Decreased expression of p73 in colorectal cancer

Introduction. The colorectal cancer (CRC) is one of the most frequent cancer in Poland and worldwide. This disease is characterized by distinct genetic alterations. p73 belongs to the p53 gene family; however, its role in the pathogenesis of CRC has not been completely understood. p73 gene encodes several mRNA variants and protein isoforms with its longest and fully functional p73a (mRNA) and TAp73a (protein) isoform. The aim of the study was to investigate p73 gene expression at the mRNA (p73a) and protein (TAp73a) levels in CRC. Material and methods. Small sections of the crc tumor tissue and macroscopically unchanged colon mucosa and submucosa from the dissection margin were collected from 23 patients diagnosed with CRC. p73 mRNA levels were measured by Real-time PCR (QPCR) method and the expression level of TAp73a protein was assessed by Western blotting (WB) and immunohistochemical (IHC) staining. Results. We found a 37% decrease in the level of p73a mRNA in neoplastically changed (tumor) compared with unchanged normal colon tissue from the surgical margin (p = 0.041). No correlations were found between mRNA levels in cancer tissue and clinical-pathological parameters. The semi-quantification of TAp73a protein revealed lower and higher TAp73a protein contents in 11/23 and 12/23 of tumor samples, respectively, when compared with the median value of TAp73a protein in normal colon tissue (p = 0.61). The level of TAp73a protein level was 5 times lower in poorly differentiated cancer cells (G3) in comparison to moderately differentiated ones (G2; p = 0.02). No statistically significant correlations were observed between the level of the TAp73a protein and clinical-pathological patients’ characteristics. The IHC analysis of TAp73a protein presence in CRC samples showed decreased immunoreactivity when compared with matched sections of the unchanged colon wall in 4/9 patients, similar intensity of the IHC reaction in 4/9 patients and increased immunoreactivity in 1/9 patients. The TAp73a protein was localized mainly in the cytoplasm of the cancer cells. No statistically significant correlations between IHC results and clinical-pathological features of the patients were found. Conclusions. The obtained results suggest that the p73 gene may play a role as a tumor suppressor in the CRC progression

Underexpression of LATS1 TSG in colorectal cancer is associated with promoter hypermethylation

AIM: To investigate large tumor suppressor 1 (LATS1) expression, promoter hypermethylation, and microsatellite instability in colorectal cancer (CRC). METHODS: RNA was isolated from tumor tissue of 142 CRC patients and 40 colon mucosal biopsies of healthy controls. After reverse transcription, quantitative polymerase chain reaction (PCR) was performed, and LATS1 expression was normalized to expression of the ACTB and RPL32 housekeeping genes. To analyze hypermethylation, genomic DNA was isolated from 44 tumor CRC biopsies, and methylation-specific PCR was performed. Microsatellite instability (MSI) status was checked with PCR using BAT26, BAT25, and BAT40 markers in the genomic DNA of 84 CRC patients, followed by denaturing gel electrophoresis. RESULTS: Decreased LATS1 expression was found in 127/142 (89.4%) CRC cases with the average ratio of the LATS1 level 10.33 ± 32.64 in CRC patients vs 32.85 ± 33.56 in healthy controls. The lowest expression was found in Dukes’ B stage tumors and G1 (well-differentiated) cells. Hypermethylation of the LATS1 promoter was present in 25/44 (57%) CRC cases analyzed. LATS1 promoter hypermethylation was strongly associated with decreased gene expression; methylated cases showed 162× lower expression of LATS1 than unmethylated cases. Although high-grade MSI (mutation in all three markers) was found in 14/84 (17%) cases and low-grade MSI (mutation in 1-2 markers) was found in 30/84 (36%) cases, we found no association with LATS1 expression. CONCLUSION: Decreased expression of LATS1 in CRC was associated with promoter hypermethylation, but not MSI status. Such reduced expression may promote progression of CRC