RESUMO
Prior to the next generation sequencing and characterization of the tumor genome landscape, mutations in the SWI/SNF chromatin remodeling complex and the KEAP1-NRF2 signaling pathway were underappreciated. While these two classes of mutations appeared to independently contribute to tumor development, recent reports have demonstrated a mechanistic link between these two regulatory mechanisms in specific cancer types and cell models. In this work, we expand upon these data by exploring the relationship between mutations in BAF and PBAF subunits of the SWI/SNF complex and activation of NRF2 signal transduction across many cancer types. ARID1A/B mutations were strongly associated with NRF2 transcriptional activity in head and neck squamous carcinomas (HNSC). Many additional tumor types showed significant association between NRF2 signaling and mutation of specific components of the SWI/SNF complex. Different effects of BAF and PBAF mutations on the polarity of NRF2 signaling were observed. Overall, our results support a context-dependent functional link between SWI/SNF and NRF2 mutations across human cancers and implicate ARID1A inactivation in HPV-negative HNSC in promoting tumor progression and survival through activation of the KEAP1-NRF2 signaling pathway. The tumor-specific effects of these mutations open a new area of study for how mutations in the KEAP1-NRF2 pathway and the SWI/SNF complex contribute to cancer.
Assuntos
Proteínas de Ligação a DNA , Neoplasias de Cabeça e Pescoço , Fator 2 Relacionado a NF-E2 , Fatores de Transcrição , Humanos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Neoplasias de Cabeça e Pescoço/genética , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Mutação , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transdução de Sinais/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Aberrant activation of the NRF2/NFE2L2 transcription factor commonly occurs in head and neck squamous cell carcinomas (HNSCC). Mouse model studies have shown that NRF2 activation alone does not result in cancer. When combined with classic oncogenes and at the right dose, NRF2 activation promotes tumor initiation and progression. Here we deleted the tumor suppressor genes p16INK4A and p53 (referred to as CP mice), which are commonly lost in human HNSCC, in the presence of a constitutively active NRF2E79Q mutant (CPN mice). NRF2E79Q expression in CPN mice resulted in squamous cell hyperplasia or dysplasia with hyperkeratosis in the esophagus, oropharynx, and forestomach. In addition, CPN mice displayed oral cavity squamous cell carcinoma (OSCC); CP mice bearing wild-type NRF2 expression did not develop oral cavity hyperplasia, dysplasia or OSCC. In both CP and CPN mice, we also observed predominantly abdominal sarcomas and carcinomas. Our data show that in the context of p53 and p16 tumor suppressor loss, NRF2 activation serves oncogenic functions to drive OSCC. CPN mice represent a new model for OSCC that closely reflects the genetics of human HNSCC. SIGNIFICANCE: Human squamous cancers frequently show constitutive NRF2 activation, associated with poorer outcomes and resistance to multiple therapies. Here, we report the first activated NRF2-driven and human-relevant mouse model of squamous cell carcinoma that develops in the background of p16 and p53 loss. The availability of this model will lead to a clearer understanding of how NRF2 contributes to the initiation, progression, and therapeutic response of OSCC.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Animais , Humanos , Camundongos , Carcinoma de Células Escamosas/genética , Modelos Animais de Doenças , Neoplasias de Cabeça e Pescoço/genética , Hiperplasia/genética , Neoplasias Bucais/genética , Fator 2 Relacionado a NF-E2/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Proteína Supressora de Tumor p53/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismoRESUMO
The human kinome, with more than 500 proteins, is crucial for cell signaling and disease. Yet, about one-third of kinases lack in-depth study. The Data and Resource Generating Center for Understudied Kinases has developed multiple resources to address this challenge including creation of a heavy amino acid peptide library for parallel reaction monitoring and quantitation of protein kinase expression, use of understudied kinases tagged with a miniTurbo-biotin ligase to determine interaction networks by proximity-dependent protein biotinylation, NanoBRET probe development for screening chemical tool target specificity in live cells, characterization of small molecule chemical tools inhibiting understudied kinases, and computational tools for defining kinome architecture. These resources are available through the Dark Kinase Knowledgebase, supporting further research into these understudied protein kinases.
Assuntos
Proteínas Quinases , Proteínas , Humanos , Proteínas Quinases/metabolismo , ProteômicaRESUMO
Squamous cell carcinomas (SCCs), including lung, head & neck, bladder, and skin SCCs often display constitutive activation of the KEAP1-NRF2 pathway. Constitutive activation is achieved through multiple mechanisms, including activating mutations in NFE2L2 (NRF2). To determine the functional consequences of Nrf2 activation on skin SCC development, we assessed the effects of mutant Nrf2E79Q expression, one of the most common activating mutations in human SCCs, on tumor promotion and progression in the mouse skin multistage carcinogenesis model using a DMBA-initiation/TPA-promotion protocol where the Hras A->T mutation (Q61L) is the canonical driver mutation. Nrf2E79Q expression was temporally and conditionally activated in the epidermis at two stages of tumor development: 1) after DMBA initiation in the epidermis but before cutaneous tumor development and 2) in pre-existing DMBA-initiated/TPA-promoted squamous papillomas. Expression of Nrf2E79Q in the epidermis after DMBA initiation but before tumor occurrence inhibited the development/promotion of 70% of squamous papillomas. However, the remaining papillomas often displayed non-canonical Hras and Kras mutations and enhanced progression to SCCs compared to control mice expressing wildtype Nrf2. Nrf2E79Q expression in pre-existing tumors caused rapid regression of 60% of papillomas. The remaining papillomas displayed the expected canonical Hras A->T mutation (Q61L) and enhanced progression to SCCs. These results demonstrate that mutant Nrf2E79Q enhances the promotion and progression of a subset of skin tumors and alters the frequency and diversity of oncogenic Ras mutations when expressed early after initiation.
RESUMO
The protection of the replication fork structure under stress conditions is essential for genome maintenance and cancer prevention. A key signaling pathway for fork protection involves TRPV2-mediated Ca2+ release from the ER, which is triggered after the generation of cytosolic DNA and the activation of cGAS/STING. This results in CaMKK2/AMPK activation and subsequent Exo1 phosphorylation, which prevent aberrant fork processing, thereby ensuring genome stability. However, it remains poorly understood how the TRPV2 channel is activated by the presence of cytosolic DNA. Here, through a genome-wide CRISPR-based screen, we identify TRPM8 channel-associated factor 1 (TCAF1) as a key factor promoting TRPV2-mediated Ca2+ release under replication stress or other conditions that activate cGAS/STING. Mechanistically, TCAF1 assists Ca2+ release by facilitating the dissociation of STING from TRPV2, thereby relieving TRPV2 repression. Consistent with this function, TCAF1 is required for fork protection, chromosomal stability, and cell survival after replication stress.
Assuntos
Cálcio , Citosol , Replicação do DNA , Proteínas de Membrana , Canais de Cátion TRPV , Humanos , Canais de Cátion TRPV/metabolismo , Canais de Cátion TRPV/genética , Cálcio/metabolismo , Citosol/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Células HEK293 , DNA/metabolismo , Células HeLa , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/genética , Fosforilação , Instabilidade Genômica , Dano ao DNA , AnimaisRESUMO
Host factors that define the cellular tropism of SARS-CoV-2 beyond the cognate ACE2 receptor are poorly defined. From a screen of human airway derived cell lines that express varying levels of ACE2/TMPRSS2, we found a subset that express comparably high endogenous levels of ACE2 but surprisingly did not support SARS-CoV-2 replication. Here we report that this resistance is mediated by a basally active cGAS-STING pathway culminating in interferon (IFN)-mediated restriction of SARS-CoV-2 replication at a post-entry step. Pharmacological inhibition of JAK1/2, depletion of the IFN-α receptor and cGAS-STING pathway effectors substantially increased SARS-CoV-2 replication in these cell models. While depletion of cGAS or STING was sufficient to reduce the preexisting levels of IFN-stimulated genes (ISGs), SARS-CoV-2 infection in STING knockout cells independently induced ISG expression. Remarkably, SARS-CoV-2-induced ISG expression in STING knockout cell as well as in primary human airway cultures was limited to uninfected bystander cells, demonstrating efficient antagonism of the type I/III IFN-pathway, but not viral sensing or IFN production, in productively infected cells. Of note, SARS-CoV-2-infected primary human airway cells also displayed markedly lower levels of STING expression, raising the possibility that SARS-CoV-2 can target STING expression or preferentially infect cells that express low levels of STING. Finally, ectopic ACE2 overexpression overcame the IFN-mediated blocks, suggesting the ability of SARS-CoV-2 to overcome these possibly saturable blocks to infection. Our study highlights that in addition to viral receptors, basal activation of the cGAS-STING pathway and innate immune defenses may contribute to defining SARS-CoV-2 cellular tropism.
RESUMO
Primary ciliary dyskinesia (PCD) is a genetic condition that results in dysmotile cilia. The repercussions of cilia dysmotility and gene variants on the multiciliated cell remain poorly understood. We used single-cell RNA sequencing, proteomics, and advanced microscopy to compare primary culture epithelial cells from patients with PCD, their heterozygous mothers, healthy individuals, and induced pluripotent stem (iPS) cells generated from a PCD patient. Transcriptomic analysis revealed unique signatures in PCD airway cells compared to their mothers and healthy individuals. Gene expression in heterozygous mothers' cells diverged from both control and PCD cells, marked by increased inflammatory and cellular stress signatures. Primary and iPS-derived PCD multiciliated cells had increased expression of glutathione-S-transferases, GSTA2 and GSTA1, as well as NRF2 target genes, accompanied by elevated levels of reactive oxygen species (ROS). Immunogold labeling in human cilia and proteomic analysis of the ciliated organism, Chlamydomonas reinhardtii, demonstrated that GSTA2 localizes to motile cilia. Loss of human GSTA2 and C. reinhardtii GSTA resulted in slowed cilia motility pointing to local cilia regulatory roles. Our findings identify cellular responses unique to PCD variants and independent of environmental stress and uncover a dedicated ciliary GSTA2 pathway essential for normal motility that may be a therapeutic target.