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Exome sequencing of a novel cervical cancer cell line

ABSTRACT: Human cancer cell lines are largely used in the searching for new antineoplastic agents. However, due to the artifacts of a long-term in culture, cell lines do not always represent the realistic tumor cell behavior. This has motivated the development of models that better mimetics the tumor tissue, among them, the establishment of primary cell cultures. In this work, we establish and characterized a low-passage cervix cancer cell line from a Brazilian patient with squamous cell carcinoma. The phenotype confirms the epithelial and tumor origin, through cytokeratins, EpCAM, and p16 staining. Whole exome sequencing showed relevant somatic mutations in several genes including BRCA2, TGFBR1, and IRX2 genes. CNV analysis by nanostring and WGS revealed amplification in genes related mainly with kinases proteins, involved in proliferation, migration and cell differentiation, such as EGFR, PIK3CA, and MAPK7. Overexpression of EGFR was confirmed by phospho RTK-array and western blot analysis. Furthermore, the cell was sensitivity to cisplatin, with IC50 13 times lower than SiHa cell line. In conclusion, this cervical cancer cell line presents molecular alterations that are an important tool for leading pre-clinical studies of new drugs that target one or more of the altered pathways.

PROVIDER: EGAS00001003343 | EGA |

REPOSITORIES: EGA

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Project description:INTRODUCTION: CDKN2A (p16) inactivation is common in lung cancer and occurs via homozygous deletions, methylation of promoter region, or point mutations. Although p16 promoter methylation has been linked to KRAS mutation and smoking, the associations between p16 inactivation mechanisms and other common genetic mutations and smoking status are still controversial or unknown. METHODS: We determined all three p16 inactivation mechanisms with the use of multiple methodologies for genomic status, methylation, RNA, and protein expression, and correlated them with EGFR, KRAS, STK11 mutations and smoking status in 40 cell lines and 45 tumor samples of primary non-small-cell lung carcinoma. We also performed meta-analyses to investigate the impact of smoke exposure on p16 inactivation. RESULTS: p16 inactivation was the major mechanism of RB pathway perturbation in non-small-cell lung carcinoma, with homozygous deletion being the most frequent method, followed by methylation and the rarer point mutations. Inactivating mechanisms were tightly correlated with loss of mRNA and protein expression. p16 inactivation occurred at comparable frequencies regardless of mutational status of EGFR, KRAS, and STK11, however, the major inactivation mechanism of p16 varied. p16 methylation was linked to KRAS mutation but was mutually exclusive with EGFR mutation. Cell lines and tumor samples demonstrated similar results. Our meta-analyses confirmed a modest positive association between p16 promoter methylation and smoking. CONCLUSION: Our results confirm that all the inactivation mechanisms are truly associated with loss of gene product and identify specific associations between p16 inactivation mechanisms and other genetic changes and smoking status. 40 NSCLC cell lines were profiled on Illumina WG6-V3 expression arrays. They consist of 4 groups of 10 cell lines each: non-smokers with EGFR mutation, non-smokers with EGFR wild-type, smokers with KRAS mutation, smokers with KRAS wild-type. Note that smoking status is not always clear.

Project description:PD-0332991 is a selective inhibitor of the CDK4/6 kinases with the ability to block retinoblastoma (Rb) phosphorylation in the low nanomolar range. Here we investigate the role of CDK4/6 inhibition in human ovarian cancer. We examined the effects of PD-0332991 on proliferation, cell-cycle, apoptosis, and Rb phosphorylation using a panel of 40 established human ovarian cancer cell lines. Molecular markers for response prediction, including p16 and Rb, were studied using gene expression profiling, Western blot, and arrayCGH. Multiple drug effect analysis was used to study interactions with chemotherapeutic drugs. Expression of p16 and Rb was studied using immunohistochemistry in a large clinical cohort ovarian cancer patients. Concentration-dependent anti-proliferative effects of PD-0332991were seen in all ovarian cancer cell lines, but varied significantly between individual lines. Rb proficient cell lines with low p16 expression were most responsive to CDK4/6 inhibition. Copy number variations of CDKN2A, Rb, CCNE1, and CCND1 were associated with response to PD-0332991. CDK4/6 inhibition induced G0/G1 cell cycle arrest, blocked Rb phosphorylation in a concentration and time dependent manner, and enhanced the effects of chemotherapy. Rb proficiency with low p16 expression was seen in 97/262 (37%) of ovarian cancer patients and associated with adverse clinical outcome (progression free survival, adjusted relative risk 1.49, 95%CI 0.99-2.22, p =0.054). PD-0332991 shows promising biologic activity in ovarian cancer cell lines. Assessment of Rb and p16 expression may help select patients most likely to benefit from CDK4/6 inhibition in ovarian cancer. Gene expression of 40 individual ovarian cell lines relative to an ovarian cell line reference mix containing equal amounts of 41 ovarian cell lines (including OCC-1 which was later identified as originating from mouse). The expression data was correllated with cell line growth response to CDK 4/6 inhibitor PD-0332991 to identify genes associated with drug sensitivity and resistance.

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Exome sequencing of a novel cervical cancer cell line

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