No abstract

The RAF/MEK/ERK (MAPK) signal transduction cascade is an important mediator of a number of cellular fates including growth, proliferation and survival. The BRAF gene, one of the human isoforms of RAF, is activated by oncogenic RAS, leading to cooperative effects in cells responding to growth factor signals. This study was performed to elucidate a possible function of BRAF in squamous cell carcinoma of the head and neck (HNSCC). Mutations of BRAF and KRAS2 were evaluated in 89 HNSCC and corresponding normal mucosa by direct DNA sequencing analyses after microdissection. The results obtained were correlated with histopathological variables. Activating BRAF missense mutations were identified in 3/89 HNSCC (3%). KRAS2 mutations were found in five out of 89 (6%) HNSCC examined. There were no mutations of KRAS2 and BRAF in non-neoplastic mucosa. We failed to observe a correlation between BRAF or KRAS2 mutations and histopathological factors. Our data indicate that BRAF gene mutations are relatively rare events in HNSCC. Although uncommon, BRAF mutations may identify a subset of patients with HNSCC sensitive to targeted therapy.

Previously, the RASSF1A, BLU and SEMAPHORIN 3B (SEMA3B) candidate tumor suppressor genes on chromosome 3p21.3 were found to be inactivated and downregulated by genetic and epigenetic changes in lung cancer. We analyzed the methylation status of RASSF1A, BLU and SEMA3B in 35 hepatocellular carcinomas (HCCs) and 15 cholangiocarcinomas (CCs) by methylationspecific PCR and loss of heterozygosity (LOH) at 3p21.3 after microdissection. The presence of mRNA transcripts was confirmed by semiquantitative PCR. SEMA3B hypermethylation was found in 29/35 HCCs (83%) and in all (15/15) patients with CC. BLU promoter hypermethylation was detected in 7/35 (20%) HCCs and 3/15 (20%) CCs. In 2 corresponding specimens of hepatitis B virus-related liver cirrhosis, BLU methylation was also observed, but not in uninvolved normal liver tissue. RASSF1A was methylated in 21/35 HCCs (60%) and in 10/15 CCs (67%). LOH at 3p21.3 occurred in 8/35 (23%) HCCs and 3/15 (20%) CCs. The presence of hypermethylation was statistically associated with LOH of SEMA3B and correlated with downregulation of mRNA transcripts. SEMA3B transcripts increased upon treatment of HCC cell lines with the demethylation compound 5-aza-2-deoxycytidine. In conclusion, our data indicate that 2-hit gene silencing of SEMA3B through epigenetic changes and allele loss is a common and important event in the carcinogenesis of malignant liver tumors. ' 2005 Wiley-Liss, Inc.Key words: hepatocellular carcinoma; cholangiocarcinoma; methylation; loss of heterozygosity; BLU; SEMA3B; RASSF1A Primary liver cancer is one of the most frequent carcinomas worldwide. Besides hepatocellular carcinoma (HCC), accounting for 80-90% of all primary liver cancers, cholangiocarcinoma (CC) is the second most common primary hepatic malignancy. The short arm of chromosome 3 has been shown to exhibit loss of heterozygosity (LOH) in several types of cancer, including ovarian, kidney, lung, nasopharyngeal and also liver cancer. 1,2 In particular, overlapping homozygous deletions in lung cancers have been identified in region 3p21.3. 3,4 In HCC, LOH of chromosome 3p occurred in about 30% of the patients. 5,6 Several genes located on chromosome 3p have been studied in HCC as well as CC and include RASSF1A on 3p21.31, FHIT at 3p14.2, RIZ1, VHL at 3p25. 7-10 These results directed an intensive search for possible tumor suppressor genes located in the 3p21 region for one or more genes that could function as gatekeepers in molecular pathogenesis of human cancers. A group of candidate tumor suppressor genes (designated BLU, SEMAPHORIN 3B, or RASSF1A) has recently been mapped to this critical gene-rich region. 2,[11][12][13] SEMAPHORIN 3B (SEMA3B) is a member of the semaphorin family, which is also located on 3p21.3. Semaphorins are a family of signaling molecules initially identified to play a role in axonal guidance and can be classified as either membrane-bound (classes 1, 4, 5 and 6) or secreted (classes 2 and 3). 14 The receptors for the class 3 semaphorins are also known as neuropilin receptors. The intr...

The BRAF gene, one of the human isoforms of RAF, is activated by ras, leading to cooperative effects in cells responsive to growth factor signals. We studied the frequency of BRAF and k-ras-2 mutations in primary neuroendocrine gastroenteropancreatic (GEP) tumors. Mutation analysis of the BRAF and k-ras-2 genes was performed in 40 primary neuroendocrine tumors of the GEP system. The expression of extracellular signaling-related kinase (ERK) 1/2, an important downstream point of convergence in the ras-RAF-mitogen-activated protein-ERK pathway was analyzed immunohistochemically. We detected one 1796 T-->A BRAF mutation that led to a substitution of valine by glutamic acid at position 599 (V599E) in 40 primary neuroendocrine GEP tumors (3%). We failed to detect specific mutation of the k-ras-2 gene. We identified constitutively activated ERK in almost all neuroendocrine tumor tissues tested irrespective of BRAF mutation or localization or functional activity. These results suggest that BRAF mutations do not have a role in tumorigenesis of neuroendocrine tumors. Nevertheless, activation of the RAF/mitogen-activated protein kinase pathway might have a causative role in the development of neuroendocrine tumors, independent of BRAF or k-ras-2 mutation.

No abstract