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1.
J Biol Chem ; 299(3): 102992, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36758799

RESUMO

The ADP-ribosylation factor (Arf) GTPases and their regulatory proteins are implicated in cancer progression. NAV-2729 was previously identified as a specific inhibitor of Arf6 that reduced progression of uveal melanoma in an orthotopic xenograft. Here, our goal was to assess the inhibitory effects of NAV-2729 on the proliferation of additional cell types. We found NAV-2729 inhibited proliferation of multiple cell lines, but Arf6 expression did not correlate with NAV-2729 sensitivity, and knockdown of Arf6 affected neither cell viability nor sensitivity to NAV-2729. Furthermore, binding to native Arf6 was not detected; however, we determined that NAV-2729 inhibited both Arf exchange factors and Arf GTPase-activating proteins. ASAP1, a GTPase-activating protein linked to cancer progression, was further investigated. We demonstrated that NAV-2729 bound to the PH domain of ASAP1 and changed ASAP1 cellular distribution. However, ASAP1 knockdown did not fully recapitulate the cytoskeletal effects of NAV-2729 nor affect cell proliferation. Finally, our screens identified 48 other possible targets of NAV-2729. These results illustrate the complexities of defining targets of small molecules and identify NAV-2729 as a model PH domain-binding inhibitor.


Assuntos
Fatores de Ribosilação do ADP , Neoplasias , Humanos , Fatores de Ribosilação do ADP/metabolismo , Clorobenzenos , Pirazóis , Proteínas Ativadoras de GTPase/metabolismo , Fator 1 de Ribosilação do ADP/metabolismo
2.
Mol Cell Proteomics ; 20: 100136, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34391887

RESUMO

Immune checkpoint inhibitors and adoptive lymphocyte transfer-based therapies have shown great therapeutic potential in cancers with high tumor mutational burden (TMB), such as melanoma, but not in cancers with low TMB, such as mutant epidermal growth factor receptor (EGFR)-driven lung adenocarcinoma. Precision immunotherapy is an unmet need for most cancers, particularly for cancers that respond inadequately to immune checkpoint inhibitors. Here, we employed large-scale MS-based proteogenomic profiling to identify potential immunogenic human leukocyte antigen (HLA) class I-presented peptides in melanoma and EGFR-mutant lung adenocarcinoma. Similar numbers of peptides were identified from both tumor types. Cell line and patient-specific databases (DBs) were constructed using variants identified from whole-exome sequencing. A de novo search algorithm was used to interrogate the HLA class I immunopeptidome MS data. We identified 12 variant peptides and several classes of tumor-associated antigen-derived peptides. We constructed a cancer germ line (CG) antigen DB with 285 antigens. This allowed us to identify 40 class I-presented CG antigen-derived peptides. The class I immunopeptidome comprised more than 1000 post-translationally modified (PTM) peptides representing 58 different PTMs, underscoring the critical role PTMs may play in HLA binding. Finally, leveraging de novo search algorithm and an annotated long noncoding RNA (lncRNA) DB, we developed a novel lncRNA-encoded peptide discovery pipeline to identify 44 lncRNA-derived peptides that are presented by class I. We validated tandem MS spectra of select variant, CG antigen, and lncRNA-derived peptides using synthetic peptides and performed HLA class I-binding assays to demonstrate binding to class I proteins. In summary, we provide direct evidence of HLA class I presentation of a large number of variant and tumor-associated peptides in both low and high TMB cancer. These results can potentially be useful for precision immunotherapies, such as vaccine or adoptive cell therapies in melanoma and EGFR-mutant lung cancers.


Assuntos
Adenocarcinoma de Pulmão/metabolismo , Antígenos de Neoplasias/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Neoplasias Pulmonares/metabolismo , Melanoma/metabolismo , Peptídeos/metabolismo , Adenocarcinoma de Pulmão/genética , Idoso , Antígenos de Neoplasias/genética , Linhagem Celular Tumoral , Receptores ErbB/genética , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Neoplasias Pulmonares/genética , Masculino , Melanoma/genética , Mutação , Peptídeos/genética , Proteogenômica
3.
Mol Cell Proteomics ; 18(4): 622-641, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30617155

RESUMO

Lung cancer is the leading cause of cancer death in both men and women. Tumor heterogeneity is an impediment to targeted treatment of all cancers, including lung cancer. Here, we sought to characterize tumor proteome and phosphoproteome changes by longitudinal, prospective collection of tumor tissue from an exceptional responder lung adenocarcinoma patient who survived with metastatic lung adenocarcinoma for over seven years while undergoing HER2-directed therapy in combination with chemotherapy. We employed "Super-SILAC" and TMT labeling strategies to quantify the proteome and phosphoproteome of a lung metastatic site and eight distinct metastatic progressive lymph nodes collected during these seven years, including five lymph nodes procured at autopsy. We identified specific signaling networks enriched in lung compared with the lymph node metastatic sites. We correlated the changes in protein abundance with changes in copy number alteration (CNA) and transcript expression. ERBB2/HER2 protein expression was higher in lung, consistent with a higher degree of ERBB2 amplification in lung compared with the lymph node metastatic sites. To further interrogate the mass spectrometry data, a patient-specific database was built by incorporating all the somatic and germline variants identified by whole genome sequencing (WGS) of genomic DNA from the lung, one lymph node metastatic site and blood. An extensive validation pipeline was built to confirm variant peptides. We validated 360 spectra corresponding to 55 germline and 6 somatic variant peptides. Targeted MRM assays revealed two novel variant somatic peptides, CDK12-G879V and FASN-R1439Q, expressed in lung and lymph node metastatic sites, respectively. The CDK12-G879V mutation likely results in a nonfunctional CDK12 kinase and chemotherapy susceptibility in lung metastatic sites. Knockdown of CDK12 in lung adenocarcinoma cells increased chemotherapy sensitivity which was rescued by wild type, but not CDK12-G879V expression, consistent with the complete resolution of the lung metastatic sites in this patient.


Assuntos
Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Quinases Ciclina-Dependentes/genética , Espectrometria de Massas/métodos , Mutação/genética , Proteômica , Adenocarcinoma de Pulmão/metabolismo , Linhagem Celular Tumoral , Variações do Número de Cópias de DNA/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Proteínas Mutantes/metabolismo , Metástase Neoplásica , Proteínas de Neoplasias/metabolismo , Peptídeos/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Reprodutibilidade dos Testes
4.
Nat Genet ; 56(4): 697-709, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38509386

RESUMO

In mice, exit from the totipotent two-cell (2C) stage embryo requires silencing of the 2C-associated transcriptional program. However, the molecular mechanisms involved in this process remain poorly understood. Here we demonstrate that the 2C-specific transcription factor double homeobox protein (DUX) mediates an essential negative feedback loop by inducing the expression of DUXBL to promote this silencing. We show that DUXBL gains accessibility to DUX-bound regions specifically upon DUX expression. Furthermore, we determine that DUXBL interacts with TRIM24 and TRIM33, members of the TRIM superfamily involved in gene silencing, and colocalizes with them in nuclear foci upon DUX expression. Importantly, DUXBL overexpression impairs 2C-associated transcription, whereas Duxbl inactivation in mouse embryonic stem cells increases DUX-dependent induction of the 2C-transcriptional program. Consequently, DUXBL deficiency in embryos results in sustained expression of 2C-associated transcripts leading to early developmental arrest. Our study identifies DUXBL as an essential regulator of totipotency exit enabling the first divergence of cell fates.


Assuntos
Genes Homeobox , Proteínas de Homeodomínio , Células-Tronco Embrionárias Murinas , Fatores de Transcrição , Animais , Camundongos , Diferenciação Celular , Regulação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo
5.
Cancers (Basel) ; 15(8)2023 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-37190191

RESUMO

Osimertinib is a third-generation epidermal growth factor receptor and tyrosine kinase inhibitor (EGFR-TKI) approved for the treatment of lung adenocarcinoma patients harboring EGFR mutations. However, acquired resistance to this targeted therapy is inevitable, leading to disease relapse within a few years. Therefore, understanding the molecular mechanisms of osimertinib resistance and identifying novel targets to overcome such resistance are unmet needs of cancer patients. Here, we investigated the efficacy of two novel CDK12/13 inhibitors, AU-15506 and AU-16770, in osimertinib-resistant EGFR mutant lung adenocarcinoma cells in culture and xenograft models in vivo. We demonstrate that these drugs, either alone or in combination with osimertinib, are potent inhibitors of osimertinib-resistant as well as -sensitive lung adenocarcinoma cells in culture. Interestingly, only the CDK12/13 inhibitor in combination with osimertinib, although not as monotherapy, suppresses the growth of resistant tumors in xenograft models in vivo. Taken together, the results of this study suggest that inhibition of CDK12/13 in combination with osimertinib has the potential to overcome osimertinib resistance in EGFR mutant lung adenocarcinoma patients.

6.
J Mol Biol ; 435(17): 168184, 2023 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-37348754

RESUMO

Hsp90 and Hsp70 are highly conserved molecular chaperones that help maintain proteostasis by participating in protein folding, unfolding, remodeling and activation of proteins. Both chaperones are also important for cellular recovery following environmental stresses. Hsp90 and Hsp70 function collaboratively for the remodeling and activation of some client proteins. Previous studies using E. coli and S. cerevisiae showed that residues in the Hsp90 middle domain directly interact with a region in the Hsp70 nucleotide binding domain, in the same region known to bind J-domain proteins. Importantly, J-domain proteins facilitate and stabilize the interaction between Hsp90 and Hsp70 both in E. coli and S. cerevisiae. To further explore the role of J-domain proteins in protein reactivation, we tested the hypothesis that J-domain proteins participate in the collaboration between Hsp90 and Hsp70 by simultaneously interacting with Hsp90 and Hsp70. Using E. coli Hsp90, Hsp70 (DnaK), and a J-domain protein (CbpA), we detected a ternary complex containing all three proteins. The interaction involved the J-domain of CbpA, the DnaK binding region of E. coli Hsp90, and the J-domain protein binding region of DnaK where Hsp90 also binds. Additionally, results show that E. coli Hsp90 interacts with E. coli J-domain proteins, DnaJ and CbpA, and that yeast Hsp90, Hsp82, interacts with a yeast J-domain protein, Ydj1. Together these results suggest that the complexes may be transient intermediates in the pathway of collaborative protein remodeling by Hsp90 and Hsp70.


Assuntos
Proteínas de Escherichia coli , Proteínas de Choque Térmico HSP70 , Proteínas de Choque Térmico HSP90 , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico HSP40/química , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Domínios Proteicos
7.
bioRxiv ; 2023 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-38076880

RESUMO

Biophysical profiling of primary tumors has revealed that individual tumor cells fall along a highly heterogeneous continuum of mechanical phenotypes. One idea is that a subset of tumor cells is "softer" to facilitate detachment and escape from the primary site, a step required to initiate metastasis. However, it has also been postulated that cells must be able to deform and generate sufficient force to exit into distant sites. Here, we aimed to dissect the mechanical changes that occur during extravasation and organ colonization. Using multiplexed methods of intravital microscopy and optical tweezer based active microrheology, we obtained longitudinal images and mechanical profiles of cells during organ colonization in vivo. We determined that cells were softer, more liquid like upon exit of the vasculature but stiffened and became more solid like once in the new organ microenvironment. We also determined that a YAP mediated mechanogenotype influenced the global dissemination in our in vivo and in vitro models and that reducing mechanical heterogeneity could reduce extravasation. Moreover, our high throughput analysis of mechanical phenotypes of patient samples revealed that this mechanics was in part regulated by the external hydrodynamic forces that the cancer cells experienced within capillary mimetics. Our findings indicate that disseminated cancer cells can keep mutating with a continuum landscape of mechano-phenotypes, governed by the YAP-mediated mechanosensing of hydrodynamic flow.

8.
Cytokine ; 54(1): 61-7, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21266308

RESUMO

The heat shock (HS) response, a phylogenetically conserved ubiquitous response to stress, is generally characterized by the induced expression of heat shock protein (HSP) genes. Our earlier studies showed that the stress-activated transcription factor, heat shock factor-1 (HSF1), activated at febrile range or HS temperatures also modified expression of non-HSP genes including cytokine and chemokine genes. We also showed by in silico analysis that 28 among 29 human and mouse CXC chemokine genes had multiple putative heat shock response elements (HSEs) present in their gene promoters. To further determine whether these potential HSEs were functional and bound HSF1, we analyzed the recruitment of HSF1 to promoters of 5 human CXC chemokine genes (CXCL-1, 2, 3, 5 and 8) by chromatin immunoprecipitation (ChIP) assay and analyzed the effect of HS exposure on tumor necrosis factor-α (TNFα)-induced expression of these genes in human lung epithelial-like A549 cells. HSF1 ChIP analysis showed that HSF1 was recruited to all but one of these CXC chemokine genes (CXCL-3) and HS caused a significant increase in recruitment of HSF1 to one or multiple HSEs present in the promoters of CXCL-1, 2, 5 and 8 genes. However, the effect of HS exposure on expression of these genes showed a variable gene-specific effect. For example, CXCL8 expression was markedly enhanced (p<0.05) whereas CXCL5 expression was significantly repressed (p<0.05) in cells exposed to HS coincident with TNFα stimulation. In contrast, expression of CXCL1 and CXCL2, despite HSF1 recruitment to their promoters, was not affected by HS exposure. Our results indicate that some, if not all, putative HSEs present in the CXC chemokine gene promoters are functional and recruit HSF1 in vivo but the effects on gene expression are variable and gene specific. We speculate, the physical proximity and interactions of other transcription factors and co-regulators with HSF1 could be critical to determining the effects of HS on the expression of these genes.


Assuntos
Quimiocinas/metabolismo , Proteínas de Ligação a DNA/biossíntese , Regulação da Expressão Gênica , Receptores CXCR/biossíntese , Fatores de Transcrição/biossíntese , Animais , Linhagem Celular Tumoral , Quimiocina CXCL1/metabolismo , Quimiocina CXCL2/metabolismo , Quimiocina CXCL5/metabolismo , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição de Choque Térmico , Humanos , Interleucina-8/metabolismo , Camundongos , Modelos Biológicos , Regiões Promotoras Genéticas , Receptores CXCR/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
9.
Cancers (Basel) ; 13(19)2021 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-34638461

RESUMO

Immune checkpoint inhibitor (ICI) therapy has been a paradigm shift in the treatment of cancer. ICI therapy results in durable responses and survival benefit for a large number of tumor types. Osimertinib, a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) has shown great efficacy treating EGFR mutant lung cancers; however, all patients eventually develop resistance. ICI therapy has not benefitted EGFR mutant lung cancer. Herein, we employed stable isotope labeling by amino acids in cell culture (SILAC) quantitative mass spectrometry-based proteomics to investigate potential immune escape molecular mechanisms in osimertinib resistant EGFR mutant lung adenocarcinoma by interrogating the alterations in the human leukocyte antigen (HLA) Class I-presented immunopeptidome, Class I-interactome, and the whole cell proteome between isogenic osimertinib-sensitive and -resistant human lung adenocarcinoma cells. Our study demonstrates an overall reduction in HLA class I-presented immunopeptidome and downregulation of antigen presentation core complex (e.g., TAP1 and ERAP1/2) and immunoproteasome in osimertinib resistant lung adenocarcinoma cells. Several key components in autophagy pathway are differentially altered. S100 proteins and SLC3A2 may play critical roles in reduced antigen presentation. Our dataset also includes ~1000 novel HLA class I interaction partners and hundreds of Class I-presented immunopeptides in EGFR mutant lung adenocarcinoma. This large-scale unbiased proteomics study provides novel insights and potential mechanisms of immune evasion of EGFR mutant lung adenocarcinoma.

10.
Oncogene ; 40(18): 3331-3346, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33850265

RESUMO

Mutations in the epidermal growth factor receptor (EGFR) tyrosine kinase domain constitutively activate EGFR resulting in lung tumorigenesis. Activated EGFR modulates downstream signaling by altering phosphorylation-driven interactions that promote growth and survival. Secretory carrier membrane proteins (SCAMPs) are a family of transmembrane proteins that regulate recycling of receptor proteins, including EGFR. The potential role of SCAMPs in mutant EGFR function and tumorigenesis has not been elucidated. Using quantitative mass-spectrometry-based phosphoproteomics, we identified SCAMP3 as a target of mutant EGFRs in lung adenocarcinoma and sought to further investigate the role of SCAMP3 in the regulation of lung tumorigenesis. Here we show that activated EGFR, either directly or indirectly phosphorylates SCAMP3 at Y86 and this phosphorylation increases the interaction of SCAMP3 with both wild-type and mutant EGFRs. SCAMP3 knockdown increases lung adenocarcinoma cell survival and increases xenograft tumor growth in vivo, demonstrating a tumor suppressor role of SCAMP3 in lung tumorigenesis. The tumor suppressor function is a result of SCAMP3 promoting EGFR degradation and attenuating MAP kinase signaling pathways. SCAMP3 knockdown also increases multinucleated cells in culture, suggesting that SCAMP3 is required for efficient cytokinesis. The enhanced growth, increased colony formation, reduced EGFR degradation and multinucleation phenotype of SCAMP3-depleted cells were reversed by re-expression of wild-type SCAMP3, but not SCAMP3 Y86F, suggesting that Y86 phosphorylation is critical for SCAMP3 function. Taken together, the results of this study demonstrate that SCAMP3 functions as a novel tumor suppressor in lung cancer by modulating EGFR signaling and cytokinesis that is partly Y86 phosphorylation-dependent.


Assuntos
Adenocarcinoma de Pulmão , Receptores ErbB , Humanos , Fosforilação
11.
Cancer Res ; 81(11): 3051-3066, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33727228

RESUMO

Lung cancer is the leading cause of cancer mortality worldwide. The treatment of patients with lung cancer harboring mutant EGFR with orally administered EGFR tyrosine kinase inhibitors (TKI) has been a paradigm shift. Osimertinib and rociletinib are third-generation irreversible EGFR TKIs targeting the EGFR T790M mutation. Osimertinib is the current standard of care for patients with EGFR mutations due to increased efficacy, lower side effects, and enhanced brain penetrance. Unfortunately, all patients develop resistance. Genomic approaches have primarily been used to interrogate resistance mechanisms. Here we characterized the proteome and phosphoproteome of a series of isogenic EGFR-mutant lung adenocarcinoma cell lines that are either sensitive or resistant to these drugs, comprising the most comprehensive proteomic dataset resource to date to investigate third generation EGFR TKI resistance in lung adenocarcinoma. Unbiased global quantitative mass spectrometry uncovered alterations in signaling pathways, revealed a proteomic signature of epithelial-mesenchymal transition, and identified kinases and phosphatases with altered expression and phosphorylation in TKI-resistant cells. Decreased tyrosine phosphorylation of key sites in the phosphatase SHP2 suggests its inhibition, resulting in subsequent inhibition of RAS/MAPK and activation of PI3K/AKT pathways. Anticorrelation analyses of this phosphoproteomic dataset with published drug-induced P100 phosphoproteomic datasets from the Library of Integrated Network-Based Cellular Signatures program predicted drugs with the potential to overcome EGFR TKI resistance. The PI3K/MTOR inhibitor dactolisib in combination with osimertinib overcame resistance both in vitro and in vivo. Taken together, this study reveals global proteomic alterations upon third generation EGFR TKI resistance and highlights potential novel approaches to overcome resistance. SIGNIFICANCE: Global quantitative proteomics reveals changes in the proteome and phosphoproteome in lung cancer cells resistant to third generation EGFR TKIs, identifying the PI3K/mTOR inhibitor dactolisib as a potential approach to overcome resistance.


Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Imidazóis/farmacologia , Fosfoproteínas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteoma/metabolismo , Quinolinas/farmacologia , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Antineoplásicos/farmacologia , Apoptose , Proliferação de Células , Receptores ErbB/antagonistas & inibidores , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Fosfatidilinositol 3-Quinases/química , Fosfoproteínas/análise , Proteoma/análise , Serina-Treonina Quinases TOR/antagonistas & inibidores , Células Tumorais Cultivadas
12.
Cell Rep Med ; 1(1)2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32483558

RESUMO

Clonal evolution of osimertinib-resistance mechanisms in EGFR mutant lung adenocarcinoma is poorly understood. Using multi-region whole-exome and RNA sequencing of prospectively collected pre- and post-osimertinib-resistant tumors, including at rapid autopsies, we identify a likely mechanism driving osimertinib resistance in all patients analyzed. The majority of patients acquire two or more resistance mechanisms either concurrently or in temporal sequence. Focal copy-number amplifications occur subclonally and are spatially and temporally separated from common resistance mutations such as EGFR C797S. MET amplification occurs in 66% (n = 6/9) of first-line osimertinib-treated patients, albeit spatially heterogeneous, often co-occurs with additional acquired focal copy-number amplifications and is associated with early progression. Noteworthy osimertinib-resistance mechanisms discovered include neuroendocrine differentiation without histologic transformation, PD-L1, KRAS amplification, and ESR1-AKAP12, MKRN1-BRAF fusions. The subclonal co-occurrence of acquired genomic alterations upon osimertinib resistance will likely require targeting multiple resistance mechanisms by combination therapies.


Assuntos
Acrilamidas/uso terapêutico , Compostos de Anilina/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas , Evolução Clonal , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Pulmonares , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Evolução Clonal/efeitos dos fármacos , Evolução Clonal/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/genética , Feminino , Heterogeneidade Genética/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Inibidores de Proteínas Quinases/uso terapêutico , Sequenciamento do Exoma , Adulto Jovem
13.
Cell Rep ; 26(10): 2651-2666.e6, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30840888

RESUMO

Intratumor mutational heterogeneity has been documented in primary non-small-cell lung cancer. Here, we elucidate mechanisms of tumor evolution and heterogeneity in metastatic thoracic tumors (lung adenocarcinoma and thymic carcinoma) using whole-exome and transcriptome sequencing, SNP array for copy-number alterations (CNAs), and mass-spectrometry-based quantitative proteomics of metastases obtained by rapid autopsy. APOBEC mutagenesis, promoted by increased expression of APOBEC3 region transcripts and associated with a high-risk APOBEC3 germline variant, correlated with mutational tumor heterogeneity. TP53 mutation status was associated with APOBEC hypermutator status. Interferon pathways were enriched in tumors with high APOBEC mutagenesis and IFN-γ-induced expression of APOBEC3B in lung adenocarcinoma cells, suggesting that the immune microenvironment may promote mutational heterogeneity. CNAs occurring late in tumor evolution correlated with downstream transcriptomic and proteomic heterogeneity, although global proteomic heterogeneity was significantly greater than transcriptomic and CNA heterogeneity. These results illustrate key mechanisms underlying multi-dimensional heterogeneity in metastatic thoracic tumors.


Assuntos
Citidina Desaminase/genética , Neoplasias Torácicas/genética , Desaminases APOBEC , Variações do Número de Cópias de DNA , Heterogeneidade Genética , Mutação em Linhagem Germinativa , Humanos , Mutagênese , Metástase Neoplásica , Proteogenômica/métodos , Neoplasias Torácicas/patologia
14.
Cold Spring Harb Mol Case Stud ; 2(6): a001263, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27900369

RESUMO

We used next-generation sequencing to identify somatic alterations in multiple metastatic sites from an "exceptional responder" lung adenocarcinoma patient during his 7-yr course of ERBB2-directed therapies. The degree of heterogeneity was unprecedented, with ∼1% similarity between somatic alterations of the lung and lymph nodes. One novel translocation, PLAG1-ACTA2, present in both sites, up-regulated ACTA2 expression. ERBB2, the predominant driver oncogene, was amplified in both sites, more pronounced in the lung, and harbored an L869R mutation in the lymph node. Functional studies showed increased proliferation, migration, metastasis, and resistance to ERBB2-directed therapy because of L869R mutation and increased migration because of ACTA2 overexpression. Within the lung, a nonfunctional CDK12, due to a novel G879V mutation, correlated with down-regulation of DNA damage response genes, causing genomic instability, and sensitivity to chemotherapy. We propose a model whereby a subclone metastasized early from the primary site and evolved independently in lymph nodes.


Assuntos
Adenocarcinoma/genética , Neoplasias Pulmonares/genética , Receptor ErbB-2/genética , Adenocarcinoma/metabolismo , Adenocarcinoma de Pulmão , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica/genética , Genes erbB-2/genética , Genômica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Neoplasias Pulmonares/metabolismo , Masculino , Pessoa de Meia-Idade , Mutação , Metástase Neoplásica/genética , Receptor ErbB-2/metabolismo , Resultado do Tratamento
15.
Cancer Discov ; 5(5): 534-49, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25735773

RESUMO

UNLABELLED: Somatic mutations in the EGFR kinase domain drive lung adenocarcinoma. We have previously identified MIG6, an inhibitor of ERBB signaling and a potential tumor suppressor, as a target for phosphorylation by mutant EGFRs. Here, we demonstrate that MIG6 is a tumor suppressor for the initiation and progression of mutant EGFR-driven lung adenocarcinoma in mouse models. Mutant EGFR-induced lung tumor formation was accelerated in Mig6-deficient mice, even with Mig6 haploinsufficiency. We demonstrate that constitutive phosphorylation of MIG6 at Y394/Y395 in EGFR-mutant human lung adenocarcinoma cell lines is associated with an increased interaction of MIG6 with mutant EGFR, which may stabilize EGFR protein. MIG6 also fails to promote mutant EGFR degradation. We propose a model whereby increased tyrosine phosphorylation of MIG6 decreases its capacity to inhibit mutant EGFR. Nonetheless, the residual inhibition is sufficient for MIG6 to delay mutant EGFR-driven tumor initiation and progression in mouse models. SIGNIFICANCE: This study demonstrates that MIG6 is a potent tumor suppressor for mutant EGFR-driven lung tumor initiation and progression in mice and provides a possible mechanism by which mutant EGFR can partially circumvent this tumor suppressor in human lung adenocarcinoma.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/deficiência , Adenocarcinoma/genética , Adenocarcinoma/patologia , Transformação Celular Neoplásica/genética , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mutação , Proteínas Supressoras de Tumor/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenocarcinoma/mortalidade , Adenocarcinoma de Pulmão , Animais , Linhagem Celular Tumoral , Progressão da Doença , Receptores ErbB/metabolismo , Deleção de Genes , Expressão Gênica , Humanos , Neoplasias Pulmonares/mortalidade , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteômica , Transdução de Sinais/efeitos dos fármacos , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
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