c-FLIP: a key regulator of colorectal cancer cell death.

c-FLIP is an inhibitor of apoptosis mediated by the death receptors Fas, DR4, and DR5 and is expressed as long (c-FLIP(L)) and short (c-FLIP(S)) splice forms. We found that small interfering RNA (siRNA)-mediated silencing of c-FLIP induced spontaneous apoptosis in a panel of p53 wild-type, mutant, and null colorectal cancer cell lines and that this apoptosis was mediated by caspase-8 and Fas-associated death domain. Further analyses indicated the involvement of DR5 and/or Fas (but not DR4) in regulating apoptosis induced by c-FLIP siRNA. Interestingly, these effects were not dependent on activation of DR5 or Fas by their ligands tumor necrosis factor-related apoptosis-inducing ligand and FasL. Overexpression of c-FLIP(L), but not c-FLIP(S), significantly decreased spontaneous and chemotherapy-induced apoptosis in HCT116 cells. Further analyses with splice form-specific siRNAs indicated that c-FLIP(L) was the more important splice form in regulating apoptosis in HCT116, H630, and LoVo cells, although specific knockdown of c-FLIP(S) induced more apoptosis in the HT29 cell line. Importantly, intratumoral delivery of c-FLIP-targeted siRNA duplexes induced apoptosis and inhibited the growth of HCT116 xenografts in BALB/c severe combined immunodeficient mice. In addition, the growth of c-FLIP(L)-overexpressing colorectal cancer xenografts was more rapid than control xenografts, an effect that was significantly enhanced in the presence of chemotherapy. These results indicate that c-FLIP inhibits spontaneous death ligand-independent, death receptor-mediated apoptosis in colorectal cancer cells and that targeting c-FLIP may have therapeutic potential for the treatment of colorectal cancer.

Cellular FLICE-inhibitory protein regulates chemotherapy-induced apoptosis in breast cancer cells.

Combination treatment regimens that include topoisomerase-II-targeted drugs, such as doxorubicin, are widely used in the treatment of breast cancer. Previously, we showed that IFN-gamma and doxorubicin cotreatment synergistically induced apoptosis in MDA435 breast cancer cells in a signal transducer and activator of transcription 1-dependent manner. In this study, we found that this synergy was caspase-8 dependent. In addition, we found that IFN-gamma down-regulated the expression of the caspase-8 inhibitor cellular FLICE-inhibitory protein (c-FLIP). Furthermore, IFN-gamma down-regulated c-FLIP in a manner that was dependent on the transcription factors signal transducer and activator of transcription 1 and IFN regulatory factor-1. However, IFN-gamma had no effect on c-FLIP mRNA levels, indicating that c-FLIP was down-regulated at a posttranscriptional level following IFN-gamma treatment. Characterization of the functional significance of c-FLIP modulation by small interfering RNA gene silencing and stable overexpression studies revealed it to be a key regulator of IFN-gamma- and doxorubicin-induced apoptosis in MDA435 cells. Analysis of a panel of breast cancer cell lines indicated that c-FLIP was an important general determinant of doxorubicin- and IFN-gamma-induced apoptosis in breast cancer cells. Furthermore, c-FLIP gene silencing sensitized MDA435 cells to other chemotherapies, including etoposide, mitoxantrone, and SN-38. These results suggest that c-FLIP plays a pivotal role in modulating drug-induced apoptosis in breast cancer cells.

Upregulation of MUC6 mucin gene expression by NFkappaB and Sp factors.

To clarify the mechanism underlying regulation of MUC6 expression, we isolated the 5' flanking region of the MUC6 gene (5'-MUC6). We determined the transcription start site of the MUC6 gene, and found a TATA box at -35 to -29bp, a putative NFkappaB consensus sequence at -173 to -164bp, and putative Sp family binding sites at -530 to -521 and -847 to -838bp. The luciferase activity of 5'-MUC6 gradually decreased with deletion of these sites. NFkappaB inhibitory factor IkappaB decreased the luciferase activity, and forced expression of NFkappaB induced MUC6 transcription. An inhibitor of Sp family binding, mithramycin A, suppressed MUC6 transcripts, and Sp1 and Sp3 overexpression up-regulated them. Binding of Sp family members to their putative sites was confirmed by electrophoretic mobility shift assays. Our results suggest that MUC6 transcription is regulated by NFkappaB and Sp family members.

Relationship between CDX2 gene methylation and dietary factors in gastric cancer patients.

Epigenetic gene silencing through DNA methylation is one of the important steps in the mechanism underlying tumorigenesis, including in the stomach. Past lifestyle factors of cancer patients, such as intake of vegetables, are very important in affecting gastric carcinogenesis. However, the relationship between DNA methylation and past dietary habits in cancer patients remains largely unknown. The CDX2 homeobox transcription factor plays a key role in intestinal development, but CDX2 is also expressed in most of the intestinal metaplasia and part of the carcinomas of the stomach. We analyzed the methylation status of the CDX2 5' CpG island in gastric cancer cell lines by methylation-specific PCR (MSP), and then CDX2 mRNA was found to be activated after 5-aza-2'-deoxycytidine treatment of the methylation-positive cells. We further examined the methylation status of CDX2 in primary gastric carcinomas by MSP and compared it with the past lifestyle of the patients, including dietary habits. Methylation of CDX2 was found in 20 (34.5%) of the 58 male patients and one (6.7%) of the 15 female patients. Since the methylation frequency was low in the female patients, the analysis was performed only on the male cases. CDX2 methylation was correlated with the decreased intake of green tea and cruciferous vegetables, and also with full or overeating habits. These findings are consistent with epidemiological observations on gastric cancer. We also analyzed the methylation status of p16/INK4a and hMLH1, but their frequencies were not associated with dietary factors or other lifestyle factors. Thus, diet could be an important factor determining the methylation status of genes such as CDX2 and the resultant aberrant expression of genes involved in carcinogenesis.

Expression of the SRY-related HMG box protein SOX2 in human gastric carcinoma.

SOX2, a SRY-related HMG box protein, is thought to be an important transcription factor during organogenesis, including the stomach although the expression and function are unclear. We investigated SOX2 protein expression to clarify its roles in differentiation and carcinogenesis of the stomach. Using polyclonal SOX2 antibodies, expression of SOX2 in gastric normal mucosae, intestinal metaplasia and carcinomas from 68 gastric carcinoma patients was studied by immuohistochemistry. SOX2 was strongly and moderately expressed in the nuclei of the foveolar epithelium and intestinal metaplasia, respectively, the expression being much higher than that in carcinomas (p<0.0001). Using antibodies to MUC5AC, MUC2 and CD10, the 68 gastric carcinomas were classified into gastric type, intestinal type, mixed gastric and intestinal type, and null type. A significant difference in SOX2 expression was observed between the gastric and intestinal types (p<0.05), with a higher expression in the former than in the latter. Moreover, over-expression of SOX2 induced the mRNA expression of endogenous MUC5AC, a specific mucin marker for the gastric type, in COS-7 cells. Our findings indicate that SOX2 may play a role in differentiation of the human gastric epithelium, and that SOX2 may be involved in gastric carcinogenesis, particularly in the gastric type.

GATA-4 and GATA-5 transcription factor genes and potential downstream antitumor target genes are epigenetically silenced in colorectal and gastric cancer.

The GATA family of transcription factors participates in gastrointestinal (GI) development. Increases in GATA-4 and -5 expression occur in differentiation and GATA-6 expression in proliferation in embryonic and adult settings. We now show that in colorectal cancer (CRC) and gastric cancer promoter hypermethylation and transcriptional silencing are frequent for GATA-4 and -5 but are never seen for GATA-6. Potential antitumor target genes upregulated by GATA-4 and -5, the trefoil factors, inhibinalpha, and disabled-2 (Dab2) are also silenced, in GI cancers, with associated methylation of the promoters. Drug or genetically induced demethylation simultaneously leads to expression, in CRC cells, of all of the GATA-4, -5, and downstream genes. Expression of exogenous GATA-5 overrides methylation at the downstream promoters to activate the target genes. Selection for silencing of both upstream transcription factors and their target genes in GI cancers could indicate that epigenetic silencing of the involved genes provides a summated contribution to tumor progression.