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-FLIPL) and short (c-FLIPS) splice forms. We found that siRNA-mediated silencing of c-FLIP induced spontaneous apoptosis in a panel of p53 wild-type, mutant and null colorectal cancer (CRC) cell lines and that this apoptosis was mediated by caspase 8 and FADD. 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 TRAIL and FasL. Overexpression of c-FLIPL, but not c-FLIPS, significantly decreased spontaneous and chemotherapy-induced apoptosis in HCT116 cells. Further analyses with splice form-specific siRNAs indicated that c-FLIPL was the more important splice form in regulating apoptosis in HCT116, H630 and LoVo cells, although specific knock down of c-FLIPS induced more apoptosis in the HT29 cell line. Importantly, intra-tumoral delivery of c-FLIP-targeted siRNA duplexes induced apoptosis and inhibited the growth of HCT116 xenografts in Balb/c SCID mice. In addition, the growth of c-FLIPL overexpressing CRC 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 CRC cells and that targeting c-FLIP may have therapeutic potential for the treatment of colorectal cancer.

Clonal characteirization of sporadic cribriform-morular variant of papillary thyroid carcinoma by laser microdissection based APC mutation analysis

Cribriform-morular variant (C-MV) of papillary thyroid carcinoma (PTC) is a rare and unusual neoplasm composed of multiple histologic components, including cribriform, papillary, solid, tall columnar, and morular patterns. Analyses of gross C-MV of PTC lesions has linked adenomatous polyposis coli (APC) mutations to its pathogenesis; however, the extent of involvement of mutations in the development Of individual components is unclear We report on bidirectional sequencing of the mutation cluster region (codons 1032-1565) of the APC gene in individually laser-microdissected components of a previously unreported C-MV of PTC. A silent Thr1493Thr gene variant was found in all tumoral components, whereas a 5-base-pair frameshift deletion at codon 1309 was identified only in the morules. Neither variant was observed in matched normal thyroid tissue. These results show the histologic components of C-MV of PTC to have some common mutational background, although additional somatic mutations may be involved in the development of morular structures.

Transcriptional response of T cells to IFN-alpha:changes induced in IFN-alpha-sensitive and resistant cutaneous T cell lymphoma

Interferon-alpha (IFN-alpha) therapy is commonly used in the treatment of neoplastic and autoimmune diseases, including cutaneous T cell lymphoma (CTCL). However, the IFN-alpha response is unpredictable, and the IFN-alpha cell targets and pathways are only partially understood. To delineate the molecular mechanisms of IFN-alpha activity, gene expression profiling was performed in a time-course experiment of both IFN-alpha sensitive and IFN-alpha-resistant variants of a CTCL cell line. These experiments revealed that IFN-alpha is responsible for the regulation of hundreds of genes in both variants and predominantly involves genes implicated in signal transduction, cell cycle control, apoptosis, and transcription regulation. Specifically, the IFN-alpha response of tumoral T cells is due to a combination of induction of apoptosis in which TNFSF10 and HSXIAPAF1 may play an important role and cell cycle arrest achieved by downregulation of CDK4 and CCNG2 and upregulation of CDKN2C and tumor suppressor genes (TSGs). Resistance to IFN-alpha appears to be associated with failure to induce IRF1 and IRF7 and deregulation of the apoptotic signals of HSXIAPAF1, TRADD, BAD, and BNIP3. Additionally, cell cycle progression is heralded by upregulation of CDC25A and CDC42. A critical role of NF-kappaB in promoting cell survival in IFN-alpha-resistant cells is indicated by the upregulation of RELB and LTB.

Mycosis fungoides shows concurrent deregulation of multiple genes involved in the TNF signaling pathway:an expression profile study

Mycosis fungoides (MF) is the most frequent type of cutaneous T-cell lymphoma, whose diagnosis and study is hampered by its morphologic similarity to inflammatory dermatoses (ID) and the low proportion of tumoral cells, which often account for only 5% to 10% of the total tissue cells. cDNA microarray studies using the CNIO OncoChip of 29 MF and 11 ID cases revealed a signature of 27 genes implicated in the tumorigenesis of MF, including tumor necrosis factor receptor (TNFR)-dependent apoptosis regulators, STAT4, CD40L, and other oncogenes and apoptosis inhibitors. Subsequently a 6-gene prediction model was constructed that is capable of distinguishing MF and ID cases with unprecedented accuracy. This model correctly predicted the class of 97% of cases in a blind test validation using 24 MF patients with low clinical stages. Unsupervised hierarchic clustering has revealed 2 major subclasses of MF, one of which tends to include more aggressive-type MF cases including tumoral MF forms. Furthermore, signatures associated with abnormal immunophenotype (11 genes) and tumor stage disease (5 genes) were identified.

Challenging the cancer molecular stratification dogma: Intratumoral heterogeneity undermines consensus molecular subtypes and potential diagnostic value in colorectal cancer

Purpose:
A number of independent gene expression profiling studies have identified transcriptional subtypes in colorectal cancer (CRC) with potential diagnostic utility, culminating in publication of a CRC Consensus Molecular Subtype classification. The worst prognostic subtype has been defined by genes associated with stem-like biology. Recently, it has been shown that the majority of genes associated with this poor prognostic group are stromal-derived. We investigated the potential for tumor misclassification into multiple diagnostic subgroups based on tumoral region sampled.

Experimental Design:
We performed multi-region tissue RNA extraction/transcriptomic analysis using Colorectal Specific Arrays on invasive front, central tumor and lymph node regions selected from tissue samples from 25 CRC patients.

Results:
We identified a consensus 30 gene list which represents the intratumoral heterogeneity within a cohort of primary CRC tumors. Using a series of online datasets, we showed that this gene list displays prognostic potential (HR=2.914 (CI 0.9286-9.162) in stage II/III CRC patients, but in addition we demonstrated that these genes are stromal derived, challenging the assumption that poor prognosis tumors with stem-like biology have undergone a widespread Epithelial Mesenchymal Transition (EMT). Most importantly, we showed that patients can be simultaneously classified into multiple diagnostically relevant subgroups based purely on the tumoral region analysed.

Conclusions:
Gene expression profiles derived from the non-malignant stromal region can influence assignment of CRC transcriptional subtypes, questioning the current molecular classification dogma and highlighting the need to consider pathology sampling region and degree of stromal infiltration when employing transcription-based classifiers to underpin clinical decision-making in CRC.