Molecular studies of pancreatic cancer: Characterization of the transforming growth factor beta signaling pathway

Sammanfattning: In the present thesis, genetic abnormalities in pancreatic cancer were studied, with special emphasis on alterations of components involved in the transforming growth factor beta (TGFB) signaling pathway. In the first study, fluorescence in situ hybridization and cytogenetic analyses revealed aberrations of chromosome 18 in all 13 pancreatic carcinoma cell lines studied, in particular frequent breaks close to the centromere of chromosome arm 18q. The results suggested the presence of at least one tumor suppressor gene (TSG) at 18q, one candidate being SMAD4, which is a key component of the TGFB signaling pathway. The second study was initiated to investigate the role of five SMAD genes involved in TGFB signaling, i.e., the potential TSGs SMAD2 (18q), SMAD3 (15q), and SMAD4 (18q), as well as the putative oncogenes SMAD6 (15q) and SMAD7 (18q). Loss of heterozygosity and mutation analyses revealed frequent loss of 18q material and SMAD4 inactivations in 5 of 12 cases, whereas no mutations of SMAD2, SMAD3, SMAD6, or SMAD7 were detected. In the third study, the TGFB receptors were investigated. No mutations of TGFBR2 or ALK5 (TGFBR1) were found, expression of TGFBR2 and TGFB3 was maintained or increased, whereas ALK5 was downregulated. The response to TGFB was analyzed in 12 pancreatic carcinoma cell lines, half of which responded by an increase in proliferation. In the fourth study, filter-based microarrays were used to study TGFB induced gene expression alterations. The results demonstrated that pancreatic cancer cell lines with SMAD4 mutations still respond to TGFB treatment. In addition, a gradual inverse gene expression pattern as compared to TGFB sensitive control cells was observed in the pancreatic carcinoma cell lines that correlated with reduced sensitivity to TGFB growth inhibition. In the fifth study, polymerase chain reaction (PCR) protocols for gene expression and gene copy analyses were developed. In summary, the results of the present thesis suggest that partial inactivation of the TGFB signaling pathway, e.g., SMAD4 inactivation and downregulated ALK5 expression, in conjunction with other genetic alterations gradually alter the ability of pancreatic tumor cells to be growth inhibited by TGFB. The most important finding was the detected ability of half of the investigated cell lines to be growth stimulated by TGFB, thus providing a basis for future investigations of the biphasic effects of TGFB observed during the progression of many different tumor types.