BACKGROUND:Due to the lack of tumor suppressor function of the fragile histidine triad (FHIT) gene product, sebaceous gland carcinomas can develop. OBJECTIVE:The model of the sebocyte cell line SZ95 was used to identify methylated CpG islands at the 5'-end of the FHIT gene and the decrease of gene expression as well as the increase of double-stranded (ds) DNA breaks were examined. MATERIAL AND METHODS:Methylation, immunofluorescence analysis, promotor sequencing and treatment of SZ95 cells with 5‑azacytidine/trichostatin A (TSA). RESULTS:The cultivation was accompanied by an increasing methylation of the CpG islands, a decrease of the FHIT gene expression and an accumulation of ds-DNA breaks. Treatment with 5‑azacytidine/TSA showed a decrease in DNA methylation and a re-expression of FHIT transcripts. DISCUSSION:Epigenetic changes in the cellular genome are caused by in vitro cell culture. Consequently, a positive selection of sebocytes with an epigenetically inactivated FHIT locus occurs.

Zusammenfassung Hintergrund Fehlt die Tumorsuppressorfunktion des fragilen Histidin-Trias( FHIT )-Genprodukts, so können Talgdrüsenkarzinome entstehen. Fragestellung Am Modell der Sebozytenlinie SZ95 sollten methylierte CpG-Inseln am 5′-Ende des FHIT -Gens identifiziert, die Abnahme der Genexpression und die Zunahme von DNA-Doppelstrang(ds)-Brüchen untersucht werden. Material und Methoden Methylierungs‑, Immunfluoreszenzanalysen, Promotorsequenzierung sowie Behandlung der SZ95-Zellen mit 5‑Azacytidin/Trichostatin A (TSA). Ergebnisse Die Kultivierung der Sebozyten verlief mit einer zunehmenden Methylierung der CpGs, einer Abnahme der FHIT -Genexpression sowie Anhäufung von DNA-ds-Brüchen. Die Behandlung mit 5‑Azacytidin/TSA zeigte eine Abnahme der DNA-Methylierung sowie Re-Expression von FHIT -Transkripten. Diskussion Durch In-vitro-Zellkultur werden epigenetische Veränderungen im zellulären Genom hervorgerufen. Es folgt eine positive Selektion von Sebozyten mit epigenetischer Inaktivierung des FHIT -Genorts.

Background Cancer metastases are the main cause of lethality. The five-year survival rate for patients diagnosed with advanced stage oral cancer is 30%. Hence, the identification of novel therapeutic targets is an urgent need. However, tumors are comprised of a heterogeneous collection of cells with distinct genetic and molecular profiles that can differentially promote metastasis making therapy development a challenging task. Here, we leveraged intratumoral heterogeneity in order to identify drivers of cancer cell motility that might be druggable targets for anti-metastasis therapy. Methods We used 2D migration and 3D matrigel-based invasion assays to characterize the invasive heterogeneity among and within four human oral cancer cell lines in vitro. Subsequently, we applied mRNA-sequencing to map the transcriptomes of poorly and strongly invasive subclones as well as primary tumors and matched metastasis. Results We identified SAS cells as a highly invasive oral cancer cell line. Clonal analysis of SAS yielded a panel of 20 subclones with different invasive capacities. Integrative gene expression analysis identified the Lymphocyte cell-specific protein-tyrosine kinase (LCK) as a druggable target gene associated with cancer cell invasion and metastasis. Inhibition of LCK using A-770041 or dasatinib blocked invasion of highly aggressive SAS cells. Interestingly, reduction of LCK activity increased the formation of adherens junctions and induced cell differentiation. Conclusion Analysis of invasive heterogeneity led to the discovery of LCK as an important regulator of motility in oral cancer cells. Hence, small molecule mediated inhibition of LCK could be a promising anti-metastasis therapy option for oral cancer patients.

BACKGROUND:Sebaceous gland carcinomas represent rare malignancies of the skin and some 60% of them demonstrate high-grade microsatellite instability on the background of a defective mismatch repair system. However, a significant fraction of periocular sebaceous gland carcinomas exhibits microsatellite stability associated with a frequent loss of the candidate tumour suppressor fragile histidine triad (FHIT).OBJECTIVES:We hypothesized that in those sebaceous gland carcinomas with microsatellite stability and loss of FHIT, effector molecules participating in homologous recombination repair (HRR), such as BRCA1/2, could be somatically inactivated.METHODS:A pilot series of 10 paraffin-embedded sebaceous gland carcinoma specimens with a defined FHIT status was studied for loss of heterozygosity (LOH) events in the genes BRCA1, BRCA2, FHIT and WWOX. We sequenced the coding exons 5-8 of the p53 gene.RESULTS:Sebaceous gland carcinomas with FHIT negativity displayed LOH and biallelic deletions of the BRCA1 gene in five of 10 (50%) of the sebaceous gland carcinoma specimens analysed. Tumour-specific genomic losses close to BRCA2 were also uncovered. A homozygous p53 R248W gain-of-function mutation as the result of a CGG to TGG transition was identified in one of seven sebaceous gland carcinomas. It has been demonstrated previously that p53 R248W mutants inactivate ATM-directed HRR. This particular sebaceous gland carcinoma presented with concomitant genomic deletions at the BRCA1 and BRCA2 loci, and also at the constitutively fragile sites FRA3B/FHIT and FRA16D/WWOX.CONCLUSIONS:Our study demonstrates for the first time that microsatellite-stable FHIT-negative sebaceous gland carcinomas accumulate mutations that target central components of the HRR network. This observation will prompt investigations in synthetic lethality of BRCA-deficient sebaceous gland carcinomas by therapeutic poly(ADP-ribose) polymerase inhibitors.

Periocular sebaceous gland carcinomas (SGCs) occur in the eyelids either sporadically or as a phenotypic feature of Muir-Torre syndrome (MTS). In knockout mice mismatch-repair (MMR) defects or inactivation of the fragile histidine triad (FHIT) gene are associated with MTS-like signs, including SGC. To dissect the genetic alterations associated with microsatellite instability (MSI) and inactivation of the FHIT gene, we studied nine periocular SGC specimens from MTS patients. Immunohistochemistry was performed for FHIT, MSH2, MLH1, and MSH6. We assessed MSI as well as loss of heterozygosity (LOH) at the FHIT locus with polymorphic markers and genomic multiplex PCR. Epigenetic silencing was detected by methylation-specific PCR (MSP) and combined bisulfite restriction analysis (COBRA). Our analyses identified two SGCs with FHIT positivity and high-grade MSI, and seven cases with loss of FHIT and microsatellite stability (MSS). MSI correlated with loss of MSH2 and MLH1 immunostaining. Loss-of-function mechanisms affecting the FHIT gene were identified as intragenic deletions eliminating the coding exons 5 and 6 on one hand, and complete biallelic methylation of the FHIT transcription regulatory region on the other hand. Germinal FHIT mutations as a predisposing factor for MTS were excluded in two index patients with cancer in three generations, including an FHIT-negative SGC. Our data suggest that either somatic inactivation of the FHIT gene associated with MSS or inactivation of the MMR system resulting in MSI contribute to the development of periocular SGCs in presumptive MTS.

BACKGROUND/AIMS:Functional deletion of the transcription factor Prospero-related homeobox 1 (PROX1) causes abnormal cellular proliferation via down-regulated expression of the cell cycle inhibitors p27(kip1) and p57(kip2). Hence, we examined whether inactivation of the PROX1 gene can be demonstrated in malignant tumors of the bilary system. METHODS:Seventeen paraffin-embedded specimens of carcinomas of the bilary system were subjected to loss-of-heterozygosity (LOH) and microsatellite instability analyses, methylation-specific polymerase-chain reaction (MSP) and immunohistochemical detection of PROX1 protein in tumor sections. RESULTS:The marker D1S213 located close to PROX1 at 1q41 indicated LOH events in 50% of informative tumor samples analyzed. In contrast to intense cytoplasmic and nuclear staining of normal bile duct epithelia, PROX1 protein was absent or drastically reduced in 10 of 16 (63%) carcinomas. MSP revealed significant PROX1 promoter hypermethylation in 8 out of 17 clinical cases (47%). A correlation between clinicopathological characteristics and reduced PROX1 expression was not observed. CONCLUSIONS:We demonstrate that mechanisms like genomic deletions and hypermethylation, which are prototypic for the inactivation of tumor suppressor genes, inactivate PROX1 in carcinomas of the bilary system. Our findings prompt the elucidation of molecular pathways involved in PROX1 dependent misregulation of differentiation and proliferation processes in bilary tract carcinomas.

BACKGROUND:Extensive exposure to ultraviolet radiation is associated with genetic alterations in basal cell carcinomas (BCCs), which represent some 75% of skin cancers.OBJECTIVES:As recent data suggested the fragile histidine triad (FHIT) gene product to participate in DNA damage responses we wished to address whether functional deletion of this tumour suppressor participates in the development of BCC. Our study focused on epigenetic inactivation of the FHIT gene.METHODS:Paraffin-embedded specimens from 17 patients with BCC were available for methylation-specific polymerase chain reaction (MSP), combined bisulphite-dependent restriction analysis (COBRA) of the FHIT gene and immunohistochemistry of its product.RESULTS:We report for the first time that 100% of BCCs are negative for FHIT by immunostaining. Aberrant methylation of the FHIT promoter occurred in a significant portion of BCCs. MSP detected hypermethylation of the FHIT/FRA3B locus in nine of nine (100%) periocular BCCs and in six of eight (75%) BCCs from other body regions. COBRA yielded similar results, confirming that some 88% of the 17 BCCs analysed harbour epigenetic silencing of the FHIT gene. Loss of FHIT protein was demonstrated immunohistochemically, confirming that promoter hypermethylation correlated with loss of gene expression.CONCLUSIONS:We have identified epigenetic silencing of the FHIT tumour suppressor gene as a frequent inactivation mechanism which is likely to contribute to functional deficiencies in DNA damage response of BCCs.

The colon cancer cell lines HT29 and SW480 were transfected with an N-terminal β-catenin binding site-deficient high mobility group (HMG)-box T-cell factor 1 (ΔN-TCF-1) construct to identify differentially expressed genes. Oligonucleotide HG-U133A microarray expression profiling revealed increased mRNA levels of carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 5, 6 and mesothelin in transfectants positive for nuclear ΔN-TCF-1B. Increased amounts of CEACAM5 (CEA) were detectable in membrane-associated compartments, particularly in cholesterol-enriched microdomains. Similarly, mesothelin was demonstrated as an uncleaved membrane-bound constituent. The identified markers were examined in specimens of 46 colorectal carcinomas (CRC) by immunohistochemistry. Patchy areas of increased CEACAM5/6 staining were seen at the tumour-host front in all samples studied. Twenty-eight (58%) of these cases showed over-expression of mesothelin in a small fraction of tumor cells displaying dedifferentiation and dissemination at the invasion front. We conclude that forced expression of ΔN-TCF-1B in HT29 and SW480 is associated with up-regulation of GPI-anchored adhesion molecules, which were assigned to the tumour-host front in CRC patients.

Aims: The transcription factor Prospero-related gene 1 (PROX1) is an early specific marker for the developing liver and pancreas in the mammalian foregut endoderm. Directed hepatocyte migration into the septum transversum during liver development requires PROX1. Intriguingly, inactivation of PROX1 caused abnormal cellular proliferation via down-regulated expression of the cell-cycle inhibitors CDKN1B and CDKN1C, i.e., p27kip1 and p57kip2, respectively. Aims: We wished to examine whether epigenetic silencing of the PROX1 gene represents a mechanism for the inactivation of this putative tumor suppressor gene in malignant tumors of the bilary system. Methods: Genomic DNA isolated from praffin-embedded cholangiocarcinomatous specimens reviewed by an experienced pathologist was subjected to bisulfite-treatment and methylation-specific polymerase-chain reaction (MSP). Results: Considerable levels of PCR products specifying methylated sequences of the PROX1 promoter were detected in 9 out of 17 clinical cases (53%) analyzed by MSP. Simultaneously, products of unmethylated DNA were obtained from all specimens suggesting hemimethylation of tumor samples or presence of unmethylated immune cells. Additionally, in 2 out of 3 carcinoma cell lines established from the biliary system we identified complete PROX1 methylation. Conclusions: Here, for the first time we present experimental evidence that an abnormal methylation state of CpG islands within the PROX1 gene promoter appears to contribute to the development of cholangiocarcinomas. Reconstitution experiments of PROX1-deficient cell lines should allow for the identification of target genes modulated by this homeodomain transcription factor. Supported by BMBF NBL3 Wilhelm-Roux-Programm (FKZ09/24).

Aims: About 10-30% of primary liver cancers represent intrahepatic cholangiocarcinomas (IHCC). Since chromosomal losses of 3p are detectable in about 40% of cholangiocarcinomas our study aimed at the identification of mechanisms leading to functional deletion of tumor suppressor genes in this region. Our efforts focussed on genomic losses and epigenetic inactivation of two tumor suppressor genes, the fragile histidine triad (FHIT) and the ras association domain family 1 (RASSF1A) genes, both located on the short arm of chromosome 3. Methods: Methylation-specific PCR (MSP) and combined bisulfite-dependent restriction analysis (COBRA) were applied to detect epigenetic silencing of gene promoters. Genomic duplex PCR was used to identify exon losses of the FHIT gene. Nineteen paraffin-embedded samples of intrahepatic cholangiocarcinomas were studied. Results: Here we report for the first time that in addition to frequent losses of the exons 5 and 6, hypermethylation of the FHIT promoter occured in a significant portion of IHCC. Methylation specific PCR (MSP) detected epigenetic inactivation of the FHIT/FRA3B locus in 8 of 19 (42%) cases. Combined bisulfite restriction analysis (COBRA) revealed that high levels of methylated FHIT promoter sequences were present in 6 of the 8 methylation positive samples. In agreement with previous reports MSP identified hypermethylation of the RASSF1A gene in 13 of 19 (68%) IHCC specimens examined. Conclusions: Epigenetic silencing of the FHIT tumor suppressor gene is a novel inactivation mechanism to be considered in the development of intrahepatic cholangiocarcinomas. However, a statistically significant inverse correlation between K-Ras activation and RASSF1A inactivation was not found.

Desmoid tumors, also known as aggressive fibromatosis, occur with an incidence of 10 to 15 percent in patients affected by familial adenomatous polyposis, an autosomal inherited disease caused by germline mutations in the APC gene. However, sporadic forms with no hereditary background exist. The aim of this study was to find out whether there are APC germline mutations in apparently sporadic desmoid tumor patients without clinical or familial signs of familial adenomatous polyposis but with a family history of colorectal carcinoma in at least one family member.

To report the frequency and clinical features of internal visceral malignancy in an unselected series of patients with sebaceous gland carcinoma (SGC).

Background and aims Intrahepatic cholangiocarcinoma is the second most common intrahepatic neoplasm, accounting for 10–30% of primary liver cancers. Since little is known about the development of this cancer, we searched for alterations to the fragile histidine triad (FHIT) gene, a putative tumour suppressor gene on chromosome 3p14.2. In addition, we investigated oncogenic mutations in β-catenin, which lead to an activated Wnt signalling pathway and microsatellite instability as a consequence of mismatch repair deficiency. Methods and results Loss of heterozygosity at the FHIT/FRA3B locus was detected in two out of ten informative cases (20%) using the marker D3S1300 and in one out of seven informative cases (14%) by marker D3S1234. Furthermore, immunohistochemistry revealed loss of Fhit expression in most of the 19 intrahepatic cholangiocarcinomas analysed, although to varying degrees. Oncogenic mutations were excluded in exon 3 of the β-catenin gene by restriction enzyme digest and DNA sequence analysis. Microsatellite instability could not be detected in the tumour specimens tested using a validated marker panel. In two out of nine informative cases (22%), loss of heterozygosity was displayed close to the adenomatous polyposis coli (APC) gene. Conclusions Alterations to FHIT/FRA3B were initially detected by allelic losses of genomic DNA in intervening sequences of this tumour suppressor gene. Immunohistochemistry extended this initial observation by demonstrating that seven of the 19 intrahepatic cholangiocarcinomas had entirely lost expression of FHIT. Loss of Fhit protein in only a subpopulation of tumour cells due to oligoclonality may explain the varying portions of negative staining observed in the other tumour samples. Microsatellite instability did not appear to contribute to the development of intrahepatic cholangiocarcinoma in the cohort of tumours we analysed. Furthermore, the Wnt pathway may be constitutively turned on by inactivation of the APC gene due to deletion of genomic DNA but not by oncogenic mutations within exon 3 of the β-catenin gene.

Molecular Pathogenesis of Hepatocellular Carcinoma – State 2003 Exogenous noxae like excessive alcohol intake and tobacco smoke, chronic infections with HBV and HCV, but also genetic predisposing conditions like hemochromatosis, α1-antitrypsin deficiency and Wilson’s disease can lead to chronic inflammatory processes accompanied by tissue damage and liver regeneration, which finally may result in the development of cirrhosis and often in hepatocellular carcinoma (HCC). The reason for this is the accumulation of genetic alterations, which could lead to a dysregulation of molecular signaling networks for growth, differentiation and genomic stability as a consequence of oxidative damage of genomic DNA and proteins. Inactivation of the tumor suppressor p53 is central to hepatocellular carcinogenesis, since mutations in this gene are common in liver cell tumors of different etiology. However, also metabolic enzymes involved either in the activation of aflatoxin B1, alcohol and tobacco smoke into mutagens or in their detoxification play a fundamental role in the development of HCC. Determination of important enzyme polymorphisms could contribute to an individualized assessment of risk for HCC in the next future.

Peutz-Jeghers syndrome is clinically characterized by mucocutaneous melanocytic pigmentation, intestinal hamartomatous polyposis and a significantly increased risk of developing cancer. Mutations in the serine/threonine kinase (STK-)11 gene, also designated LKB1, are found in approximately 60% of cases of Peutz-Jeghers syndrome. There is evidence that genetic heterogeneity exists and gene(s) that have not yet been discovered may be responsible for the disease. Since most mutations in Peutz-Jeghers syndrome are null alleles and are dispersed throughout the entire STK11/LKB1 gene, the mutation screening strategies that combine approaches at both the DNA and RNA level are favored. Based upon the identification of novel mutational mechanisms, the impact of RNA-based screening for germinal STV11/LKB1 mutations in Peutz-Jeghers syndrome are specifically discussed.

PURPOSE: To report fragile histidine triad expression and microsatellite instability in periocular sebaceous gland carcinoma.DESIGN: Interventional case series.METHODS: Biopsy specimens of periocular sebaceous gland carcinoma obtained from six patients (mean age, 60 +/- 17 years; range, 38 to 83 years, 5 male, 1 female) with Muir-Torre syndrome and histopathologically proven sebaceous gland carcinoma were studied immunohistochemically for the presence of fragile histidine triad protein. Polymerase chain reaction-based analysis of the markers BAT25, BAT26, D2S123, D5S346, and D17S250 was performed for microsatellite instability in tumorous and matching normal tissues.RESULTS: Fragile histidine triad protein was detectable in the sebaceous gland carcinoma from one patient with microsatellite instability. It was not detectable in sebaceous gland carcinoma specimens from five patients without any evidence of microsatellite instability.CONCLUSION: Inactivation of fragile histidine triad tumor suppressor gene or inactivation of the mismatch-repair system resulting in microsatellite instability may contribute to the development of periocular sebaceous gland carcinoma in Muir-Torre syndrome. (C) 2002 by Elsevier Science Inc. All rights reserved.