RESUMO
OBJECTIVE: Microsatellite instability (MSI) is detected in approximately 15% of all colorectal cancers (CRC) and virtually in all cases with Lynch syndrome. The MSI phenotype is caused by dysfunctional mismatch repair (MMR) and leads to accumulation of DNA replication errors. Sporadic MSI CRC often harbours BRAF(V600E); however, no consistent data exist regarding targeted treatment approaches in BRAF(wt) MSI CRC. DESIGN: Mutations and quantitative MSI were analysed by deep sequencing in 196 formalin fixed paraffin embedded (FFPE) specimens comprising Lynch and Lynch-like CRCs from the German Hereditary Nonpolyposis Colorectal Cancer registry. Functional relevance of recurrent ERBB2/HER2 mutations was investigated in CRC cell lines using reversible and irreversible HER-targeting inhibitors, EGFR-directed antibody cetuximab, HER2-directed antibody trastuzumab and siRNA-mediated ERBB2/HER2 knockdown. RESULTS: Quantification of nucleotide loss in non-coding mononucleotide repeats distinguished microsatellite status with very high accuracy (area under curve=0.9998) and demonstrated progressive losses with deeper invasion of MMR-deficient colorectal neoplasms (p=0.008). Characterisation of BRAF(wt) MSI CRC revealed hot-spot mutations in well-known oncogenic drivers, including KRAS (38.7%), PIK3CA (36.5%), and ERBB2 (15.0%). L755S and V842I substitutions in ERBB2 were highly recurrent. Functional analyses in ERBB2-mutated MSI CRC cell lines revealed a differential response to HER-targeting compounds and superiority of irreversible pan-HER inhibitors. CONCLUSIONS: We developed a high-throughput deep sequencing approach for concomitant MSI and mutational analyses in FFPE specimens. We provided novel insights into clinically relevant alterations in MSI CRC and a rationale for targeting ERBB2/HER2 mutations in Lynch and Lynch-like CRC.
Assuntos
Cetuximab/farmacologia , Neoplasias Colorretais Hereditárias sem Polipose , Neoplasias Colorretais , Receptores ErbB , Receptor ErbB-2 , Trastuzumab/farmacologia , Antineoplásicos/farmacologia , Técnicas de Cultura de Células , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais Hereditárias sem Polipose/tratamento farmacológico , Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Reparo de Erro de Pareamento de DNA , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Feminino , Humanos , Masculino , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Testes Farmacogenômicos/métodos , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/genéticaRESUMO
Small cell lung cancers (SCLCs) and extrapulmonary small cell cancers (SCCs) are very aggressive tumors arising de novo as primary small cell cancer with characteristic genetic lesions in RB1 and TP53. Based on murine models, neuroendocrine stem cells of the terminal bronchioli have been postulated as the cellular origin of primary SCLC. However, both in lung and many other organs, combined small cell/non-small cell tumors and secondary transitions from non-small cell carcinomas upon cancer therapy to neuroendocrine and small cell tumors occur. We define features of "small cell-ness" based on neuroendocrine markers, characteristic RB1 and TP53 mutations and small cell morphology. Furthermore, here we identify a pathway driving the pathogenesis of secondary SCLC involving inactivating NOTCH mutations, activation of the NOTCH target ASCL1 and canonical WNT-signaling in the context of mutual bi-allelic RB1 and TP53 lesions. Additionally, we explored ASCL1 dependent RB inactivation by phosphorylation, which is reversible by CDK5 inhibition. We experimentally verify the NOTCH-ASCL1-RB-p53 signaling axis in vitro and validate its activation by genetic alterations in vivo. We analyzed clinical tumor samples including SCLC, SCC and pulmonary large cell neuroendocrine carcinomas and adenocarcinomas using amplicon-based Next Generation Sequencing, immunohistochemistry and fluorescence in situ hybridization. In conclusion, we identified a novel pathway underlying rare secondary SCLC which may drive small cell carcinomas in organs other than lung, as well.
Assuntos
Carcinoma Neuroendócrino/genética , Carcinoma Neuroendócrino/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Transdução de Sinais , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/patologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Análise Mutacional de DNA , Citometria de Fluxo , Imunofluorescência , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Receptores Notch/genética , Receptores Notch/metabolismo , Proteína do Retinoblastoma/genética , Transfecção , Proteína Supressora de Tumor p53/genéticaRESUMO
We identified the four-and-a-half LIM domain protein 2 (FHL2) as a novel regulator of CCL19-induced dendritic cell (DC) migration. Initiation of migration is a hallmark of DC function and plays a central role in the induction and regulation of immune responses. In vivo, DCs continuously acquire Ag in the periphery and migrate to draining lymph nodes, under the influence of local environmental chemotactic factors like CCL19/21 or sphingosine 1-phosphate (S1P). We investigated the role of S1P- and RhoA-regulated FHL2 in this process. We found reduced nuclear localization of FHL2 in mature bone marrow-derived DCs (BMDCs), compared with immature BMDCs, following stimulation with CCL19. Furthermore, in vitro-generated murine FHL2(-/-) BMDCs displayed a significantly increased migratory speed, directionality, and migratory persistence toward the chemokine CCL19 compared with wild-type BMDCs. Moreover, in vivo, FHL2(-/-) BMDCs showed increased migration toward lymphoid organs. FHL2(-/-) BMDCs increased the expression of S1PR1, which was associated with greater Rac activation. An S1PR1 antagonist and knock-down of S1PR1 abrogated the increased migratory speed of FHL2(-/-) BMDCs. Our results identify FHL2 as an important novel regulator of DC migration via regulation of their sensitivity toward environmental migratory cues like S1P and CCL19.
Assuntos
Movimento Celular/imunologia , Quimiocina CCL19/fisiologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Proteínas de Homeodomínio/fisiologia , Proteínas Musculares/fisiologia , Receptores de Lisoesfingolipídeo/metabolismo , Fatores de Transcrição/fisiologia , Animais , Células da Medula Óssea/enzimologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Movimento Celular/genética , Núcleo Celular/genética , Núcleo Celular/imunologia , Núcleo Celular/metabolismo , Células Cultivadas , Células Dendríticas/enzimologia , Proteínas de Homeodomínio/genética , Imunofenotipagem , Proteínas com Homeodomínio LIM , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Musculares/deficiência , Proteínas Musculares/genética , Receptores de Lisoesfingolipídeo/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptores de Esfingosina-1-Fosfato , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Regulação para Cima/genética , Regulação para Cima/imunologia , Proteínas rac de Ligação ao GTP/metabolismoRESUMO
LIN28B is a RNA-binding protein regulating predominantly let-7 microRNAs with essential functions in inflammation, wound healing, embryonic stem cells, and cancer. LIN28B expression is associated with tumor initiation, progression, resistance, and poor outcome in several solid cancers, including lung cancer. However, the functional role of LIN28B, especially in non-small cell lung adenocarcinomas, remains elusive. Here, we investigated the effects of LIN28B expression on lung tumorigenesis using LIN28B transgenic overexpression in an autochthonous KRASG12V-driven mouse model. We found that LIN28B overexpression significantly increased the number of CD44+/CD326+ tumor cells, upregulated VEGF-A and miR-21 and promoted tumor angiogenesis and epithelial-to-mesenchymal transition (EMT) accompanied by enhanced AKT phosphorylation and nuclear translocation of c-MYC. Moreover, LIN28B accelerated tumor initiation and enhanced proliferation which led to a shortened overall survival. In addition, we analyzed lung adenocarcinomas of the Cancer Genome Atlas (TCGA) and found LIN28B expression in 24% of KRAS-mutated cases, which underscore the relevance of our model.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas de Ligação a RNA/genética , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Molécula de Adesão da Célula Epitelial/metabolismo , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Humanos , Receptores de Hialuronatos/metabolismo , Neoplasias Pulmonares/genética , Camundongos , Camundongos Transgênicos , Neoplasias Experimentais/genética , Análise de SobrevidaRESUMO
INTRODUCTION: The Network Genomic Medicine Lung Cancer was set up to rapidly translate scientific advances into early clinical trials of targeted therapies in lung cancer performing molecular analyses of more than 3500 patients annually. Because sequential analysis of the relevant driver mutations on fixated samples is challenging in terms of workload, tissue availability, and cost, we established multiplex parallel sequencing in routine diagnostics. The aim was to analyze all therapeutically relevant mutations in lung cancer samples in a high-throughput fashion while significantly reducing turnaround time and amount of input DNA compared with conventional dideoxy sequencing of single polymerase chain reaction amplicons. METHODS: In this study, we demonstrate the feasibility of a 102 amplicon multiplex polymerase chain reaction followed by sequencing on an Illumina sequencer on formalin-fixed paraffin-embedded tissue in routine diagnostics. Analysis of a validation cohort of 180 samples showed this approach to require significantly less input material and to be more reliable, robust, and cost-effective than conventional dideoxy sequencing. Subsequently, 2657 lung cancer patients were analyzed. RESULTS: We observed that comprehensive biomarker testing provided novel information in addition to histological diagnosis and clinical staging. In 2657 consecutively analyzed lung cancer samples, we identified driver mutations at the expected prevalence. Furthermore we found potentially targetable DDR2 mutations at a frequency of 3% in both adenocarcinomas and squamous cell carcinomas. CONCLUSION: Overall, our data demonstrate the utility of systematic sequencing analysis in a clinical routine setting and highlight the dramatic impact of such an approach on the availability of therapeutic strategies for the targeted treatment of individual cancer patients.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/terapia , Neoplasias Pulmonares/genética , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Estudos de Coortes , DNA de Neoplasias/análise , DNA de Neoplasias/genética , DNA de Neoplasias/isolamento & purificação , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/terapia , Reação em Cadeia da Polimerase/métodos , Análise de Sequência de DNA/métodosRESUMO
Epithelial-to-mesenchymal transition (EMT), the phenotypical change of cells from an epithelial to a mesenchymal type, is thought to be a key event in invasion and metastasis of adenocarcinomas. These changes involve loss of keratin expression as well as loss of cell polarity and adhesion. We here aimed to determine whether the loss of keratin expression itself drives increased invasion and metastasis in adenocarcinomas and whether keratin loss leads to the phenotypic changes associated with EMT. Therefore, we employed a recently described murine model in which conditional deletion of the Keratin cluster II by Cre-recombinase leads to the loss of the entire keratinmultiprotein family. These mice were crossed into a newly generated Cre-recombinase inducible KRAS-driven murine lung cancer model to examine the effect of keratin loss on morphology, invasion and metastasis as well as expression of EMT related genes in the resulting tumors. We here clearly show that loss of a functional keratin cytoskeleton did not significantly alter tumor morphology or biology in terms of invasion, metastasis, proliferation or tumor burden and did not lead to induction of EMT. Further, tumor cells did not induce synchronously expression of vimentin, which is often seen in EMT, to compensate for keratin loss. In summary, our data suggest that changes in cell shape and migration that underlie EMT are dependent on changes in signaling pathways that cause secondary changes in keratin expression and organization. Thus, we conclude that loss of the keratin cytoskeleton per se is not sufficient to causally drive EMT in this tumor model.