Mutations in a novel gene lead to kidney tumors, lung wall defects, and benign tumors of the hair follicle in patients with the Birt-Hogg-Dubé syndrome.

Birt-Hogg-Dubé (BHD) syndrome is a rare inherited genodermatosis characterized by hair follicle hamartomas, kidney tumors, and spontaneous pneumothorax. Recombination mapping in BHD families delineated the susceptibility locus to 700 kb on chromosome 17p11.2. Protein-truncating mutations were identified in a novel candidate gene in a panel of BHD families, with a 44% frequency of insertion/deletion mutations within a hypermutable C(8) tract. Tissue expression of the 3.8 kb transcript was widespread, including kidney, lung, and skin. The full-length BHD sequence predicted a novel protein, folliculin, that was highly conserved across species. Discovery of disease-causing mutations in BHD, a novel kidney cancer gene associated with renal oncocytoma or chromophobe renal cancer, will contribute to understanding the role of folliculin in pathways common to skin, lung, and kidney development.

Regression of human kidney cancer following allogeneic stem cell transplantation is associated with recognition of an HERV-E antigen by T cells.

Transplanted donor lymphocytes infused during hematopoietic stem cell transplantation (HSCT) have been shown to cure patients with hematological malignancies. However, less is known about the effects of HSCT on metastatic solid tumors. Thus, a better understanding of the immune cells and their target antigens that mediate tumor regression is urgently needed to develop more effective HSCT approaches for solid tumors. Here we report regression of metastatic renal cell carcinoma (RCC) in patients following nonmyeloablative HSCT consistent with a graft-versus-tumor effect. We detected RCC-reactive donor-derived CD8(+) T cells in the blood of patients following nonmyeloablative HSCT. Using cDNA expression cloning, we identified a 10-mer peptide (CT-RCC-1) as a target antigen of RCC-specific CD8(+) T cells. The genes encoding this antigen were found to be derived from human endogenous retrovirus (HERV) type E and were expressed in RCC cell lines and fresh RCC tissue but not in normal kidney or other tissues. We believe this to be the first solid tumor antigen identified using allogeneic T cells from a patient undergoing HSCT. These data suggest that HERV-E is activated in RCC and that it encodes an overexpressed immunogenic antigen, therefore providing a potential target for cellular immunity.

Simultaneous down-regulation of tumor suppressor genes RBSP3/CTDSPL, NPRL2/G21 and RASSF1A in primary non-small cell lung cancer.

BACKGROUND: The short arm of human chromosome 3 is involved in the development of many cancers including lung cancer. Three bona fide lung cancer tumor suppressor genes namely RBSP3 (AP20 region),NPRL2 and RASSF1A (LUCA region) were identified in the 3p21.3 region. We have shown previously that homozygous deletions in AP20 and LUCA sub-regions often occurred in the same tumor (P < 10-6). METHODS: We estimated the quantity of RBSP3, NPRL2, RASSF1A, GAPDH, RPN1 mRNA and RBSP3 DNA copy number in 59 primary non-small cell lung cancers, including 41 squamous cell and 18 adenocarcinomas by real-time reverse transcription-polymerase chain reaction based on TaqMan technology and relative quantification. RESULTS: We evaluated the relationship between mRNA level and clinicopathologic characteristics in non-small cell lung cancer. A significant expression decrease (> or =2) was found for all three genes early in tumor development: in 85% of cases for RBSP3; 73% for NPRL2 and 67% for RASSF1A (P < 0.001), more strongly pronounced in squamous cell than in adenocarcinomas. Strong suppression of both, NPRL2 and RBSP3 was seen in 100% of cases already at Stage I of squamous cell carcinomas. Deregulation of RASSF1A correlated with tumor progression of squamous cell (P = 0.196) and adenocarcinomas (P < 0.05). Most likely, genetic and epigenetic mechanisms might be responsible for transcriptional inactivation of RBSP3 in non-small cell lung cancers as promoter methylation of RBSP3 according to NotI microarrays data was detected in 80% of squamous cell and in 38% of adenocarcinomas. With NotI microarrays we tested how often LUCA (NPRL2, RASSF1A) and AP20 (RBSP3) regions were deleted or methylated in the same tumor sample and found that this occured in 39% of all studied samples (P < 0.05). CONCLUSION: Our data support the hypothesis that these TSG are involved in tumorigenesis of NSCLC. Both genetic and epigenetic mechanisms contribute to down-regulation of these three genes representing two tumor suppressor clusters in 3p21.3. Most importantly expression of RBSP3, NPRL2 and RASSF1A was simultaneously decreased in the same sample of primary NSCLC: in 39% of cases all these three genes showed reduced expression (P < 0.05).

Expression of transmembrane carbonic anhydrases, CAIX and CAXII, in human development.

BACKGROUND: Transmembrane CAIX and CAXII are members of the alpha carbonic anhydrase (CA) family. They play a crucial role in differentiation, proliferation, and pH regulation. Expression of CAIX and CAXII proteins in tumor tissues is primarily induced by hypoxia and this is particularly true for CAIX, which is regulated by the transcription factor, hypoxia inducible factor-1 (HIF-1). Their distributions in normal adult human tissues are restricted to highly specialized cells that are not always hypoxic. The human fetus exists in a relatively hypoxic environment. We examined expression of CAIX, CAXII and HIF-1alpha in the developing human fetus and postnatal tissues to determine whether expression of CAIX and CAXII is exclusively regulated by HIF-1. RESULTS: The co-localization of CAIX and HIF-1alpha was limited to certain cell types in embryonic and early fetal tissues. Those cells comprised the primitive mesenchyma or involved chondrogenesis and skin development. Transient CAIX expression was limited to immature tissues of mesodermal origin and the skin and ependymal cells. The only tissues that persistently expressed CAIX protein were coelomic epithelium (mesothelium) and its remnants, the epithelium of the stomach and biliary tree, glands and crypt cells of duodenum and small intestine, and the cells located at those sites previously identified as harboring adult stem cells in, for example, the skin and large intestine. In many instances co-localization of CAIX and HIF-1alpha was not evident. CAXII expression is restricted to cells involved in secretion and water absorption such as parietal cells of the stomach, acinar cells of the salivary glands and pancreas, epithelium of the large intestine, and renal tubules. Co-localization of CAXII with CAIX or HIF-1alpha was not observed. CONCLUSION: The study has showed that: 1) HIF-1alpha and CAIX expression co- localized in many, but not all, of the embryonic and early fetal tissues; 2) There is no evidence of co-localization of CAIX and CAXII; 3) CAIX and CAXII expression is closely related to cell origin and secretory activity involving proton transport, respectively. The intriguing finding of rare CAIX-expressing cells in those sites corresponding to stem cell niches requires further investigation.

Two novel VHL targets, TGFBI (BIGH3) and its transactivator KLF10, are up-regulated in renal clear cell carcinoma and other tumors.

Mutations in the VHL gene are associated with highly vascular tumors of kidney, brain, retina, and adrenal gland. The inability of the mutant VHL protein to destabilize HIF-1 plays a crucial role in malignant angiogenesis. VHL is also associated with ECM assembly but the molecular mechanisms of this activity remain unclear. We used expression arrays and cell lines with different VHL status to identify ECM-associated genes controlled by VHL. One of them, adhesion-associated TGFBI, was repressed by VHL and overexpressed in renal, gastrointestinal, brain, and other tumors. Analyzing the mechanism of TGFBI up-regulation in clear cell carcinoma, we identified a novel VHL target, a Kruppel-like transcriptional factor 10 (KLF10). The TGFBI promoter, which we isolated and studied in Luc-reporter assay, was induced by KLF10 but not hypoxia. These data provide the molecular basis for the observed VHL effect on TGFBI and stimulate further research into the KLF10 and TGFBI roles in cancer.

Reduced expression of RASSF1A in esophageal and nasopharyngeal carcinomas significantly correlates with tumor stage.

Reduced expression or loss of tumor suppressor genes play a key role in many cancers. In this study, we investigated the role of RASSF1A in the pathogenesis of esophageal squamous cell carcinoma (ESCC) and nasopharyngeal carcinoma (NPC). We detected the down-regulated expression of both RASSF1A transcripts and protein in tumor tissues using RT-PCR and tissue microarray immunohistochemical staining analyses. Down-regulated expression of RASSF1A showed a significant association with WHO grade, tumor status, and lymph node metastasis, showing its possible utility as a biomarker for clinical specimens.

DNA damage is a prerequisite for p53-mediated proteasomal degradation of HIF-1alpha in hypoxic cells and downregulation of the hypoxia marker carbonic anhydrase IX.

We investigated the relationship between the tumor suppressor p53 and the hypoxia-inducible factor-1 (HIF-1)-dependent expression of the hypoxia marker, carbonic anhydrase IX (CAIX). MCF-7 (wt p53) and Saos-2 (p53-null) cells displayed similar induction of CAIX expression and CA9 promoter activity under hypoxic conditions. Activation of p53 by the DNA damaging agent mitomycin C (MC) was accompanied by a potent repression of CAIX expression and the CA9 promoter in MCF-7 but not in Saos-2 cells. The activated p53 mediated increased proteasomal degradation of HIF-1alpha protein, resulting in considerably lower steady-state levels of HIF-1alpha protein in hypoxic MCF-7 cells but not in Saos-2 cells. Overexpression of HIF-1alpha relieved the MC-induced repression in MCF-7 cells, confirming regulation at the HIF-1alpha level. Similarly, CA9 promoter activity was downregulated by MC in HCT 116 p53(+/+) but not the isogenic p53(-/-) cells. Activated p53 decreased HIF-1alpha protein levels by accelerated proteasome-dependent degradation without affecting significantly HIF-1alpha transcription. In summary, our results demonstrate that the presence of wtp53 under hypoxic conditions has an insignificant effect on the stabilization of HIF-1alpha protein and HIF-1-dependent expression of CAIX. However, upon activation by DNA damage, wt p53 mediates an accelerated degradation of HIF-1alpha protein, resulting in reduced activation of CA9 transcription and, correspondingly, decreased levels of CAIX protein. A model outlining the quantitative relationship between p53, HIF-1alpha, and CAIX is presented.

Transcriptional regulator CTCF controls human interleukin 1 receptor-associated kinase 2 promoter.

Immune responses to invading pathogens are mediated largely through a family of transmembrane Toll-like receptors and modulated by a number of downstream effectors. In particular, a family of four interleukin 1 receptor-associated kinases (IRAK) regulates responsiveness to bacterial endotoxins. Pharmacological targeting of particular IRAK components may be beneficial for treatment of bacterial infections. Here, we studied transcriptional regulation of the human IRAK2 gene. Analysis of the IRAK2 promoter region reveals putative binding sites for several transcriptional factors, including ZIP (EGR1 and SP1), CTCF and AP-2beta. Deletion of the ZIP or AP-2 sites did not significantly affect IRAK2 promoter activity in naive and endotoxin-treated mononuclear cells, in dormant and activated Jurkat T-cells, in lung and kidney cells. In contrast, we found that CTCF plays a major role in IRAK2 transcription. An electrophoretic mobility shift assay of the DNA fragments containing the IRAK2 CpG island, revealed a single high-affinity binding site for the transcriptional regulator and a chromatin insulator protein, CTCF. This assay revealed a CTCF-binding site within the mouse Irak2 promoter. The presence of the CTCF protein in human IRAK2 promoter was confirmed by chromatin immunoprecipitation assay. Specific residues that interacted with the CTCF protein, were identified by methylation interference assay. In all cell lines analyzed, including cells of lung, renal, monocytic and T-cell origin, the IRAK2 luciferase reporter construct, containing an intact CTCF-binding site, showed strong promoter activity. However, IRAK2 promoter activity was decreased dramatically for the constructs with a mutated CTCF-binding site.

Expression of candidate chromosome 3p21.3 tumor suppressor genes and down-regulation of BLU in some esophageal squamous cell carcinomas.

The expression of six chromosome 3p21.3 candidate tumor suppressor genes (BLU, FUS2, HYAL2, NPRL2, RASSF1A, and SEMA3B) in esophageal squamous cell carcinoma (ESCC) has been investigated. Reduced expression of BLU was detected in some ESCC cell lines and tumor tissues and the difference was quantitated by real-time quantitative polymerase chain reaction. Methylation specific-PCR revealed the down-regulation of BLU by epigenetic inactivation. However, exogenous expression of BLU did not functionally suppress tumorigenicity in nude mice. These results suggest that over-expression of BLU alone is not sufficient to inhibit tumorigenicity. Further studies on BLU interacting proteins are required to elucidate the possible role of BLU in the development of ESCC.

An integrated physical and gene map of the 3.5-Mb chromosome 3p21.3 (AP20) region implicated in major human epithelial malignancies.

To facilitate the identification of tumor suppressor genes in the chromosome 3p21.3-p22 AP20 subregion, we constructed a 3.5-Mb physical and gene map of this segment (between markers D3S4285 and D3S3873) that spans the distance from 124.4cR3000 to 133.5 cR3000 of the GB4 genetic map. We used NotI-linking and -jumping clones, sequence-tagged site PCR marker analysis, and multicolor and fiber fluorescence in situ hybridization to confirm the sequence order and map orientation. An integrated clone contig composed of 5 yeast artificial chromosome, 15 bacterial artificial chromosome, 5 P1 artificial chromosome, and 8 NotI-linking clones provided the physical base of the map. We unequivocally established the order of 28 sequence-tagged sites and 35 genes in the region. Gaps between published bacterial artificial chromosome contigs were determined and covered by our own sequence data. Furthermore, three new genes were isolated, namely the human homologue to the rat Golgi peripheral membrane protein p65, GOLPH5 (GORASP1), the gene for stress-inducible protein, STI2, and the AP20-region gene 1, APRG1. The tumor suppressor gene candidate APRG1 was positioned close to the border of the homozygous deletion in a small cell lung cancer cell line ACC-LC5. Expression analysis with a tissue-specific panel of cDNA revealed seven distinct tissue-specific splice variants (A-G) of the message (size range, 1.0-1.8 kb). Although the gene was expressed at a low level in all tested tissues, comparatively higher expression was detected in pancreas (splice forms B and D), kidney (A) and placenta (B and C). The APRG1 gene encoded a predicted protein of 170 amino acids (isoform B), which had an NH2-terminal part conserved among members of the eukaryotic translation factor 6 gene family. A Prosite pattern corresponding to the cell attachment sequence Arg-Gly-Asp was also found. The presence of this domain raised the intriguing possibility that APRG1B may be directly involved in membrane interactions and cell adhesion. We showed that the AP20 region was duplicated during mammalian evolution and homologous gene clusters were present in human chromosome 2 and syntenic mouse regions on chromosomes 1, 2, and 9. Interestingly, the HYA22 gene (human ortholog of the yeast YA22 gene) was located at the borders of both breakpoints, evolutionarily conserved gene cluster and homozygous deletions detected in lung, kidney and other cancers. NotI digestion revealed that the AP20 region was frequently and extensively methylated in renal carcinoma cell lines and tumor biopsies.

Inactivation of RAS association domain family 1A gene in cervical carcinomas and the role of human papillomavirus infection.

Recently, we have identified a new putative tumor suppressor gene, RASSF1A (Ras association domain family 1A gene), located at human chromosome 3p21.3, the segment that is often lost in many types of human cancers. The RASSF1A promoter was shown to be frequently hypermethylated in various epithelial tumors, including small cell lung, breast, bladder, prostate, gastric, and renal cell carcinomas. In this study, we have analyzed the methylation status of the RASSF1A gene in primary human cervical cancers and in eight cervical cancer cell lines. The RASSF1A promoter is hypermethylated in 4 of 42 (= 10%) of squamous cell carcinomas, in 4 of 19 (= 21%) of adenosquamous carcinomas, and in 8 of 34 (= 24%) of cervical adenocarcinomas. Although in adenocarcinomas, methylation of RASSF1A and presence of human papillomavirus (HPV) type 16 or 18 sometimes coexisted, not a single case of HPV-16/18-positive squamous cell carcinomas had RASSF1A methylation. Similarly, in all eight analyzed cervical cell lines, RASSF1A inactivation and HPV infection were mutually exclusive (Fisher's exact test; P = 0.0357): two HPV-negative cervical cancer cell lines had a methylated and silenced RASSF1A promoter (C-33A and HT-3), whereas the other six HPV-positive lines expressed RASSF1A mRNA (ME 180, MS751, SiHa, C-4I, HeLa, and CaSki). For cervical tumors and cell lines combined, the Pearson's chi(2) test (chi(2) = 3.99; P

Lowered oxygen tension induces expression of the hypoxia marker MN/carbonic anhydrase IX in the absence of hypoxia-inducible factor 1 alpha stabilization: a role for phosphatidylinositol 3'-kinase.

Transcription of the gene coding for the tumor-associated antigen MN/carbonic anhydrase IX (CAIX) is regulated by hypoxia-inducible factor 1 (HIF-1). Previous studies identified CAIX expression in areas adjacent to hypoxic regions in solid tumors and suggested supplementary/alternative modes of regulation. To better understand the mechanisms activating CAIX expression, we characterized the cell density-dependent induction of CAIX in HeLa cells. This process is anchorage and serum independent and is not mediated by a soluble factor, decreased pH, or lowered glucose concentration. Stabilization of HIF-1 alpha was not observed in dense cultures. In contrast to sparse cell culture conditions, phosphatidylinositol 3'-kinase (PI3K) activity was significantly increased in dense HeLa cultures. The PI3K inhibitors LY294002 and wortmannin inhibited CAIX expression in dense cultures in a dose-dependent manner, specifically targeting the CA9 promoter (-173/+31 region) that was transactivated by constitutively active p110 PI3K subunit. The mechanism controlling CAIX expression in dense cultures is, however, dependent on lowered O(2) tension because stirring abrogates induction of CAIX expression. Hypoxia- and cell density-induced CAIX expressions were mediated by two seemingly independent mechanisms, as documented by the additive effect of increased cell density and treatment with the hypoxia-mimic CoCl(2) on levels of CAIX expression. The minimal cell density-dependent region within the CA9 promoter consists of the juxtaposed protected region 1 and hypoxia-response elements. However cell density-dependent CAIX expression was abrogated in the HIF-1 alpha-deficient Kal3.5 cells, suggesting an important role of HIF-1 in the corresponding mechanism. Thus, induction of CAIX in high-density cultures requires separate but interdependent pathways of PI3K activation and a minimal level of HIF-1 alpha. These interdependent pathways function at a lowered O(2) concentration that is, however, above that necessary for HIF-1 alpha stabilization.

Functional characterization of the candidate tumor suppressor gene NPRL2/G21 located in 3p21.3C.

Initial analysis identified the NPRL2/G21 gene located in 3p21.3C, the lung cancer region, as a strong candidate tumor suppressor gene. Here we provide additional evidence of the tumor suppressor function of NPRL2/G21. The gene has highly conserved homologs/orthologs ranging from yeast to humans. The yeast ortholog, NPR2, shows three highly conserved regions with 32 to 36% identity over the whole length. By sequence analysis, the main product of NPRL2/G21 encodes a soluble protein that has a bipartite nuclear localization signal, a protein-binding domain, similarity to the MutS core domain, and a newly identified nitrogen permease regulator 2 domain with unknown function. The gene is highly expressed in many tissues. We report inactivating mutations in a variety of tumors and cancer cell lines, growth suppression of tumor cells with tet-controlled NPRL2/G21 transgenes on plastic Petri dishes, and suppression of tumor formation in SCID mice. Screening of 7 renal, 5 lung, and 7 cervical carcinoma cell lines showed homozygous deletions in the 3' end of NPRL2 in 2 renal, 3 lung, and 1 cervical (HeLa) cell line. Deletions in the 3' part of NPRL2 could result in improper splicing, leading to the loss of the 1.8 kb functional NPRL2 mRNA. We speculate that the NPRL2/G21 nuclear protein may be involved in mismatch repair, cell cycle checkpoint signaling, and activation of apoptotic pathway(s). The yeast NPR2 was shown to be a target of cisplatin, suggesting that the human NPRL2/G21 may play a similar role. At least two homozygous deletions of NPRL2/G21 were detected in 6 tumor biopsies from various locations and with microsatellite instability. This study, together with previously obtained results, indicates that NPRL2 is a multiple tumor suppressor gene.

Expression of several genes in the human chromosome 3p21.3 homozygous deletion region by an adenovirus vector results in tumor suppressor activities in vitro and in vivo.

A group of candidate tumor suppressor genes (designated CACNA2D2, PL6, 101F6, NPRL2, BLU, RASSF1, FUS1, HYAL2, and HYAL1) has been identified in a 120-kb critical tumor homozygous deletion region (found in lung and breast cancers) of human chromosome 3p21.3. We studied the effects of six of these 3p21.3 genes (101F6, NPRL2, BLU, FUS1, HYAL2, and HYAL1) on tumor cell proliferation and apoptosis in human lung cancer cells by recombinant adenovirus-mediated gene transfer in vitro and in vivo. We found that forced expression of wild-type FUS1, 101F6, and NPRL2 genes significantly inhibited tumor cell growth by induction of apoptosis and alteration of cell cycle processes in 3p21.3 120-kb region-deficient (homozygous) H1299 and A549 cells but not in the 3p21.3 120-kb region-heterozygous H358 and the normal human bronchial epithelial cells. Intratumoral injection of Ad-101F6, Ad-FUS1, Ad-NPRL2, and Ad-HYAL2 vectors or systemic administration of protamine-complexed vectors significantly suppressed growth of H1299 and A549 tumor xenografts and inhibited A549 experimental lung metastases in nu/nu mice. Together, our results, coupled with other studies demonstrating a tumor suppressor role for the RASSSF1A isoform, suggest that multiple contiguous genes in the 3p21.3 120-kb chromosomal region may exhibit tumor suppressor activity in vitro and in vivo.

The RASSF1A tumor suppressor gene is inactivated in prostate tumors and suppresses growth of prostate carcinoma cells.

We analyzed expression status of the recently identified tumor suppressor geneRASSF1A in primary prostate carcinomas and in prostate cell lines. We found complete methylation of the RASSF1A promoter in 63% of primary microdissected prostate carcinomas (7 of 11 samples). The remaining 4 samples (37%) were partially methylated, possibly because of contamination with normal cells. No promoter methylation was observed in matching normal prostate tissues. High levels of RASSF1A transcript and no methylation of RASSF1A promoter were found in explanted primary normal prostate epithelial and stromal cells. Complete silencing and methylation of RASSF1A promoter was observed in five widely used prostate carcinoma cell lines, which acquired the ability to grow in culture spontaneously, including LNCaP, PC-3, ND-1, DU-145, 22Rv1, and one primary prostate carcinoma immortalized by overexpression of the human telomerase catalytic subunit (RC-58T/hTERT). However, no silencing of RASSF1A was found in four other prostate carcinoma cell lines, which were adapted for cell culture after transformation with human papillomaviral DNA. Suppression of cell growth in vitro was demonstrated after the reintroduction of RASSF1A-expressing construct into LNCaP prostate carcinoma cells. Our data implicate the RASSF1A gene in human prostate tumorigenesis.

Detection of an Immunogenic HERV-E Envelope with Selective Expression in Clear Cell Kidney Cancer.

VHL-deficient clear cell renal cell carcinomas (ccRCC), the most common form of kidney cancer, express transcripts derived from the novel human endogenous retrovirus HERV-E (named CT-RCC HERV-E). In this study, we define a transcript encoding the entire envelope gene of HERV-E as expressed selectively in ccRCC tumors, as distinct from normal kidney tissues or other tumor types. Sequence analysis of this envelope transcript revealed long open reading frames encoding putative surface and transmembrane envelope proteins. Retroviral envelopes are known to be capable of eliciting immunity in humans. Accordingly, we found that HLA-A*0201-restricted peptides predicted to be products of the CT-RCC HERV-E envelope transcript-stimulated CD8(+) T cells, which could recognize HLA-A*0201-positive HERV-E-expressing kidney tumor cells. Overall, our results offer evidence of unique HERV-E envelope peptides presented on the surface of ccRCC cells, offering potentially useful tumor-restricted targets for T-cell-based immunotherapy of kidney cancer. Cancer Res; 76(8); 2177-85. ©2016 AACR.

Tumor suppressor genes on chromosome 3p involved in the pathogenesis of lung and other cancers.

Loss of heterozygosity (LOH) involving several chromosome 3p regions accompanied by chromosome 3p deletions are detected in almost 100% of small (SCLCs) and more than 90% of non-small (NSCLCs) cell lung cancers. In addition, these changes appear early in the pathogenesis of lung cancer and are found as clonal lesions in the smoking damaged respiratory epithelium including histologically normal epithelium as well as in epithelium showing histologic changes of preneoplasia. These 3p genetic alterations lead to the conclusion that the short arm of human chromosome 3 contains several tumor suppressor gene(s) (TSG(s)). Although the first data suggesting that 3p alterations were involved in lung carcinogenesis were published more than 10 years ago, only recently has significant progress been achieved in identifying the candidate TSGs and beginning to demonstrate their functional role in tumor pathogenesis. Some of the striking results of these findings has been the discovery of multiple 3p TSGs and the importance of tumor acquired promoter DNA methylation as an epigenetic mechanism for inactivating the expression of these genes in lung cancer. This progress, combined with the well known role of smoking as an environmental causative risk factor in lung cancer pathogenesis, is leading to the development of new diagnostic and therapeutic strategies which can be translated into the clinic to combat and prevent the lung cancer epidemic. It is clear now that genetic and epigenetic abnormalities of several genes residing in chromosome region 3p are important for the development of lung cancers but it is still obscure how many of them exist and which of the numerous candidate TSGs are the key players in lung cancer pathogenesis. We review herein our current knowledge and describe the most credible candidate genes.

Inactivation of RASSF1C during in vivo tumor growth identifies it as a tumor suppressor gene.

RASSF1A, a major member of the RASSF1 gene family, is silenced by promoter methylation at a high frequency in a large number of human solid tumors. Controlled expression of RASSF1A reverts the tumorigenic phenotype of several human cancer cell lines. Here we investigated another main isoform, RASSF1C, and compared it with RASSF1A in the gene inactivation test (GIT), based on a tetracycline regulation system. In the small-cell lung cancer (SCLC) line U2020, only RASSF1A has shown growth inhibitory activity in vitro, while in the prostate cell line LNCaP and renal cell carcinoma (RCC) line KRC/Y both RASSF1A and RASSF1C showed similar (approximately 90%) suppressing activity in vitro. Both RASSF1C and RASSF1A suppressed the tumorigenicity of the KRC/Y RCC cell line in SCID mice. Mutations, deletions and loss of expression of RASSF1A and RASSF1C transgenes were identified in all 15 grown SCID tumors. In contrast, the mutant RASSF1A containing Cys65Arg and Val211Ala had reduced growth suppression activity both in vitro and in vivo and did not show any further changes in four grown SCID tumors. In addition, RASSF1C was shown to induce cell cycle arrest in KRC/Y cells. These results strongly imply that like RASSF1A the RASSF1C gene could serve a tumor suppressor function.

Deletion mapping using quantitative real-time PCR identifies two distinct 3p21.3 regions affected in most cervical carcinomas.

We report chromosome 3p deletion mapping of 32 cervical carcinoma (CC) biopsies using 26 microsatellite markers located in frequently deleted 3p regions to detect loss of heterozygosity and homozygous loss. In addition, two STS markers (NLJ-003 and NL3-001) located in the 3p21.3 telomeric (3p21.3T) and 3p21.3 centromeric (3p21.3C) regions, respectively, were used for quantitative real-time PCR as TaqMan probes. We show that quantitative real-time PCR is reliable and sensitive and allows discriminating between 0, 1 and 2 marker copies per human genome. For the first time, frequent (five of 32 cases, i.e. 15.6%) homozygous deletions were demonstrated in CCs in both 3p21.3T and 3p21.3C regions. The smallest region homozygously deleted in 3p21.3C was located between D3S1568 (CACNA2D2 gene) and D3S4604 (SEMA3F gene) and contains 17 genes previously defined as lung cancer candidate Tumor suppressor genes (TSG(s)). The smallest region homozygously deleted in 3p21.3T was flanked by D3S1298 and NL1-024 (D3S4285), excluding DLEC1 and MYD88 as candidate TSGs involved in cervical carcinogenesis. Overall, this region contains five potential candidates, namely GOLGA4, APRG1, ITGA9, HYA22 and VILL, which need to be analysed. The data showed that aberrations of either NLJ-003 or NL3-001 were detected in 29 cases (90.6%) and most likely have a synergistic effect (P<0.01). The study also demonstrated that aberrations in 3p21.3 were complex and in addition to deletions, may involve gene amplification as well. The results strongly suggest that 3p21.3T and 3p21.3C regions harbor genes involved in the origin and/or development of CCs and imply that those genes might be multiple TSG(s).

Discovery of frequent homozygous deletions in chromosome 3p21.3 LUCA and AP20 regions in renal, lung and breast carcinomas.

We searched for chromosome 3p homo- and hemizygous losses in 23 lung cancer cell lines, 53 renal cell and 22 breast carcinoma biopsies using 31 microsatellite markers located in frequently deleted 3p regions. In addition, two sequence-tagged site markers (NLJ-003 and NL3-001) located in the Alu-PCR clone 20 region (AP20) and lung cancer (LUCA) regions, respectively, were used for quantitative real-time PCR (QPCR). We found frequent (10-18%) homozygous deletions (HDs) in both 3p21.3 regions in the biopsies and lung cancer cell lines. In addition, we discovered that amplification of 3p is a very common (15-42.5%) event in these cancers and probably in other epithelial malignancies. QPCR showed that aberrations of either NLJ-003 or NL3-001 were detected in more than 90% of all studied cases. HDs were frequently detected simultaneously both in NLJ-003 or NL3-001 loci in the same tumour (P<3-10(-7)). This observation suggests that tumour suppressor genes (TSG) in these regions could have a synergistic effect. The exceptionally high frequency of chromosome aberrations in NLJ-003 and NL3-001 loci suggests that multiple TSG(s) involved in different malignancies are located very near to these markers. Precise mapping of 15 independent HDs in the LUCA region allowed us to establish the smallest HD region in 3p21.3C located between D3S1568 (CACNA2D2 gene) and D3S4604 (SEMA3F gene). This region contains 17 genes. Mapping of 19 HDs in the AP20 region resulted in the localization of the minimal region to the interval flanked by D3S1298 and D3S3623 markers. Only four genes were discovered in this interval, namely, APRG1, ITGA9, HYA22 and VILL.