated with low EGFR pathway activity. While ineffective on predicted resistant PDXs, in vivo administration of pevonedistat statistically significantly impaired growth of five out of six predicted sensitive models (P < .01). In samples from CRC patients, transcriptional prediction of pevonedistat sensitivity was associated with poor prognosis after surgery (hazard ratio [HR] = 2.49, 95% confidence interval [CI] = 1.34 to 4.62, P = .003) and early progression under cetuximab treatment (HR = 3.59, 95% CI = 1.60 to 8.04, P < .001). Histological and immunohistochemical analyses revealed that the pevonedistat sensitivity signature captures transcriptional traits of poor differentiation and high-grade mucinous adenocarcinoma. Conclusions: These results highlight NEDD8-pathway inhibition by pevonedistat as a potentially effective treatment for poorly differentiated, clinically aggressive CRC.
Assuntos
Adenocarcinoma Mucinoso/tratamento farmacológico , Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Ciclopentanos/farmacologia , Pirimidinas/farmacologia , Transcriptoma , Ubiquitinas/antagonistas & inibidores , Adenocarcinoma Mucinoso/genética , Adenocarcinoma Mucinoso/patologia , Animais , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Fator de Transcrição CDX2/genética , Caderinas/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cetuximab/uso terapêutico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Ciclopentanos/uso terapêutico , Replicação do DNA/efeitos dos fármacos , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Proteínas de Homeodomínio/genética , Humanos , Queratina-20/genética , Camundongos , Proteína NEDD8 , Gradação de Tumores , Transplante de Neoplasias , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Pirimidinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Ubiquitinas/metabolismoRESUMO
Cancer stem cells (CSCs) are key players in bone metastasis. In some renal tumors CSCs overexpress the HGF receptor c-MET, speculating that c-MET targeting could lead to bone metastasis inhibition. To address this hypothesis we isolated renal CD105+/CD24-CSCs, expressing c-MET receptor from a primary renal carcinoma. Then, to study their ability to metastasize to bone, we injected renal CSCs in NOD/SCID mice implanted with a human bone and we tested the effect of a c-MET inhibitor (JNJ-38877605) on bone metastasis development. JNJ-38877605 inhibited the formation of metastases at bone implant site. We showed that JNJ-38877605 inhibited the activation of osteoclasts induced by RCC stem cells and it stimulated osteoblast activity, finally resulting in a reduction of bone turnover consistent with the inhibition of bone metastases. We measured the circulating levels of osteotropic factors induced by RCC stem cells in the sera of mice treated with c-Met inhibitor, showing that IL-11 and CCL20 were reduced in mice treated with JNJ-38877605, strongly supporting the involvement of c-MET in the regulation of this process. To address the clinical relevance of c-MET upregulation during tumor progression, we analysed c-MET in renal cancer patients detecting an increased expression in the bone metastatic lesions by IHC. Then, we dosed CCL20 serum levels resulting significantly increased in patients with bone metastases compared to non-metastatic ones. Collectively, our data highlight the importance of the c-MET pathway in the pathogenesis of bone metastases induced by RCC stem cells in mice and humans.
Assuntos
Neoplasias Ósseas/secundário , Carcinoma de Células Renais/secundário , Neoplasias Renais/patologia , Células-Tronco Neoplásicas/patologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Antineoplásicos/farmacologia , Neoplasias Ósseas/metabolismo , Carcinoma de Células Renais/metabolismo , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Humanos , Neoplasias Renais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/metabolismo , Pirazóis/farmacologia , Piridazinas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Colorectal cancer is the third most common cancer worldwide, with 1.2 million patients diagnosed annually. In late-stage colorectal cancer, the most commonly used targeted therapies are the monoclonal antibodies cetuximab and panitumumab, which prevent epidermal growth factor receptor (EGFR) activation. Recent studies have identified alterations in KRAS and other genes as likely mechanisms of primary and secondary resistance to anti-EGFR antibody therapy. Despite these efforts, additional mechanisms of resistance to EGFR blockade are thought to be present in colorectal cancer and little is known about determinants of sensitivity to this therapy. To examine the effect of somatic genetic changes in colorectal cancer on response to anti-EGFR antibody therapy, here we perform complete exome sequence and copy number analyses of 129 patient-derived tumour grafts and targeted genomic analyses of 55 patient tumours, all of which were KRAS wild-type. We analysed the response of tumours to anti-EGFR antibody blockade in tumour graft models and in clinical settings and functionally linked therapeutic responses to mutational data. In addition to previously identified genes, we detected mutations in ERBB2, EGFR, FGFR1, PDGFRA, and MAP2K1 as potential mechanisms of primary resistance to this therapy. Novel alterations in the ectodomain of EGFR were identified in patients with acquired resistance to EGFR blockade. Amplifications and sequence changes in the tyrosine kinase receptor adaptor gene IRS2 were identified in tumours with increased sensitivity to anti-EGFR therapy. Therapeutic resistance to EGFR blockade could be overcome in tumour graft models through combinatorial therapies targeting actionable genes. These analyses provide a systematic approach to evaluating response to targeted therapies in human cancer, highlight new mechanisms of responsiveness to anti-EGFR therapies, and delineate new avenues for intervention in managing colorectal cancer.
Assuntos
Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/antagonistas & inibidores , Genoma Humano/genética , Genômica , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Cetuximab/farmacologia , Cetuximab/uso terapêutico , Neoplasias Colorretais/metabolismo , Variações do Número de Cópias de DNA/genética , Receptores ErbB/química , Receptores ErbB/genética , Exoma/genética , Feminino , Humanos , Proteínas Substratos do Receptor de Insulina/genética , MAP Quinase Quinase 1/genética , Camundongos , Terapia de Alvo Molecular , Mutação/genética , Panitumumabe , Proteínas Proto-Oncogênicas p21(ras)/genética , Receptor ErbB-2/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Preclinical studies in HER2-amplified gastrointestinal cancer models have shown that cotargeting HER2 with a monoclonal antibody and a small molecule is superior to monotherapy with either inhibitor, but the underlying cooperative mechanisms remain unexplored. We investigated the molecular underpinnings of this synergy to identify key vulnerabilities susceptible to alternative therapeutic opportunities. EXPERIMENTAL DESIGN: The phosphorylation/activation of HER2, HER3, EGFR (HER receptors), and downstream transducers was evaluated in HER2-overexpressing colorectal and gastric cancer cell lines by Western blotting and/or multiplex phosphoproteomics. The in vivo outcome of antibody-mediated HER2 blockade by trastuzumab, reversible HER2 inhibition by lapatinib, and irreversible HER2 inhibition by afatinib was assessed in patient-derived tumorgrafts and cell-line xenografts by monitoring tumor growth curves and by using antibody-based proximity assays. RESULTS: Trastuzumab monotherapy reduced HER3 phosphorylation, with minor consequences on downstream transducers. Lapatinib alone acutely inhibited all HER receptors and effectors but led to delayed rephosphorylation of HER3 and EGFR and partial restoration of ERK and AKT activity. When combined with lapatinib, trastuzumab prevented HER3/EGFR reactivation and caused prolonged inhibition of ERK/AKT. Afatinib alone was also very effective in counteracting the reinstatement of HER3, EGFR, and downstream signaling activation. In vivo, the combination of trastuzumab and lapatinib-or, importantly, monotherapy with afatinib-resulted in overt tumor shrinkage. CONCLUSIONS: Only prolonged inhibition of HER3 and EGFR, achievable by dual blockade with trastuzumab and lapatinib or irreversible HER2 inhibition by single-agent afatinib, led to regression of HER2-amplified gastrointestinal carcinomas. Clin Cancer Res; 21(24); 5519-31. ©2015 AACR.
Assuntos
Antineoplásicos/farmacologia , Carcinoma/genética , Carcinoma/metabolismo , Receptores ErbB/antagonistas & inibidores , Neoplasias Gastrointestinais/genética , Neoplasias Gastrointestinais/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Receptor ErbB-2/genética , Receptor ErbB-3/antagonistas & inibidores , Afatinib , Animais , Carcinoma/tratamento farmacológico , Carcinoma/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Sinergismo Farmacológico , Quimioterapia Combinada , Receptores ErbB/metabolismo , Neoplasias Gastrointestinais/tratamento farmacológico , Neoplasias Gastrointestinais/patologia , Amplificação de Genes , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Lapatinib , Camundongos , Fosforilação , Quinazolinas/administração & dosagem , Quinazolinas/farmacologia , Receptor ErbB-2/metabolismo , Receptor ErbB-3/genética , Receptor ErbB-3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Trastuzumab/administração & dosagem , Trastuzumab/farmacologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
UNLABELLED: The Cancer Genome Atlas project identified HER2 somatic mutations and gene amplification in 7% of patients with colorectal cancer. Introduction of the HER2 mutations S310F, L755S, V777L, V842I, and L866M into colon epithelial cells increased signaling pathways and anchorage-independent cell growth, indicating that they are activating mutations. Introduction of these HER2 activating mutations into colorectal cancer cell lines produced resistance to cetuximab and panitumumab by sustaining MAPK phosphorylation. HER2 mutants are potently inhibited by low nanomolar doses of the irreversible tyrosine kinase inhibitors neratinib and afatinib. HER2 gene sequencing of 48 cetuximab-resistant, quadruple (KRAS, NRAS, BRAF, and PIK3CA) wild-type (WT) colorectal cancer patient-derived xenografts (PDX) identified 4 PDXs with HER2 mutations. HER2-targeted therapies were tested on two PDXs. Treatment with a single HER2-targeted drug (trastuzumab, neratinib, or lapatinib) delayed tumor growth, but dual HER2-targeted therapy with trastuzumab plus tyrosine kinase inhibitors produced regression of these HER2-mutated PDXs. SIGNIFICANCE: HER2 activating mutations cause EGFR antibody resistance in colorectal cell lines, and PDXs with HER2 mutations show durable tumor regression when treated with dual HER2-targeted therapy. These data provide a strong preclinical rationale for clinical trials targeting HER2 activating mutations in metastatic colorectal cancer.
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Neoplasias Colorretais/genética , Mutação , Receptor ErbB-2/genética , Afatinib , Animais , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Transformação Celular Neoplásica/genética , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/tratamento farmacológico , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Receptores ErbB/antagonistas & inibidores , Humanos , Terapia de Alvo Molecular , Mucosa/metabolismo , Mucosa/patologia , Quinazolinas/farmacologia , Quinolinas/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Initially described as an endogenous inhibitor of proteases, the tissue inhibitor of metalloproteinases 1 (TIMP-1) also displays cytokine-like functions. TIMP-1 is a soluble protein whose levels are increased under inflammatory conditions. We recently found that TIMP-1(-/-) mice have decreased bone marrow (BM) cellularity and that the engraftment capability of TIMP-1(-/-) hematopoietic stem cells (HSCs) is impaired, owing to proliferation defects. Here, we investigated the role of recombinant human TIMP-1 (rhTIMP-1) in human hematopoietic stem/progenitor cells (HSPCs) and elucidated the downstream pathway ignited by rhTIMP-1. We found that rhTIMP-1 affects in vitro cell survival, proliferation, and particularly clonogenic expansion of CD34(+) HSPCs without compromising their short-term engraftment potential after transplantation into immunodeficient mice. These effects are independent on matrix metalloproteinase (MMP) inhibition and rely on TIMP-1's binding to the tetraspanin membrane receptor CD63. Further investigation indicated that rhTIMP-1 stimulation induces phosphatidylinositol 3-kinase (PI3K) recruitment and Akt phosphorylation, both presiding over survival/proliferation pathways in HSPCs. Downstream targets of phosphorylated Akt (pAkt) are also modulated, including the proliferation marker cyclin D1 (CycD1), whose levels are increased upon exposure to rhTIMP-1. These findings indicate that rhTIMP-1 promotes clonogenic expansion and survival in human progenitors via the activation of the CD63/PI3K/pAkt signaling pathway, suggesting that TIMP-1 might be a key player in the network of proinflammatory factors modulating HSPC functions.
Assuntos
Células-Tronco Hematopoéticas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Tetraspanina 30/metabolismo , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Animais , Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Ciclina D1/genética , Ciclina D1/metabolismo , Feminino , Células-Tronco Hematopoéticas/citologia , Humanos , Masculino , Camundongos , Camundongos Knockout , Fosfatidilinositol 3-Quinases/genética , Fosforilação/fisiologia , Proteínas Proto-Oncogênicas c-akt/genética , Tetraspanina 30/genética , Inibidor Tecidual de Metaloproteinase-1/genéticaRESUMO
Among patients with colorectal cancer who benefit from therapy targeted to the epidermal growth factor receptor (EGFR), stable disease (SD) occurs more frequently than massive regressions. Exploring the mechanisms of this incomplete sensitivity to devise more efficacious treatments will likely improve patients' outcomes. We tested therapies tailored around hypothesis-generating molecular features in patient-derived xenografts ("xenopatients"), which originated from 125 independent samples that did not harbor established resistance-conferring mutations. Samples from xenopatients that responded to cetuximab, an anti-EGFR agent, with disease stabilization displayed high levels of EGFR family ligands and receptors, indicating high EGFR pathway activity. Five of 21 SD models (23.8%) characterized by particularly high expression of EGFR and EGFR family members regressed after intensified EGFR blockade by cetuximab and a small-molecule inhibitor. In addition, a subset of cases in which enhanced EGFR inhibition was unproductive (6 of 16, 37.5%) exhibited marked overexpression of insulin-like growth factor 2 (IGF2). Enrichment of IGF2 overexpressors among cases with SD was demonstrated in the entire xenopatient collection and was confirmed in patients by mining clinical gene expression data sets. In functional studies, IGF2 overproduction attenuated the efficacy of cetuximab. Conversely, interception of IGF2-dependent signaling in IGF2-overexpressing xenopatients potentiated the effects of cetuximab. The clinical implementation of IGF inhibitors awaits reliable predictors of response, but the results of this study suggest rational combination therapies for colorectal cancer and provide evidence for IGF2 as a biomarker of reduced tumor sensitivity to anti-EGFR therapy and a determinant of response to combined IGF2/EGFR targeting.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Receptores ErbB/química , Fator de Crescimento Insulin-Like II/química , Animais , Anticorpos Monoclonais Humanizados/uso terapêutico , Biomarcadores Tumorais/metabolismo , Cetuximab , Ensaios Clínicos como Assunto , Resistencia a Medicamentos Antineoplásicos , Éxons , Humanos , Imuno-Histoquímica , Ligantes , Camundongos , Mutação , Transplante de NeoplasiasRESUMO
The hepatocyte growth factor (HGF) also known as scatter factor activates cancer cell invasion and metastasis. We show that in ovarian cancer cells HGF induced the phosphorylation of the small heat shock protein of 27 kDa (HSP27) by activating the p38MAPK. HSP27 is increased in many cancers at advanced stage including ovarian cancer and associated with cancer resistance to therapy and poor patients' survival. The phosphorylation of HSP27 regulates both its chaperone activity and its control of cytoskeletal stability. We show that HSP27 was necessary for the remodeling of actin filaments induced by HGF and that motility in vitro depended on the p38MAPK-MK2 axis. In vivo, HSP27 silencing impaired the ability of the highly metastatic, HGF-secreting ovarian cancer cells to give rise to spontaneous metastases. This was due to defective motility across the vessel wall and reduced growth. Indeed, HSP27 silencing impaired the ability of circulating ovarian cancer cells to home to the lungs and to form experimental hematogenous metastases and the capability of cancer cells to grow as subcutaneous xenografts. Moreover, HSP27 suppression resulted in the sensitization of xenografts to low doses of the chemotherapeutic paclitaxel, likely because HSP27 protected microtubules from bundling caused by the drug. Altogether, these data show that the HSP27 is required for the proinvasive and prometastatic activity of HGF and suggest that HSP27 might be not only a marker of progression of ovarian cancer, but also a suitable target for therapy.
Assuntos
Movimento Celular , Resistencia a Medicamentos Antineoplásicos , Proteínas de Choque Térmico HSP27/metabolismo , Fator de Crescimento de Hepatócito/farmacologia , Neoplasias Pulmonares/secundário , Neoplasias Ovarianas/patologia , Animais , Antineoplásicos Fitogênicos/farmacologia , Apoptose , Western Blotting , Proliferação de Células , Feminino , Imunofluorescência , Proteínas de Choque Térmico HSP27/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Paclitaxel/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
EGF receptor (EGFR)-targeted monoclonal antibodies are effective in a subset of metastatic colorectal cancers. Inevitably, all patients develop resistance, which occurs through emergence of KRAS mutations in approximately 50% of the cases. We show that amplification of the MET proto-oncogene is associated with acquired resistance in tumors that do not develop KRAS mutations during anti-EGFR therapy. Amplification of the MET locus was present in circulating tumor DNA before relapse was clinically evident. Functional studies show that MET activation confers resistance to anti-EGFR therapy both in vitro and in vivo. Notably, in patient-derived colorectal cancer xenografts, MET amplification correlated with resistance to EGFR blockade, which could be overcome by MET kinase inhibitors. These results highlight the role of MET in mediating primary and secondary resistance to anti-EGFR therapies in colorectal cancer and encourage the use of MET inhibitors in patients displaying resistance as a result of MET amplification.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Receptores ErbB/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/farmacologia , Animais , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais Humanizados/efeitos adversos , Antineoplásicos/efeitos adversos , Cetuximab , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/genética , Genes ras , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Panitumumabe , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Distribuição Aleatória , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Gene mutations along the Ras pathway (KRAS, NRAS, BRAF, PIK3CA) occur in approximately 50% of colorectal cancers (CRC) and correlate with poor response to anti-EGF receptor (EGFR) therapies. We assessed the effects of mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK) and phosphoinositide 3-kinase (PI3K)/mTOR inhibitors, which neutralize the major Ras effectors, in patient-derived xenografts from RAS/RAF/PIK3CA-mutant metastatic CRCs (mCRC). EXPERIMENTAL DESIGN: Forty mCRC specimens harboring KRAS, NRAS, BRAF, and/or PIK3CA mutations were implanted in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Each xenograft was expanded into four treatment arms: placebo, the MEK inhibitor AZD6244, the PI3K/mTOR inhibitor, BEZ235, or AZD6244 + BEZ235. Cases initially treated with placebo crossed over to AZD6244, BEZ235, and the anti-EGFR monoclonal antibody cetuximab. RESULTS: At the 3-week evaluation time point, cotreatment of established tumors with AZD6244 + BEZ235 induced disease stabilization in the majority of cases (70%) but did not lead to overt tumor regression. Monotherapy was less effective, with BEZ235 displaying higher activity than AZD6244 (disease control rates, DCRs: AZD6244, 27.5%; BEZ235, 42.5%). Triple therapy with cetuximab provided further advantage (DCR, 88%). The extent of disease control declined at the 6-week evaluation time point (DCRs: AZD6244, 13.9%; BEZ235, 16.2%; AZD6244 + BEZ235, 34%). Cross-analysis of mice harboring xenografts from the same original tumor and treated with each of the different modalities revealed subgroups with preferential sensitivity to AZD6244 (12.5%), BEZ235 (35%), or AZD6244 + BEZ235 (42.5%); another subgroup (10%) showed equivalent response to any treatment. CONCLUSIONS: The prevalent growth-suppressive effects produced by MEK and PI3K/mTOR inhibition suggest that this strategy may retard disease progression in patients. However, data offer cautionary evidence against the occurrence of durable responses.
Assuntos
1-Fosfatidilinositol 4-Quinase/antagonistas & inibidores , Neoplasias Colorretais/prevenção & controle , Genes ras , Neoplasias Hepáticas/prevenção & controle , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Mutação/genética , Serina-Treonina Quinases TOR/antagonistas & inibidores , 1-Fosfatidilinositol 4-Quinase/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Estudos de Casos e Controles , Linhagem Celular Tumoral , Colo/metabolismo , Colo/patologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Técnicas Imunoenzimáticas , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundário , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/metabolismo , Reto/metabolismo , Reto/patologia , Serina-Treonina Quinases TOR/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Biliary tract carcinoma (BTC) has a poor prognosis due to limited treatment options. There is, therefore, an urgent need to identify new targets and to design innovative therapeutic approaches. Among potential candidate molecules, we evaluated the nonreceptor tyrosine kinase Src, observing promising antitumor effects of its small-molecule inhibitor saracatinib in BTC preclinical models. The presence of an active Src protein was investigated by immunohistochemistry in 19 surgical samples from patients with BTC. Upon saracatinib treatment, the phosphorylation of Src and of its downstream transducers was evaluated in the BTC cell lines TFK-1, EGI-1, HuH28, and TGBC1-TKB. The effect of saracatinib on proliferation and migration was analyzed in these same cell lines, and its antitumor activity was essayed in EGI-1 mouse xenografts. Saracatinib-modulated transcriptome was profiled in EGI-1 cells and in tumor samples of the xenograft model. Src was activated in about 80% of the human BTC samples. In cultured BTC cell lines, low-dose saracatinib counteracted the activation of Src and of its downstream effectors, increased the fraction of cells in G(0)-G(1) phase, and inhibited cell migration. At high concentrations (median dose from 2.26-6.99 µmol/L), saracatinib was also capable of inhibiting BTC cell proliferation. In vivo, saracatinib treatment resulted in delayed tumor growth, associated with an impaired vascular network. Here, we provide a demonstration that the targeted inhibition of Src kinase by saracatinib is of therapeutic benefit in preclinical models of BTC. We propose our results as a basis for the design of saracatinib-based clinical applications.
Assuntos
Antineoplásicos/farmacologia , Benzodioxóis/farmacologia , Neoplasias do Sistema Biliar/metabolismo , Carcinoma/metabolismo , Quinazolinas/farmacologia , Quinases da Família src/antagonistas & inibidores , Idoso , Animais , Antineoplásicos/administração & dosagem , Benzodioxóis/administração & dosagem , Neoplasias do Sistema Biliar/tratamento farmacológico , Neoplasias do Sistema Biliar/genética , Carcinoma/tratamento farmacológico , Carcinoma/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Neovascularização Patológica/tratamento farmacológico , Fosforilação , Quinazolinas/administração & dosagem , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Extracellular ATP and UTP nucleotides increase the proliferation and engraftment potential of normal human hematopoietic stem cells via the engagement of purinergic receptors (P2Rs). In the present study, we show that ATP and UTP have strikingly opposite effects on human acute myeloblastic leukemia (AML) cells. Leukemic cells express P2Rs. ATP-stimulated leukemic cells, but not normal CD34+ cells, undergo down-regulation of genes involved in cell proliferation and migration, whereas cell-cycle inhibitors are up-regulated. Functionally, ATP induced the inhibition of proliferation and accumulation of AML cells, but not of normal cells, in the G0 phase of the cell cycle. Exposure to ATP or UTP inhibited AML-cell migration in vitro. In vivo, xenotransplantation experiments demonstrated that the homing and engraftment capacity of AML blasts and CD34+CD38- cells to immunodeficient mice BM was significantly inhibited by pretreatment with nucleotides. P2R-expression analysis and pharmacologic profiling suggested that the inhibition of proliferation by ATP was mediated by the down-regulation of the P2X7R, which is up-regulated on untreated blasts, whereas the inhibition of chemotaxis was mainly mediated via P2Y2R and P2Y4R subtypes. We conclude that, unlike normal cells, P2R signaling inhibits leukemic cells and therefore its pharmacologic modulation may represent a novel therapeutic strategy.
Assuntos
Trifosfato de Adenosina/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Transplante de Células , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Receptores Purinérgicos/metabolismo , Uridina Trifosfato/farmacologia , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Transdução de SinaisRESUMO
BACKGROUND: Activating mutations of the epidermal growth factor receptor (EGFR) confer sensitivity to the tyrosine kinase inhibitors (TKi), gefitinib and erlotinib. We analysed EGFR expression, EGFR mutation status and gene expression profiles of prostate cancer (PC) to supply a rationale for EGFR targeted therapies in this disease. METHODS: Mutational analysis of EGFR TK domain (exons from 18 to 21) and immunohistochemistry for EGFR were performed on tumour tissues derived from radical prostatectomy from 100 PC patients. Gene expression profiling using oligo-microarrays was also carried out in 51 of the PC samples. RESULTS: EGFR protein overexpression (EGFRhigh) was found in 36% of the tumour samples, and mutations were found in 13% of samples. Patients with EGFRhigh tumours experienced a significantly increased risk of biochemical relapse (hazard ratio-HR 2.52, p=0.02) compared with patients with tumours expressing low levels of EGFR (EGFRlow). Microarray analysis did not reveal any differences in gene expression between EGFRhigh and EGFRlow tumours. Conversely, in EGFRhigh tumours, we were able to identify a 79 gene signature distinguishing mutated from non-mutated tumours. Additionally, 29 genes were found to be differentially expressed between mutated/EGFRhigh (n=3) and mutated/EGFRlow tumours (n=5). Four of the down-regulated genes, U19/EAF2, ABCC4, KLK3 and ANXA3 and one of the up-regulated genes, FOXC1, are involved in PC progression. CONCLUSIONS: Based on our findings, we hypothesize that accurate definition of the EGFR status could improve prognostic stratification and we suggest a possible role for EGFR-directed therapies in PC patients. Having been generated in a relatively small sample of patients, our results warrant confirmation in larger series.
Assuntos
Receptores ErbB/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Mutação , Neoplasias da Próstata/genética , Idoso , Análise Mutacional de DNA , Progressão da Doença , Receptores ErbB/metabolismo , Fatores de Transcrição Forkhead/genética , Predisposição Genética para Doença/genética , Humanos , Imuno-Histoquímica , Calicreínas/genética , Masculino , Pessoa de Meia-Idade , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Estadiamento de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Antígeno Prostático Específico , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genéticaRESUMO
Trastuzumab has changed the prognosis of HER2 positive breast cancers. Despite this progress, resistance to trastuzumab occurs in most patients. Newer anti-HER2 therapies, like the dual tyrosine-kinase inhibitor (TKI) lapatinib, show significant antitumor activity, indicating that HER2 can be still exploited as a target after trastuzumab failure. However, since a high proportion of patients fail to respond to these alternative strategies, it is possible that cell escape from HER2 targeting may rely on HER2 independent pathways. The knowledge of these pathways deserve to be exploited to develop new therapies. We characterized two human HER2 overexpressing breast cancer cell lines resistant to trastuzumab and lapatinib (T100 and JIMT-1) from a molecular and biological point of view. Indeed, we assessed both in vitro and in vivo the activity of the multitarget inhibitor sorafenib. In both cell lines, the previously proposed mechanisms did not explain resistance to HER2 inhibitors. Notably, silencing HER2 by shRNA did not affect the growth of our cells, suggesting loss of reliance upon HER2. Moreover, we identified alterations in two antiapoptotic proteins Mcl-1 and Survivin which are known to be targets of the multikinase inhibitor sorafenib. Moreover, sorafenib, strongly inhibited the in vitro growth of T100 and JIMT-1 cells, through the downregulation of both Mcl-1 and Survivin. Similar results were obtained in JIMT-1 xenografts subcutaneously injected in NOD SCID mice. We provide preclinical evidence that tumor cells resistant to trastuzumab and lapatinib may rely on HER2 independent pathways that can be efficiently inhibited by sorafenib.