Abstract B33: Flexibility of the exome of the LNCaP cell line(s): Differences in heterozygocity and heterogeneity
Authors: Lien Spans, Zeynep Kalender, Stein Aerts, Frank Claessens
Journal: Cancer Research (2012)

Abstract The LNCaP cell line is a frequently used model for basic and preclinical studies of human prostate adenocarcinoma. We used next-generation sequencing to identify Single Nucleotide Variants (SNVs) and indels in the exome from this cell line. Rigorous filtering of more than 25 million reads and elimination of known SNPs led to a list of 1,886 SNVs that cause an amino acid change and a list of 51 indels. Due to the lack of the normal genome of the patient from which the LNCaP line was derived, we can't determine all SNPs. Indeed, the number of SNVs remains surprisingly high, certainly if we compare it with the number of variants that has recently been reported to occur in prostate cancer samples. The known LNCaP point mutation in the AR gene and the deletion in the PTEN gene were present, and over 40 new SNVs and SNPS have been confirmed with genomic PCR and conventional Sanger sequencing. We used open-source software to predict which of the SNVs most likely affect protein functions. An application of Endavour software incorporated three predictive software packages to calculate the effects of the variations on protein structure, as well as data annotations, protein-protein interactions, LNCaP expression levels according to published gene array datasets, and a PubMed search linking the changed protein with prostate cancer. This has led to a prioritization of the SNVs. While the top 9 SNVs reside in protein kinase genes, several others are likely to affect the function of proteins that are involved in the androgen response. We also checked whether there are SNVs present in 115 genes that have been reported in the literature to be linked with the development of prostate cancer. In this group, we detected 14 SNVs (APC, AR, ARAF, CDH1, CDK4, CHEK2, ERBB2, MSH6, MYC, NF1, NOTCH1, PLXNB1, SMO and TERT) and 12 SNPs. While none of the 14 SNV were present in other prostate cancer cell lines (DU145, PC-3, 22Rv1, and VCaP) as expected, some of the SNPs were. Moreover, for most SNVs and SNPS, two alternative bases were detected in the LNCaP genome. For each of the cancer gene SNVs and SNPs, both variants could be detected in the LNCaP cDNA, indicating that in those cases the wild type and the mutated gene are expressed simultaniously. Nine of the fourteen variants that we detected in the exome of our LNCaP cells were found back in the genomes of LNCaP cells from other laboratories as well as in monoclonal sublines, like LNCaP-TR2 and C4-2B. This indicates that LNCaP cells are heterozygous rather than heterogenous for these SNVs. Surprisingly, four of the thirteen variants that we tested for (CDH1, CDK4, NOTCH1 and PLXNB1) were absent in the genome of some of the LNCaP sublines. Hence, there are few, but important genetic variations between LNCaP cell lines used in different laboratories. This indicates some genetic flexibility, which might explain why cell lines in different laboratories react differently. In conclusion, our data show that the LNCaP cell line has a surprising degree of flexibility in its exome sequence. It is unknown yet whether the same is true for other cell lines. We speculate that part of the LNCaP flexibility arises from differences in selective forces due to the many variations in LNCaP subculturing protocols and media between different laboratories. Citation Format: Lien Spans, Zeynep Kalender, Stein Aerts, Frank Claessens. Flexibility of the exome of the LNCaP cell line(s): Differences in heterozygocity and heterogeneity [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B33.