Genome-wide CRISPR screens of oral squamous cell carcinoma reveal fitness genes in the Hippo pathway.
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ABSTRACT:
SUBMITTER: Chai AWY
PROVIDER: S-EPMC7591259 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundChemoresistance is one of the major factors for treatment failure in OSCC. Identifying key resistance triggering molecules will be useful strategy for developing novel treatment methods.MethodsTo identify the causative factors of chemoresistance, we performed RNA sequencing and global proteomic profiling of human OSCC lines presenting with sensitive, early and late cisplatin-resistance patterns.ResultsFrom the common set of dysregulated genes from both the analysis, RRBP1 was identified to be upregulated in both early and late cisplatin-resistant cells with respect to the sensitive counterpart. Analysis of OSCC patient sample indicates that RRBP1 expression is upregulated in chemotherapy-non-responder tumours as compared to chemotherapy-responder tumours. Genetic (knockout) or pharmacological (Radezolid, represses expression of RRBP1) inhibition of RRBP1 restores cisplatin-mediated cell death in chemo-resistant OSCC. Mechanistically, RRBP1 regulates Yes-associated protein1 (YAP1), a key protein in the Hippo pathway to induce chemoresistance. The PDC xenograft data suggests that knockout of RRBP1 induces cisplatin-mediated cell death and facilitates a significant reduction of tumour burden.ConclusionOverall, our data suggests that (I) RRBP1 is a major driver of cisplatin-resistance in OSCC, (II) RRBP1 regulates YAP1 expression to mediate cisplatin-resistance, (III) Radezolid represses RRBP1 expression and (IV) targeting RRBP1 reverses cisplatin-induced chemoresistance in advanced OSCC.
| S-EPMC8184829 | biostudies-literature
Project description:The ability to perturb genes in human cells is crucial for elucidating gene function and holds great potential for finding therapeutic targets for diseases such as cancer. To extend the catalog of human core and context- dependent fitness genes, we have developed a high-complexity second-generation genome-scale CRISPR-Cas9 gRNA library and applied it to fitness screens in five human cell lines. Using an improved Bayesian analytical approach, we consistently discover 5-fold more fitness genes than were previously observed. We present a list of 1,580 human core fitness genes and describe their general properties. Moreover, we demonstrate that context-dependent fitness genes accurately recapitulate pathway-specific genetic vulnerabilities induced by known oncogenes and reveal cell-type-specific dependencies for specific receptor tyrosine kinases, even in oncogenic KRAS backgrounds. Thus, rigorous identification of human cell line fitness genes using a high-complexity CRISPR-Cas9 library affords a high-resolution view of the genetic vulnerabilities of a cell. Additional data can be found at tko.ccbr.utoronto.ca RNAseq of five human cell lines with Cas9 knock-ins.
2015-11-25 | E-GEOD-75189 | biostudies-arrayexpress
Project description:Early diagnosis of nasopharyngeal carcinoma (NPC) is difficult because of a lack of specific symptoms. Many patients have advanced disease at diagnosis, and these patients respond poorly to treatment. New treatments are therefore needed to improve the outcome of NPC. To better understand the molecular pathogenesis of NPC, here we used an NPC cell line in a genome-wide CRISPR-based knockout screen to identify the cellular factors and pathways essential for NPC (i.e. dependence factors). This screen identified the Moz, Ybf2/Sas3, Sas2, Tip60 histone acetyl transferase complex, NF-κB signaling, purine synthesis, and linear ubiquitination pathways; and MDM2 proto-oncogene as NPC dependence factors/pathways. Using gene knock out, complementary DNA rescue, and inhibitor assays, we found that perturbation of these pathways greatly reduces the growth of NPC cell lines but does not affect growth of SV40-immortalized normal nasopharyngeal epithelial cells. These results suggest that targeting these pathways/proteins may hold promise for achieving better treatment of patients with NPC.
| S-EPMC6597810 | biostudies-literature
Project description:Oral tongue squamous cell carcinoma (OTSCC) is a highly malignant type of tumor. The 5-year survival rate of patients with advanced tongue squamous cell carcinoma is only ~50%. Pyruvate kinase M2 (PKM2) is the key rate-limiting enzyme of glycolysis, maintaining the Warburg effect in tumor cells. The present study aimed to investigate the relationship between PKM2 expression and the poor prognosis of patients with OTSCC and to determine oral squamous carcinoma tumor cell proliferation and apoptosis. Reverse transcription-quantitative (RT-q) PCR, western blotting and immunohistochemistry were used to analyze the expression levels of PKM2 in OTSCC, and the clinicopathological characteristics and prognosis of patients with OTSCC were further analyzed by statistical analysis. The results from RT-qPCR and immunohistochemistry demonstrated that PKM2 was upregulated in OTSCC tissues and highly expressed in advanced stage OTSCC tissues compared with paired adjacent tissues and lower stage OTSCC tissues. Patients with OTSCC and high PKM2 expression had shorter overall survival (OS) compared with those with low PKM2 expression. Furthermore, high expression of PKM2 was significantly associated with Tumor-Node-Metastasis (TNM) stage. TNM stage and PKM2 expression were independent predictive factors for OS in patients with OTSCC. In addition, PKM2 knockdown inhibited the proliferation and increased the apoptosis of oral squamous carcinoma tumor cells. Furthermore, PKM2 knockdown could regulate the expression of cell cycle and apoptosis-related proteins by activating Hippo signaling pathway, as confirmed by the decreased expression of yes-associated protein 1 (YAP), Bcl-2 and Ki-67 and the increased expression of large tumor suppressor kinase 1, phosphorylated YAP and Bax. Taken together, the findings from this study demonstrated that PKM2 may be considered as a potential target for the diagnosis and treatment of OTSCC.
| S-EPMC8063272 | biostudies-literature
Project description:Most cancer cells are exposed to extracellular environments, such as extracellular matrix, which commonly becomes stiffer along with transformation. It is conceivable that tumorous extracellular environments would be changing to affect tumor cell behavior. It has been reported that Hippo pathway responds to the extracellular environments and induces the nuclear localization of transcription activator, yes-associated protein (YAP), resulting in stimulating cell proliferation. Its pathway also regulates gene expression, but the precise molecule to meditate cell proliferating effect of Hippo pathway in oral squamous cell carcinoma (OSCC) is not well understood. Here, we examined the effects of YAP-mediated Hippo pathway in OSCC tumorigenesis. Loss-of-function experiments using siRNA or an inhibitor, and immunohistochemical analyses of tissue specimens obtained from OSCC specimens demonstrated that YAP-mediated Hippo pathway was involved in OSCC cell proliferation. We identified Piezo-type mechanosensitive ion channel component 1 (PIEZO1), a Ca2+ channel, as a downstream molecule of Hippo pathway and showed that elevated PIEZO1 expression was required for PIEZO1 agonist-dependent Ca2+ entry and cell proliferation in OSCC cells. Furthermore, the experiments using three-dimensional culture and suspension culture revealed that PIEZO1, of which expression was regulated by YAP-mediated Hippo pathway, was involved in OSCC cellular growth. Immunohistochemically, YAP overexpression with nucleus and/or cytoplasm was detected with both PIEZO1 and Ki-67 expression at high frequencies in tumor lesion, but not in non-tumor region, of OSCC specimens. These results suggest that the YAP-mediated Hippo/PIEZO1 axis, which might be activated by the tumorous extracellular environments, promotes OSCC tumor cell growth.
2020-05-08 | GSE150044 | GEO
Project description:In mammalian cells, nutrients and growth factors signal through an array of upstream proteins to regulate the mTORC1 growth control pathway. Because the full complement of these proteins has not been systematically identified, we developed a FACS-based CRISPR-Cas9 genetic screening strategy to pinpoint genes that regulate mTORC1 activity. Along with almost all known positive components of the mTORC1 pathway, we identified many genes that impact mTORC1 activity, including DCAF7, CSNK2B, SRSF2, IRS4, CCDC43, and HSD17B10 Using the genome-wide screening data, we generated a focused sublibrary containing single guide RNAs (sgRNAs) targeting hundreds of genes and carried out epistasis screens in cells lacking nutrient- and stress-responsive mTORC1 modulators, including GATOR1, AMPK, GCN2, and ATF4. From these data, we pinpointed mitochondrial function as a particularly important input into mTORC1 signaling. While it is well appreciated that mitochondria signal to mTORC1, the mechanisms are not completely clear. We find that the kinases AMPK and HRI signal, with varying kinetics, mitochondrial distress to mTORC1, and that HRI acts through the ATF4-dependent up-regulation of both Sestrin2 and Redd1. Loss of both AMPK and HRI is sufficient to render mTORC1 signaling largely resistant to mitochondrial dysfunction induced by the ATP synthase inhibitor oligomycin as well as the electron transport chain inhibitors piericidin and antimycin. Taken together, our data reveal a catalog of genes that impact the mTORC1 pathway and clarify the multifaceted ways in which mTORC1 senses mitochondrial dysfunction.
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Project description:Poly (ADP-ribose) polymerase inhibitor (PARPi)-based therapies initially reduce tumor burden but eventually lead to acquired resistance in cancer patients with BRCA1 or BRCA2 mutation. To understand the potential PARPi resistance mechanisms, we performed whole-genome CRISPR screens to discover genetic alterations that change the gene essentiality in cells with inducible depletion of BRCA2. We identified that several RNA Polymerase II transcription Mediator complex components, especially Cyclin C (CCNC) as synthetic survival targets upon BRCA2 loss. Total mRNA sequencing demonstrated that loss of CCNC could activate the transforming growth factor (TGF)-beta signaling pathway and extracellular matrix (ECM)-receptor interaction pathway, however the inhibition of these pathways could not reverse cell survival in BRCA2 depleted CCNC-knockout cells, indicating that the activation of these pathways is not required for the resistance. Moreover, we showed that the improved survival is not due to restoration of homologous recombination repair although decreased DNA damage signaling was observed. Interestingly, loss of CCNC could restore replication fork stability in BRCA2 deficient cells, which may contribute to PARPi resistance. Taken together, our data reveal CCNC as a critical genetic determinant upon BRCA2 loss of function, which may help the development of novel therapeutic strategies that overcome PARPi resistance.
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Project description:Ataxia telangiectasia mutated and RAD3 related (ATR) protein kinase plays critical roles in ensuring DNA replication, DNA repair, and cell cycle control in response to replication stress, making ATR inhibition a promising therapeutic strategy for cancer treatment. To identify genes whose loss makes tumor cells hypersensitive to ATR inhibition, we performed CRISPR/Cas9-based whole-genome screens in 3 independent cell lines treated with a highly selective ATR inhibitor, AZD6738. These screens uncovered a comprehensive genome-wide profile of ATR inhibitor sensitivity. From the candidate genes, we demonstrated that RNASEH2 deficiency is synthetic lethal with ATR inhibition both in vitro and in vivo. RNASEH2-deficient cells exhibited elevated levels of DNA damage and, when treated with AZD6738, underwent apoptosis (short-time treated) or senescence (long-time treated). Notably, RNASEH2 deficiency is frequently found in prostate adenocarcinoma; we found decreased RNASEH2B protein levels in prostate adenocarcinoma patient-derived xenograft (PDX) samples. Our findings suggest that ATR inhibition may be beneficial for cancer patients with reduced levels of RNASEH2 and that RNASEH2 merits further exploration as a potential biomarker for ATR inhibitor-based therapy.
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Project description:Identification of host genes essential for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may reveal novel therapeutic targets and inform our understanding of coronavirus disease 2019 (COVID-19) pathogenesis. Here we performed genome-wide CRISPR screens in Vero-E6 cells with SARS-CoV-2, Middle East respiratory syndrome CoV (MERS-CoV), bat CoV HKU5 expressing the SARS-CoV-1 spike, and vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike. We identified known SARS-CoV-2 host factors, including the receptor ACE2 and protease Cathepsin L. We additionally discovered pro-viral genes and pathways, including HMGB1 and the SWI/SNF chromatin remodeling complex, that are SARS lineage and pan-coronavirus specific, respectively. We show that HMGB1 regulates ACE2 expression and is critical for entry of SARS-CoV-2, SARS-CoV-1, and NL63. We also show that small-molecule antagonists of identified gene products inhibited SARS-CoV-2 infection in monkey and human cells, demonstrating the conserved role of these genetic hits across species. This identifies potential therapeutic targets for SARS-CoV-2 and reveals SARS lineage-specific and pan-CoV host factors that regulate susceptibility to highly pathogenic CoVs.
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Project description:Oral squamous cell carcinoma (OSCC) is one of the most common malignancies and has a poor prognosis. Circular RNA (circRNA) has been increasingly recognized as a crucial contributor to carcinogenesis. circRNA_0000140 has been aberrantly expressed in OSCC, but its role in tumor growth and metastasis remains largely unclear. Sanger sequencing, actinomycin D, and RNase R treatments were used to confirm head-to-tail junction sequences and the stability of circ_0000140. In vitro cell activities, including proliferation, migration, invasion, and apoptosis, were determined by colony formation, transwell, and flow cytometry assays. The expression levels of circ_0000140, Hippo signaling pathway, and serial epithelial-mesenchymal transition (EMT) markers were measured by quantitative real-time PCR, western blotting, immunofluorescence, and immunohistochemistry. Dual luciferase reporter assays and Argonaute 2-RNA immunoprecipitation assays were performed to explore the interplay among circ_0000140, miR-31, and LATS2. Subcutaneous tumor growth was observed in nude mice, in which in vivo metastasis was observed following tail vein injection of OSCC cells. circ_0000140 is derived from exons 7 to 10 of the KIAA0907 gene. It was down-regulated in OSCC tissues and cell lines, and correlated negatively with poor prognostic outcomes in OSCC patients. Gain-of-function experiments demonstrated that circ_0000140 enhancement suppressed cell proliferation, migration, and invasion, and facilitated cell apoptosis in vitro. In xenograft mouse models, overexpression of circ_0000140 was able to repress tumor growth and lung metastasis. Furthermore, mechanistic studies showed that circ_0000140 could bind with miR-31 and up-regulate its target gene LATS2, thus affecting OSCC cellular EMT. Our findings demonstrated the roles of circ_0000140 in OSCC tumorigenesis as well as in metastasis, and circ_0000140 exerts its tumor-suppressing effect through miR-31/LATS2 axis of Hippo signaling pathway in OSCC.
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