Thymidylate synthase locus LOH in combination with genotype has prognostic and predictive significance in colorectal cancer.

The aim of the current study was to investigate the prognostic and predictive significance of polymorphisms in the thymidylate synthase (TS) gene, alongside the loss of heterozygocity (LOH) at this gene locus in patients with colorectal cancer. Genotyping was carried out for a variable number tandem repeat (VNTR) polymorphism in the TS 5'-untranslated region, a G/C single nucleotide polymorphism (SNP) located within this VNTR, and for TS LOH status in 246 colorectal cancer and paired normal DNA samples. The results were analyzed in relation to clinicopathological features, including the prognostic and predictive significance of TS genotype in patients who underwent curative surgery. Complete VNTR, SNP and LOH information for TS was obtained in 226 cases. No significant associations were observed between normal tissue TS genotype status and clinicopathological features. LOH of TS was observed in 58% of tumor samples and was associated with poor prognosis independently of clinical stage. Cases exhibiting TS LOH were classified into the three groups of 2R/loss, 3G/loss and 3C/loss. Patients with 3C/loss genotype status had poor outcomes when treated by surgery alone, but their survival was similar to patients with other genotypes following Fluorouracil (5-FU)-based adjuvant chemotherapy. The results suggested that LOH of the TS locus may be a significant prognostic factor in colorectal cancer, with the genotype of the residual allele also demonstrating an influence on prognosis. In conclusion, LOH status should be considered when TS genotype is explored as a potential prognostic and predictive marker for 5-FU-based adjuvant chemotherapy in colorectal cancer.

[A Case of Abdominal Fullness Caused by a Retroperitoneal Bulky Tumor Treated with Epidural Analgesia].

We presented the case of a 63-year-old woman with severe abdominal distention due to recurrent retroperitoneal sarcoma. Further, the rapid progression of the tumor made it difficult to relieve the abdominal distention. Titrated intravenous morphine was administered. Although the dose of morphine was escalated and the patient was sedated, she continued to experience pain. The addition of a continuous epidural analgesic lidocaine to manage the abdominal distention was effective. This case report describes a stepwise approach with continuous epidural analgesia of lidocaine for a bulky tumor- related abdominal distention.

KRAS mutation analysis of single circulating tumor cells from patients with metastatic colorectal cancer.

BACKGROUND: The molecular profiles of tumors may inform the selection of appropriate targeted therapies. Circulating tumor cells (CTCs) reflect the real-time status of tumor genotypes. CTCs exhibit high genetic heterogeneity within a patient; accordingly, the analysis of individual CTCs, including their heterogeneity, may enable more precise treatments. We analyzed KRAS mutations in single CTCs from patients with metastatic colorectal cancer (mCRC) using a new single-cell picking system. METHODS: Blood samples were obtained from 61 patients with mCRC. CTCs were enriched and fluorescently labeled using the CellSearch® System. They were recovered using the single-cell picking system based on the fluorescence intensity of marker dyes. Single CTCs and tumor tissue samples were examined for mutations in codons 12 and 13 of the KRAS gene. RESULTS: CTCs were detected in 27 of 61 patients with mCRC. We isolated at least two CTCs from 15 of 27 patients. KRAS genotype was evaluated in a total of 284 CTCs from 11 patients, and 15 cells with mutations were identified in four patients. In 10 of 11 patients, the KRAS status was the same in the primary tumor and CTCs. In one patient, the KRAS status was discordant between the primary tumor and CTCs. In two patients, different KRAS mutations were found among individual CTCs. CONCLUSIONS: We successfully isolated single CTCs and detected KRAS mutations in individual cells from clinical samples using a novel application of single-cell isolation system. Using the system, we detected CTC heterozygosity and heterogeneity in KRAS status among CTCs within a patient and between CTCs and tumor tissues.

Phase II study of bevacizumab and irinotecan as second-line therapy for patients with metastatic colorectal cancer previously treated with fluoropyrimidines, oxaliplatin, and bevacizumab.

PURPOSE: Fluorouracil and folinic acid with irinotecan (FOLFIRI) plus bevacizumab (BV) is widely used as second-line chemotherapy for patients with metastatic colorectal cancer (mCRC) previously treated with fluoropyrimidines, oxaliplatin, and BV. FOLFIRI requires a CV catheter and an infusion pump, which are inconvenient for patients. Sufficient data are not available for characterizing the effectiveness of fluoropyrimidines beyond first disease progression. In this study, we evaluated the efficacy and safety of irinotecan (CPT-11) plus BV as second-line therapy. METHODS: Patients with mCRC previously treated with at least four courses of a fluoropyrimidine, oxaliplatin, and BV were designated to receive 150 mg/m2 of CPT-11 and 10 mg/kg of BV every 2 weeks as second-line therapy. The primary endpoint was progression-free survival (PFS), and secondary endpoints included response rate (RR), overall survival (OS), and adverse events. RESULTS: Thirty patients from six institutes were enrolled from March 2011 to January 2014. The median PFS was 5.7 months (95% CI 4.2-7.3 months), and the RR was 6.7% (range 0.8-22.1%). Grades 3-4 adverse events included leucopenia (36.7%), neutropenia (50%), thrombocytopenia (26.7%), anemia (30%), diarrhea (3.3%), anorexia (6.7%), and hypertension (3.3%). Relative dose intensities were 94.5 and 96.3% for CPT-11 and BV, respectively. The median OS was 11.8 months (6.3 months-not reached). CONCLUSIONS: Administration of CPT-11 plus BV to patients with mCRC achieved comparable efficacies with relatively lower toxicities compared with the results of previous studies using FOLFIRI plus BV as second-line therapy. The dose intensity of CPT-11 was judged as satisfactory. CLINICAL TRIAL INFORMATION: UMIN000005228.

Glycogen synthase kinase 3ß inhibition sensitizes human glioblastoma cells to temozolomide by affecting O6-methylguanine DNA methyltransferase promoter methylation via c-Myc signaling.

Glycogen synthase kinase 3ß (GSK3ß) is a serine/threonine protein kinase involved in human cancers including glioblastoma. We have previously demonstrated that GSK3ß inhibition enhances temozolomide effect in glioma cells. In this report, we investigated the molecular mechanisms of sensitization of glioblastoma cells to temozolomide by GSK3ß inhibition, focusing on O(6)-methylguanine DNA methyltransferase (MGMT) gene silencing. Glioblastoma tissues from patients treated with the GSK3ß-inhibiting drugs were subjected to immunohistochemistry and methylation-specific PCR assay. Human glioblastoma cell lines T98G, U138, U251 and U87 were treated with a small-molecule GSK3ß inhibitor, AR-A014418 or GSK3ß-specific small interfering RNA. The combined effect of temozolomide and AR-A014418 on cell proliferation was determined by AlamarBlue assay and an isobologram method. MGMT promoter methylation was estimated by methylation-specific PCR and MethyLight assay. MGMT gene expression was evaluated by real-time quantitative reverse transcriptase-PCR. c-Myc and DNA (cytosine-5)-methyltransferase 3A binding to the MGMT promoter was estimated by chromatin immunoprecipitation assay. GSK3ß inhibition decreased phosphorylation of glycogen synthase and reduced MGMT expression and increased MGMT promoter methylation in clinical tumors. In glioblastoma cell lines, GSK3ß inhibition decreased cell viability, enhanced temozolomide effect and downregulated MGMT expression with relevant changes in the methylation levels of the MGMT promoter. Here, we showed for the first time that c-Myc binds to the MGMT promoter with consequent recruitment of DNA (cytosine-5)-methyltransferase 3A, regulating the levels of MGMT promoter methylation. The results of this study suggest that GSK3ß inhibition enhances temozolomide effect by silencing MGMT expression via c-Myc-mediated promoter methylation.

Aberrant glycogen synthase kinase 3ß is involved in pancreatic cancer cell invasion and resistance to therapy.

BACKGROUND AND PURPOSE: The major obstacles to treatment of pancreatic cancer are the highly invasive capacity and resistance to chemo- and radiotherapy. Glycogen synthase kinase 3ß (GSK3ß) regulates multiple cellular pathways and is implicated in various diseases including cancer. Here we investigate a pathological role for GSK3ß in the invasive and treatment resistant phenotype of pancreatic cancer. METHODS: Pancreatic cancer cells were examined for GSK3ß expression, phosphorylation and activity using Western blotting and in vitro kinase assay. The effects of GSK3ß inhibition on cancer cell survival, proliferation, invasive ability and susceptibility to gemcitabine and radiation were examined following treatment with a pharmacological inhibitor or by RNA interference. Effects of GSK3ß inhibition on cancer cell xenografts were also examined. RESULTS: Pancreatic cancer cells showed higher expression and activity of GSK3ß than non-neoplastic cells, which were associated with changes in its differential phosphorylation. Inhibition of GSK3ß significantly reduced the proliferation and survival of cancer cells, sensitized them to gemcitabine and ionizing radiation, and attenuated their migration and invasion. These effects were associated with decreases in cyclin D1 expression and Rb phosphorylation. Inhibition of GSK3ß also altered the subcellular localization of Rac1 and F-actin and the cellular microarchitecture, including lamellipodia. Coincident with these changes were the reduced secretion of matrix metalloproteinase-2 (MMP-2) and decreased phosphorylation of focal adhesion kinase (FAK). The effects of GSK3ß inhibition on tumor invasion, susceptibility to gemcitabine, MMP-2 expression and FAK phosphorylation were observed in tumor xenografts. CONCLUSION: The targeting of GSK3ß represents an effective strategy to overcome the dual challenges of invasiveness and treatment resistance in pancreatic cancer.

LINE-1 methylation shows little intra-patient heterogeneity in primary and synchronous metastatic colorectal cancer.

BACKGROUND: Long interspersed nucleotide element 1 (LINE-1) hypomethylation is suggested to play a role in the progression of colorectal cancer (CRC). To assess intra-patient heterogeneity of LINE-1 methylation in CRC and to understand its biological relevance in invasion and metastasis, we evaluated the LINE-1 methylation at multiple tumor sites. In addition, the influence of stromal cell content on the measurement of LINE-1 methylation in tumor tissue was analyzed. METHODS: Formalin-fixed paraffin-embedded primary tumor tissue was obtained from 48 CRC patients. Matched adjacent normal colon tissue, lymph node metastases and distant metastases were obtained from 12, 18 and 7 of these patients, respectively. Three different areas were microdissected from each primary tumor and included the tumor center and invasive front. Normal mucosal and stromal cells were also microdissected for comparison with the tumor cells. The microdissected samples were compared in LINE-1 methylation level measured by multicolor MethyLight assay. The assay results were also compared between microdissected and macrodissected tissue samples. RESULTS: LINE-1 methylation within primary tumors showed no significant intra-tumoral heterogeneity, with the tumor center and invasive front showing identical methylation levels. Moreover, no difference in LINE-1 methylation was observed between the primary tumor and lymph node and distant metastases from the same patient. Tumor cells showed significantly less LINE-1 methylation compared to adjacent stromal and normal mucosal epithelial cells. Consequently, LINE-1 methylation was significantly lower in microdissected samples compared to macrodissected samples. A trend for less LINE-1 methylation was also observed in more advanced stages of CRC. CONCLUSIONS: LINE-1 methylation shows little intra-patient tumor heterogeneity, indicating the suitability of its use for molecular diagnosis in CRC. The methylation is relatively stable during CRC progression, leading us to propose a new concept for the association between LINE-1 methylation and disease stage.

AP1B plays an important role in intestinal tumorigenesis with the truncating mutation of an APC gene.

Recent evidence has suggested that carcinoma is accompanied by the loss of cell polarity. An epithelial cell-specific form of the AP-1 clathrin adaptor complex, AP1B, is involved in the polarized transport of membrane proteins to the basolateral surface of epithelial cells. In our study, we investigated whether AP1B is involved in intestinal tumorigenesis. The cellular polarity of intestinal tumor cells was examined using APC(Min/+) mice as an in vivo model and SW480 cells with a truncating mutation in the adenomatous polyposis coli (APC) gene as an in vitro model by confocal microscopy. Next, the expression of AP1B in intestinal tumor cells was examined by real-time polymerase chain reaction (PCR) and Western blotting. The localization of ß-catenin and the expression of AP1B in the tumor tissue of patients with colorectal cancer were evaluated by confocal microscopy and real-time PCR, respectively, and the relationships among cell polarity, AP1B expression and intestinal tumorigenesis were examined. Cellular polarity was lost in intestinal tumor cells, and the expression of AP1B was downregulated. In addition, the reduction in the expression level of AP1B correlated with the nuclear localization of ß-catenin in human colorectal cancer. Our study indicates the close associations between AP1B, intestinal tumorigenesis and mutations in the APC gene. This is the first report to reveal the relationships among AP1B, cellular polarity and intestinal tumorigenesis, and achieving a detailed understanding of AP1B will hopefully lead to discovery of therapeutic targets and novel biomarkers for intestinal cancer.

Glycogen synthase kinase 3ß inhibition sensitizes pancreatic cancer cells to gemcitabine.

BACKGROUND: Pancreatic cancer is obstinate and resistant to gemcitabine, a standard chemotherapeutic agent for the disease. We previously showed a therapeutic effect of glycogen synthase kinase-3ß (GSK3ß) inhibition against gastrointestinal cancer and glioblastoma. Here, we investigated the effect of GSK3ß inhibition on pancreatic cancer cell sensitivity to gemcitabine and the underlying molecular mechanism. METHODS: Expression, phosphorylation, and activity of GSK3ß in pancreatic cancer cells (PANC-1) were examined by Western immunoblotting and in vitro kinase assay. The combined effect of gemcitabine and a GSK3ß inhibitor (AR-A014418) against PANC-1 cells was examined by isobologram and PANC-1 xenografts in mice. Changes in gene expression in PANC-1 cells following GSK3ß inhibition were studied by cDNA microarray and reverse transcription (RT)-PCR. RESULTS: PANC-1 cells showed increased GSK3ß expression, phosphorylation at tyrosine 216 (active form), and activity compared with non-neoplastic HEK293 cells. Administration of AR-A014418 at pharmacological doses attenuated proliferation of PANC-1 cells and xenografts, and significantly sensitized them to gemcitabine. Isobologram analysis determined that the combined effect was synergistic. DNA microarray analysis detected GSK3ß inhibition-associated changes in gene expression in gemcitabine-treated PANC-1 cells. Among these changes, RT-PCR and Western blotting showed that expression of tumor protein 53-induced nuclear protein 1, a gene regulating cell death and DNA repair, was increased by gemcitabine treatment and substantially decreased by GSK3ß inhibition. CONCLUSIONS: The results indicate that GSK3ß inhibition sensitizes pancreatic cancer cells to gemcitabine with altered expression of genes involved in DNA repair. This study provides insight into the molecular mechanism of gemcitabine resistance and thus a new strategy for pancreatic cancer chemotherapy.

Long interspersed nuclear element-1 hypomethylation is a potential biomarker for the prediction of response to oral fluoropyrimidines in microsatellite stable and CpG island methylator phenotype-negative colorectal cancer.

We investigated the clinical value of methylation of long interspersed nuclear element-1 (LINE-1) for the prognosis of colorectal cancer (CRC) and for the survival benefit from adjuvant chemotherapy with oral fluoropyrimidines. LINE-1 methylation in tumor DNA was measured by quantitative methylation-specific PCR in 155 samples of stage II and stage III CRC. The presence of microsatellite instability and CpG island methylator phenotype (CIMP) were assessed and 131 microsatellite stable/CIMP- cases were selected for survival analysis, of which 77 patients had received postoperative adjuvant chemotherapy with oral fluoropyrimidines. The CRC cell lines were used to investigate possible mechanistic links between LINE-1 methylation and effects of 5-fluorouracil (5-FU). High LINE-1 methylation was a marker for better prognosis in patients treated by surgery alone. Patients with low LINE-1 methylation who were treated with adjuvant chemotherapy survived longer than those treated by surgery alone, suggestive of a survival benefit from the use of oral fluoropyrimidines. In contrast, a survival benefit from chemotherapy was not observed for patients with high LINE-1 methylation. The CRC cell lines treated with 5-FU showed increased expression of LINE-1 mRNA. This was associated with upregulation of the phospho-histone H2A.X in cells with low LINE-1 methylation, but not in cells with high LINE-1 methylation. The 5-FU-mediated induction of phospho-histone H2A.X, a marker of DNA damage, was inhibited by knockdown of LINE-1. These results suggest that LINE-1 methylation is a novel predictive marker for survival benefit from adjuvant chemotherapy with oral fluoropyrimidines in CRC patients. This finding could be important for achieving personalized chemotherapy.

Inhibition of gastric carcinogenesis by the hormone gastrin is mediated by suppression of TFF1 epigenetic silencing.

BACKGROUND & AIMS: Epigenetic alterations have been correlated with field cancerization in human patients, but evidence from experimental models that specific epigenetic changes can initiate cancer has been lacking. Although hormones have been associated with cancer risk, the mechanisms have not been determined. The peptide hormone gastrin exerts a suppressive effect on antral gastric carcinogenesis. METHODS: N-methyl-N-nitrosourea (MNU)-dependent gastric cancer was investigated in hypergastrinemic (INS-GAS), gastrin-deficient (GAS(-/-)), Tff1-deficient (Tff1(+/-)), and wild-type (WT) mice. Epigenetic alterations of the trefoil factor 1 (TFF1) tumor suppressor gene were evaluated in vitro and in vivo. RESULTS: Human intestinal-type gastric cancers in the antrum exhibited progressive TFF1 repression and promoter hypermethylation. Mice treated with MNU exhibited a field defect characterized by widespread Tff1 repression associated with histone H3 lysine 9 methylation and H3 deacetylation at the Tff1 promoter in epithelial cells. In MNU-induced advanced cancers, DNA methylation at the Tff1 promoter was observed. Tumor induction and Tff1 repression were increased in MNU-treated mice by Helicobacter infection. Hypergastrinemia suppressed MNU-dependent tumor initiation and progression in a manner that correlated with gene silencing and epigenetic alterations of Tff1. In contrast, homozygous gastrin-deficient and heterozygous Tff1-deficient mice showed enhanced MNU-dependent field defects and cancer initiation compared with WT mice. In gastric cancer cells, gastrin stimulation partially reversed the epigenetic silencing in the TFF1 promoter. CONCLUSIONS: Initiation of antral gastric cancer is associated with progressive epigenetic silencing of TFF1, which can be suppressed by the hormone gastrin.

Metabolic disorder, inflammation, and deregulated molecular pathways converging in pancreatic cancer development: implications for new therapeutic strategies.

Pancreatic cancer develops and progresses through complex, cumulative biological processes involving metabolic disorder, local inflammation, and deregulated molecular pathways. The resulting tumor aggressiveness hampers surgical intervention and renders pancreatic cancer resistant to standard chemotherapy and radiation therapy. Based on these pathologic properties, several therapeutic strategies are being developed to reverse refractory pancreatic cancer. Here, we outline molecular targeting therapies, which are primarily directed against growth factor receptor-type tyrosine kinases deregulated in tumors, but have failed to improve the survival of pancreatic cancer patients. Glycogen synthase kinase-3ß (GSK3ß) is a member of a serine/threonine protein kinase family that plays a critical role in various cellular pathways. GSK3ß has also emerged as a mediator of pathological states, including glucose intolerance, inflammation, and various cancers (e.g., pancreatic cancer). We review recent studies that demonstrate the anti-tumor effects of GSK3ß inhibition alone or in combination with chemotherapy and radiation. GSK3ß inhibition may exert indirect anti-tumor actions in pancreatic cancer by modulating metabolic disorder and inflammation.

[DNA methylation and cancer metastasis].

Cancer cells acquire metastatic phenotypes by accumulating genetic and epigenetic abnormalities. DNA methylation is involved in epigenetic regulation of gene transcription and frequently altered with carcinogenesis. Two modes of aberrant DNA methylation, promoter hypermethylation and global hypomethylation, play a role in cancer metastasis with different mechanisms. Here, we discuss how the aberrant DNA methylation contributes to acquisition of metastatic phenotypes in cancer and review the recent trials for molecular diagnosis of cancer metastasis using methylation markers.

Helicobacter pylori infection promotes methylation and silencing of trefoil factor 2, leading to gastric tumor development in mice and humans.

BACKGROUND & AIMS: Trefoil factors (TFFs) regulate mucosal repair and suppress tumor formation in the stomach. Tff1 deficiency results in gastric cancer, whereas Tff2 deficiency increases gastric inflammation. TFF2 expression is frequently lost in gastric neoplasms, but the nature of the silencing mechanism and associated impact on tumorigenesis have not been determined. METHODS: We investigated the epigenetic silencing of TFF2 in gastric biopsy specimens from individuals with Helicobacter pylori-positive gastritis, intestinal metaplasia, gastric cancer, and disease-free controls. TFF2 function and methylation were manipulated in gastric cancer cell lines. The effects of Tff2 deficiency on tumor growth were investigated in the gp130(F/F) mouse model of gastric cancer. RESULTS: In human tissue samples, DNA methylation at the TFF2 promoter began at the time of H pylori infection and increased throughout gastric tumor progression. TFF2 methylation levels were inversely correlated with TFF2 messenger RNA levels and could be used to discriminate between disease-free controls, H pylori-infected, and tumor tissues. Genome demethylation restored TFF2 expression in gastric cancer cell lines, so TFF2 silencing requires methylation. In Tff2-deficient gp130(F/F)/Tff2(-/-) mice, proliferation of mucosal cells and release of T helper cell type-1 (Th-1) 1 cytokines increased, whereas expression of gastric tumor suppressor genes and Th-2 cytokines were reduced, compared with gp130(F/F)controls. The fundus of gp130(F/F)/Tff2(-/-) mice displayed glandular atrophy and metaplasia, indicating accelerated preneoplasia. Experimental H pylori infection in wild-type mice reduced antral expression of Tff2 by increased promoter methylation. CONCLUSIONS: TFF2 negatively regulates preneoplastic progression and subsequent tumor development in the stomach, a role that is subverted by promoter methylation during H pylori infection.

Induction of HITS, a newly identified family with sequence similarity 107 protein (FAM107B), in cancer cells by heat shock stimulation.

The Family with sequence similarity 107 (FAM107) possesses an N-terminal domain of unknown function (DUF1151) that is highly conserved beyond species. In human, FAM107A termed TU3A/DRR1 has been reported as a candidate tumor suppressor gene which expression is downregulated in several types of cancer, however no studies have investigated the other family protein, FAM107B. In the present study, we designated FAM107B as heat shock-inducible tumor small protein (HITS) and studied its expression and functional properties in cancer. HITS is an 18-kDa nuclear protein expressed in a variety of tissues including stomach, colon, lung and lymphoid organs. In human gastric and colorectal cancers and a mouse model of colon cancer, its expression in tumor cells was much lower than normal epithelial cells, while expression pattern and intensity varied among different histological types of cancer. In functional analysis in vitro, forced expression of this protein suppresses the cellular responses to growth factors. Furthermore, HITS gene carries the promoter region providing heat shock transcription factor (HSF) binding sites and amplifying the transcription of HITS by heat shock or hyperthermia treatment both in vitro and in vivo. Thus HITS would be a potential tumor suppressor gene similar to TU3A containing heat responding elements, which contrasts with previously described oncogenic activities of other heat shock proteins such as HSP70 and HSP90.

Long interspersed nuclear element 1 hypomethylation is a marker of poor prognosis in stage IA non-small cell lung cancer.

PURPOSE: Global hypomethylation and the hypermethylation of gene promoter regions are common events in tumor DNA. The aim of this study was to evaluate the prognostic significance of both global hypomethylation and gene promoter hypermethylation in DNA from non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: Genomic DNA was obtained from the tumor tissue of 379 NSCLC patients who underwent surgery. Methylation levels were measured by real-time PCR following bisulfite modification of DNA and were correlated with clinicopathologic parameters and patient prognosis. Methylation of long interspersed nuclear element 1 (LINE-1) was used as a surrogate marker for global methylation. Hypermethylation of the APC, CDH13, and RASSF1 promoter regions was also evaluated. RESULTS: Tumor tissue showed significantly higher CDH13 and RASSF1 methylation levels compared with normal lung tissue, but lower LINE-1 methylation levels. APC, RASSF1, and LINE-1 methylation levels were significant prognostic factors in univariate analysis of an initial cohort of 234 cases. APC and LINE-1 methylation remained significant prognostic factors in multivariate analysis that included age, gender, smoking history, histologic type, and pathologic stage. LINE-1 methylation showed marginally significant prognostic value in stage IA and IB disease. Expansion of the study cohort to 364 cases revealed that LINE-1 methylation had significant prognostic value for stage IA NSCLC patients in multivariate analysis. CONCLUSIONS: LINE-1 hypomethylation was an independent marker of poor prognosis in stage IA NSCLC. Validation of this finding in additional tumor cohorts could have clinical relevance for the management of early-stage NSCLC.

An emerging strategy for cancer treatment targeting aberrant glycogen synthase kinase 3 beta.

Improvement in the outcome of cancer patients who are refractory to currently available treatments relies on the development of target-directed therapies. One group of molecular targets with potential clinical relevance is a set of protein tyrosine kinases encoded mostly by proto-oncogenes and that are frequently deregulated in cancer. Glycogen synthase kinase 3beta (GSK3beta), a serine/threonine protein kinase, has emerged as a therapeutic target for common chronic diseases including type 2 diabetes mellitus, neurodegenerative disorders, inflammation and osteoporosis. This is based on its currently known functions and primary pathologic causalities. GSK3beta has well characterized roles in the regulation of gene transcription and in oncogenic signaling. We have shown that deregulated GSK3beta promotes gastrointestinal, pancreatic and liver cancers and glioblastomas. Furthermore, we have demonstrated that inhibition of GSK3beta attenuates cell survival and proliferation, induces cell senescence and apoptosis and sensitizes tumor cells to chemotherapeutic agents and ionizing radiation. This has led us to propose GSK3beta as a potential therapeutic target in cancer. The anti-tumor effects of GSK3beta inhibition are mediated by changes in the expression and phosphorylation of molecules critical to the regulation of cell cycling, proliferation and apoptosis and underlie the pathological role for GSK3beta in cancer. Investigation of the mechanisms responsible for deregulation of GSK3beta and the consequent downstream pathologic effects in cancer cells has shed light on the molecular pathways leading to tumorigenesis. This will allow exploration of novel therapeutic strategies for cancer that target aberrant GSK3beta.

Wnt signaling stimulates transcriptional outcome of the Hedgehog pathway by stabilizing GLI1 mRNA.

Wnt and Hedgehog signaling pathways play central roles in embryogenesis, stem cell maintenance, and tumorigenesis. However, the mechanisms by which these two pathways interact are not well understood. Here, we identified a novel mechanism by which Wnt signaling pathway stimulates the transcriptional output of Hedgehog signaling. Wnt/beta-catenin signaling induces expression of an RNA-binding protein, CRD-BP, which in turn binds and stabilizes GLI1 mRNA, causing an elevation of GLI1 expression and transcriptional activity. The newly described mode of regulation of GLI1 seems to be important to several functions of Wnt, including survival and proliferation of colorectal cancer cells.

Deregulated GSK3{beta} sustains gastrointestinal cancer cells survival by modulating human telomerase reverse transcriptase and telomerase.

PURPOSE: Glycogen synthase kinase-3beta (GSK3beta) regulates multiple cell signaling pathways and has been implicated in glucose intolerance, neurodegenerative disorders, and inflammation. We investigated the expression, activity, and putative pathologic role of GSK3beta in gastrointestinal, pancreatic, and liver cancers. EXPERIMENTAL DESIGN: Colon, stomach, pancreatic, and liver cancer cell lines; nonneoplastic HEK293 cells; and matched pairs of normal and tumor tissues of stomach and colon cancer patients were examined for GSK3beta expression and its phosphorylation at serine 9 (inactive form) and tyrosine 216 (active form) by Western immunoblotting and for GSK3beta activity by in vitro kinase assay. The effects of small-molecule GSK3beta inhibitors and of RNA interference on cell survival, proliferation, and apoptosis were examined in vitro and on human colon cancer cell xenografts in athymic mice. The effects of GSK3beta inhibition on human telomerase reverse transcriptase (hTERT) expression and telomerase activity were compared between colon cancer and HEK293 cells. RESULTS: Cancer cell lines and most cancer tissues showed increased GSK3beta expression and increased tyrosine 216 phosphorylation and activity but decreased serine 9 phosphorylation compared with HEK293 cells and nonneoplastic tissues. Inhibition of GSK3beta resulted in attenuated cell survival and proliferation and increased apoptosis in most cancer cell lines and in HT-29 xenografts in rodents but not in HEK293 cells. GSK3beta inhibition in colon cancer cells was associated with decreased hTERT expression and telomerase activity. CONCLUSION: The results indicate that deregulated GSK3beta sustains gastrointestinal cancer cells survival through modulation of hTERT and telomerase.

Different histological types of non-small cell lung cancer have distinct folate and DNA methylation levels.

Aberrant DNA methylation is a commonly observed epigenetic change in lung cancer. Folate has been suggested to play a role in the homeostasis of DNA methylation and has also been implicated in cancer chemotherapy. We investigated a possible role for folate in DNA methylation by measuring folate concentrations in tumors and adjacent normal tissues from 72 non-small cell lung cancer (NSCLC) patients. These were compared to DNA methylation levels and to clinicopathological features. Folate concentrations were determined as the sum of 5,10-methylenetetrahydrofolate and tetrahydrofolate. The MethyLight assay was used to quantitate methylation in promoter regions of P16(CDKN2A), APC, CDH13, RARB, RASSF1, RUNX3, and MYOD1. Methylation of LINE-1 repeats was used as a surrogate for global methylation. Folate levels in tumors correlated positively with LINE-1, CDH13, and RUNX3 methylation. Folate concentrations and methylation of LINE-1, RASSF1, and RUNX3 were significantly higher in adenocarcinoma compared to squamous cell carcinoma (SCC). Two sets of array-based data retrieved from the Gene Expression Omnibus consistently showed that expression of FOLR1, a folate transport enzyme, and GGH, an enzyme that prevents folate retention, were higher and lower, respectively, in adenocarcinomas compared to SCC. This was independently validated by quantitative RT-PCR in 26 adenocarcinomas and 13 SCC. Our results suggest that folate metabolism plays a role in aberrant DNA methylation in NSCLC. The histological subtype differences in folate concentration and DNA methylation observed here were associated with distinct expression patterns for folate metabolizing enzymes. These findings may have clinical applications for histology-directed chemotherapy with fluoropyrimidine and anti-folates in NSCLC.