Genomische analyses van hoog-risico prostaatkanker

Abstract

PCa is the second most frequently diagnosed cancer in males worldwide. A wide range of genomic alterations, including point mutations, copy number changes and rearrangements, can lead to the development of cancer. Due to the heterogeneity of PCa, it still remains a clinical challenge to differentiate indolent from very aggressive tumors. A better molecular profiling of the primary tumors should enable a better classification of the disease, ultimately providing information that could direct a more personalized treatment. One approach is to study the contribution of somatic base pair substitutions to the oncogenic process.Cancer cell lines are commonly used as laboratory resources to study basic molecular and cellular biology. For prostate cancer (PCa), LNCaPs are the most commonly used cells. However, information on protein-changing mutations, genetic heterogeneity and genetic (in)stability is largely lacking for these cells. In a first part, we used exome-sequencing to focus on missense and nonsense single nucleotide variants and short insertions and deletions. We detected 1802 non-synonymous point mutations and 218 small insertions and deletions. While most detected mutations were undescribed so far, we confirmed the known mutations in the androgen receptor and the PTEN gene. Surprisingly, we confirmed 38 out of 42 mutations in DNA and RNA from different monoclonal and polyclonal LNCaP derivatives. From this, we deduced that LNCaP cells are heterozygous for a large number of variants and that both the variant and the wild type allele can be simultaneously expressed as mRNA. The fact that mutations in the E-cadherin, CDK4, Notch1 and PlexinB1 genes are absent in some subclones, strongly indicates a degree of genetic instability. Finally, to help identify the mutations that are most likely drivers of the oncogenic process, we developed an in silico protocol, which can be adopted for other exome analyses. We provided an extensive database of genetic variations in the exome of LNCaP cells, and these should be taken into consideration when using LNCaPs as a model for PCa. The progression of PCa from androgen-dependent to androgen-independent poses an important clinical question, as the mechanisms leading to metastatic PCa are not well understood. C4-2B cells are derivatives of LNCaP cells, as they were derived from a bone metastasis that grew in nude mice after inoculation with the LNCaP-derived, castration-resistant C4-2 cells. The combination of LNCaP and C4-2B cells thus forms an excellent preclinical model to study the development of metastatic castration-resistant PCa. Because of the importance of this progression model, a second part of this study characterized both cell lines more thoroughly using exome and transcriptome sequencing to obtain point mutations and differential expression patterns. Exome sequencing detected 2188 and 3840 mutations in LNCaP and C4-2B cells respectively, of which 1784 were found in both cell lines. The use of more recent algorithms resulted in a higher sensitivity to detect point mutations, increasing the number of mutations detected in LNCaP from 1802 to 2188. Surprisingly, the parental LNCaP cells contained more than 400 mutations that were not found in the C4-2B exome. Moreover, more than half of the mutations found in the exomes of both cell lines were confirmed by analyzing the transcriptome sequencing data. The transcriptome data also revealed that 457 genes show increased expression and 246 genes show decreased expression in C4-2B cells as compared to LNCaP cells. Based on the list of C4-2B-specific point mutations and the list of differentially expressed genes, we detected changes in the focal adhesion and ECM-receptor interaction pathways which converged on the myosin light chain kinase gene. Whether this contributes to the metastatic potential of C4-2B cells remains to be investigated. To conclude, we provide lists of mutated genes and differentially expressed genes in the LNCaP and C4-2B PCa cell lines to all researchers interested in using these cells as preclinical models.A final component of this project used tissue from 27 patients with high-risk primary PCa. We performed exome sequencing and copy number profiling of 27 primary prostate tumors and their normal tissue pairs. Tumors having a PSA > 20 ng/ml, or Gleason score ≥ 8 or clinical stage ≥ T2c are known to have a high risk on disease recurrence after treatment. In addition to amplifications and deletions that were described before, we identified a novel recurrent amplification on 7p22.3. Exome sequencing revealed one hypermutated sample containing 451 mutations, compared to an average of 19 mutations in the other samples, indicating that DNA repair is compromised in this sample. This hypermutated tumor indeed harbored a mutation in the DNA-repair gene Replication Factor C. This mutation is predicted to affect the interaction with PCNA and hence the recruitment of DNA polymerase to PCNA. In a second tumor, we detected a novel point mutation in the TET1 gene. This methylcytosine dioxygenase converts 5-methylcytosine to 5-hydroxymethylcytosine, which leads to demethylation of cytosines and might lead to changes in gene expression or chromatin organization. Immunoprecipitation of methylated and hydroxymethylated DNA followed by deep-sequencing performed on the tumor sample containing the A1908S TET1 mutation demonstrated an overall hypo-hydroxymethylation and hypermethylation at specific genomic loci, when compared to two other tumor samples without mutation in TET1. This effect was corroborated by the in vitro effect of the mutation on the dioxygenase activity as assessed by dot blot assays. We further showed that there is an overlap between groups of androgen-regulated genes and the TET1-regulated genes. Moreover, in cotransfection experiments, TET1 seems to act as a coactivator of the androgen receptor. From the above data, we conclude that the A1908S TET1 mutation as detected in a primary PCa leads to partial loss of TET1 tumor suppressor activity.nrpages: 145status: publishe