Mechanisms of cancer initiation/progression: an investigation of chromosomal "hot spots" in South African cancer patients

• Main Author: Willem-Belot, Pascale Sylvie
• Format: Others
• Language: en
• Published: 2009
• Subjects: chromosomal hot spots; cancer
• Online Access: http://hdl.handle.net/10539/7352

Ph. D., Faculty of Health Sciences, University of the Witwatersrand, 2008. === The human chromosome complement contains nonrandom genomic regions that are prone to breaks and recurrently altered in tumors. These chromosomal “hot spots” are preferentially involved in early events of genomic instability and point to genes in their vicinity that may participate to carcinogenesis. Amplicons and deletions are frequently generated at “hot spots”, including common fragile sites (CFS), and are thought to host cancer genes whose rearrangements drive cell proliferation and promote the initiation and progression of cancer. Chromosomal “hot spots” in South African cancer patients, were investigated in this study with a view to characterizing underlying gene alterations. A chromosome 12p amplicon was mapped and array comparative genomic hybridization (aCGH) was pioneered to identify candidate genes in the amplicon. Genes of the 12p stem cell gene cluster (NANOG, STELLAR and GDF3) were involved in striking similarity to what has been reported in testicular germ cell tumors and suggest that they may be more commonly involved in different types of cancer. Two tumor suppressor genes, FHIT and WWOX, are located at the two most commonly expressed fragile sites, FRA3B and FRA16D respectively. Alterations in these fragile site associated genes have been reported in a variety of tumors including lung, esophageal, gastric, breast and cervical cancers most frequently as a result of submicroscopic deletions. Genomic deletions at CFS have been mostly investigated using loss of heterozygosity assays that do not necessarily inform on gene exon deletions. A new method was developed based on multiplex ligation-dependent probe amplification (MLPA) that screens for exon deletions/amplifications of genes at CFS. The assay was validated on five esophageal squamous carcinoma cell lines and showed deletions in the FRA3B-associated gene FHIT in four of the cell lines. Two geographically distinct South African cohorts of esophageal squamous cell carcinoma were then screened for FHIT/WWOX exon deletions and a visual basic (VBA) encoded program was written to automate MPLA products analysis. A high frequency of intragenic deletions in FHIT and/or WWOX (73%) was observed in the Eastern Cape cohort. FHIT deletions were seen in 27% of specimens from the Gauteng cohort, which by contrast did not show WWOX deletions. This difference may however reflect a difference in sampling collection. The breakpoints of a translocation t(3;11)(p14;p15.1) present in an ovarian carcinoma cell line was characterized using the above MLPA assay, aCGH and the polymerase chain reaction. The translocation was found to interrupt the FHIT gene making it the 5th cancer associated translocation involving FHIT. The evaluation of gene relative copy number by aCGH and MLPA were highly correlated further validating the power of the MLPA assay in fresh tissue. The involvement of critical genes at “hot spots” in SA cancer patients was high in the context of this study raising questions about the possible role of environmental exposure. The new MLPA assay may assist to expand the screening of critical genes at fragile sites in the future.