VHL TSG (3p25) To analyze the function(s) of the VHL gene and its carcinogenic pathway(s) we obtained the entire genomic sequence of the gene including the promoter, introns, and flanks, and constructed a set of VHL minigenes (wild type and mutant) and a complete intronless VHL gene driven by the VHL promoter. We then set out to discover target genes controlled by pVHL. The differential display technology was employed to discover these genes using the UMRC6 and 786-0 cells stably transfected with wt and mutant VHL minigenes. To date (September 2002) six down regulated genes were identified, namely, NOTCH2 and STRA13, that specify cell fate determination and may have oncogenic potential, two transmembrane type carbonic anhydrases, CA9 and CA12, and two new unknown genes. The CA9 and CA12 genes are overexpressed in many tumor types due to hypoxia causing the loss of functional pVHL. The CAIX/XII enzymes could sense the intracellular pH and control the acidity (extracellular pH) of the miliew surrounding the cancer cells and thus create a microenvironment conducive to tumor growth and spread. They also play fundamental roles in normal physiology such as production of eye humor, brain and kidney functions etc. The STRA13 a bHLH transcription factor is negatively controlled by the VHL gene and hypoxia (a condition prevalent in cancer tissues) in many human tumors. We have now shown that STRA13 is a powerful regulator of the transcriptional factor STAT3 that is activated in many cancers. Because the STAT3 protein is fundamentally involved in regulating cancer cell growth and survival it would be beneficial to disrupt its function in cancer cells to block tumor progression. Analysis of the methylation of the VHL promoter in renal carcinoma cells carrying a methylated VHL endogene by monochromosome gene transfer, cell fusion, and VHL gene transfections showed that the meth+ phenotype is dominant in the UOK 21 cells probably resulting from changes in cis-acting elements of the VHL locus.We then created a mouse transgenic model expressing the human genomic VHL locus and demonstrated that human VHL methylation pattern was reproduced during mouse development and was very similar to that of the mouse VHL gene. This model would allow studying the local methylation protection mechanisms in the VHL locus and the effect of chromosomal context on de novo methylation of various elements of the VHL locus, such as repetitive sequences and the VHL CpG promoter. The future work will be focused (i) on the role of carbonic anhydrases (CAs) in the regulation of tumor pH and its impact on cancer growth, (ii) discovery of new specific inhibitors of these enzymes to treat cancer, (iii) on development of CAIX/XII cDNA based vaccines to treat cancer, (iv) on the design of inhibitors that could disrupt the STRA13-STAT3 signals to directly target STRA13 and/or STAT3 to improve treatment of cancer; and (v) on the nature of the cis-acting elements in the VHL locus involved in de novo aberrant methylation. The 3p21.3 TSG We used overlapping and nested homozygous deletions, contig building, genomic sequencing, physical, and transcript mapping to further define a ~630-kb lung cancer homozygous deletion region harboring one or more tumor suppressor gene(s) (TSGs) on chromosome 3p21.3. This location was identified through somatic genetic mapping in cancers, cancer cell lines and pre-malignant lesions of the lung and breast including the discovery of several homozygous deletions. The combination of molecular manual methods and computational predictions permitted us to detect, isolate, characterize and annotate a set of 25 genes which likely constitute the complete set of protein-coding genes residing in this ~630-kb sequence. A subset of 19 of these genes were found within the deleted overlap region of ~370-kb. This region was further subdivided by a nesting 200-kb breast cancer homozygous deletion into two gene sets: 8 genes lying in the proximal ~120-kb segment and 11 genes lying in the distal ~250-kb segment. These 19 genes were analyzed extensively by computational methods and were tested by manual methods for loss of expression and mutations in lung cancers to identify candidate TSGs from within this group. Several genes showed loss-of-expression or reduced mRNA levels in non-small cell lung cancer (NSCLC) (CACNA2D2/ (a2d-2), SEMA3B (formerly SEMA(V),) BLU, RASSF1/A (formerly 123F2), and HYAL1) or small cell lung cancer (SCLC) (SEMA3B, BLU, RASSF1/A (formerly 123F2), and HYAL1) cell lines. We found six of the genes to have 2 or more amino acid sequence altering mutations including: BLU, NPRL2/Gene21, FUS1, HYAL1, FUS2, and SEMA3B. However, none of the 19 genes tested for mutation showed a frequent (>10%) mutation rate in lung cancer samples. This led us to exclude several of the genes in the region as classical tumor suppressors for sporadic lung cancer. On the other hand, we now also have to consider the newly recognized class of haploinsufficient TSGs, where the absence of only one wild-type allele facilitates tumorigenesis. Functional testing of the critical genes by gene transfer and gene disruption strategies is under way and will permit the identification of the putative lung cancer TSG(s), LUCA. To date (September, 2002) we identified the RASSF1/A gene as a multiple TSG involved in many tumors, including lung, breast, ,prostate, kidney, head & neck, uterine cervix and others. The HYAL2 gene was identified as a GPI-anchored receptor for the sheep lung cancer retrovirus, JSRV and a sequestration mechanism inactivating HYAL2 product was demonstrated. The Env gene of JSRV was shown to transform human bronchial epithelial cells in vitro and sequester the HYAL2 product which allow to study the signal transduction pathways leading to carcinogenesis in this system. We have now identified the oncogenic pathway negatively controlled by the HYAL2 product. The RON receptor tyrosine kinase is activated in human lung cancer. We have now shown that in SCLC the promoter of RON is hypermethylated resulting in silencing of the gene and simultaneous activation of an internal promoter; the shorter transcript originating from this internal promoter encodes only the cytoplasmic portion of the receptor that functions as an oncogene. A new FAS2 gene cDNA polymorphism was shown to be associated with NPC with predictive value in Asian populations. Current work is focused on (I) the detection and isolation of the putative human retrovirus that may cause a rapidly rising form of human lung cancer namely bronchioloalveolar adenocarcinoma (BAC), (ii) analysis of the oncogenic pathway activated by the truncated form of RON. The 3p12 TSG Cytogenetic deletions and LOH at human 3p12 are a consistent feature of lung cancer specimens and suggest the presence of a tumor suppressor gene(s) (TSG) at this location. Only one gene (DUTT1, Deleted in U Twenty Twenty) was so far cloned from the overlapping region deleted in several lung and breast cancer cell lines (U2020, NCI H2198, HCC38). DUTT1 is the human ortholog of the fly gene ROBO that has homology to NCAM proteins. Extensive analyses of DUTT1 in lung cancer did not reveal any mutations, suggesting another gene(s) at this location could be associated with lung cancer initiation and/or development. We discovered in the overlapping critical region a new small (~230kb), nested homozygous deletion in the SCLC cell line GLC20. This deletion has been PCR-characterized using several polymorphic markers. P1 library screening produced three overlapping clones that cover the whole region and flanks. These clones were used to define by fiber-FISH the location and size of the deletion. Recently several BAC clones covering this region were sequenced by the MIT genome sequencing center providing a genomic tool to discover in silico the resident genes. Several genes represented by EST clusters were detected in the deletion and are being isolated. Subsequent mutation and functional studies will identify the potential 3p12 lung/breast cancer TSG.
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