Overexpression of Dicer predicts poor survival in colorectal cancer.

AIMS: The RNASE III endonuclease Dicer is one of the key enzymes of microRNA biogenesis. The influence of Dicer-expression in tumour cells on the prognosis of patients with several cancers has been studied with controversial results among different cancer types. To date no one has examined the effect of this biomarker on survival in colorectal carcinoma. Thus, we aimed to study the influence of Dicer expression on survival in colorectal cancer. METHODS: We performed immunohistochemical analyses on formalin-fixed paraffin embedded (FFPE) cancer tissue with an antibody against the Dicer protein. Tumour material from 237 cases was available from patients with colorectal adeonocarcinomas with moderate differentiation (G2) and without evidence of lymph-node (N0) or distant metastasis (M0). Sixty-four cases were in T2 and 173 in T3 stages. A tissue microarray (TMA) was constructed with each tumour in triplicate. Each tumour was assigned to a scoring scale of 0-3, depending on the cytoplasmatic expression of Dicer. A Kaplan-Maier analysis was performed and the log-rank test was used for significance levels by using SPSS v.17 software. RESULTS: The expression of Dicer in colorectal carcinoma shows a strong association with poor survival (cancer specific survival=CSS, p<0,001) as well as with reduced progression free survival (PFS, p<0,001). In the group with no Dicer staining there was no recorded relapse (0/15) compared with 10/18 relapses in the group with the strongest staining of Dicer. CONCLUSIONS: Strong expression of the central microRNA biosynthesis enzyme Dicer predicts poor prognosis in patients with colorectal cancer. This is in line with investigations on prostate cancer. Contradictory, in breast, lung and ovary cancer Dicer has been shown to be a marker of good prognosis. Further studies on the cellular functions of Dicer need to address these issues.

Epithelial-mesenchymal and mesenchymal-epithelial transitions during cancer progression.

Invasion by colorectal carcinomas is characterized by an epithelial-mesenchymal transition (EMT)-like de-differentiation of the tumor cells. However a re-differentiation towards an epithelial phenotype, resembling a mesenchymal-epithelial transition (MET) is detectable in metastases. This indicates that malignant progression is based on dynamic processes, which can not be explained solely by irreversible genetic alterations, but must be additionally regulated by the tumor environment. The main oncoprotein in colorectal cancer is the Wnt-pathway effector beta-catenin, which is overexpressed due to mutations in the APC tumor suppressor in most cases. EMT of the tumor cells is associated with a nuclear accumulation of the transcriptional activator beta-catenin, which is reversed in metastases. Nuclear beta-catenin is involved in two fundamental processes in embryonic development: EMT and stem cell formation. Accumulating data demonstrate that aberrant nuclear expression of beta-catenin can confere these two abilites also to tumor cells. The unusual combination of EMT with stem cell competence might result in a migrating tumor stem cell, which drives tumor invasion and metastasis.

Securin (hPTTG1) expression is regulated by beta-catenin/TCF in human colorectal carcinoma.

Overexpression of the transcriptional activator beta-catenin, mostly owing to loss-of-function mutations of the adenomatous polyposis coli (APC) tumour suppressor gene, is crucial for the initiation and progression of human colorectal carcinogenesis. Securin is a regulator of chromosome separation and its overexpression has been shown to be involved in different tumour-promoting processes, like transformation, hyperproliferation and angiogenesis, and correlates with tumour cell invasion. However, the molecular mechanism leading to securin overexpression in human colorectal cancer is unknown. Here we show a correlated high expression of beta-catenin and securin (hPTTG1) in colorectal adenomas and carcinomas and further demonstrate that securin is a target of beta-catenin transcriptional activation. This implies that deregulation of the beta-catenin/T-cell factor-signalling pathway leads to overexpression of securin in human colorectal cancer, which subsequently may contribute to tumour progression.

Expression of the invasion factor laminin gamma2 in colorectal carcinomas is regulated by beta-catenin.

The migration-inducing gamma2 chain of laminin-5, one of the best known invasion markers, is strongly overexpressed in disseminating and infiltrating tumor cells at the invasive front of colorectal carcinomas. The same tumor cells show nuclear accumulation of the oncoprotein beta-catenin, which together with T-cell factor-DNA-binding proteins, functions as transcriptional activator of genes involved in tumor progression. Here we show that beta-catenin activates the human laminin-5 gamma2 gene through two T-cell factor-binding elements in a synergistic manner together with hepatocyte growth factor and conclude that laminin-5 gamma2 is another important target gene of nuclear beta-catenin during tumor progression.

Variable beta-catenin expression in colorectal cancers indicates tumor progression driven by the tumor environment.

Invasion and dissemination of well-differentiated carcinomas are often associated with loss of epithelial differentiation and gain of mesenchyme-like capabilities of the tumor cells at the invasive front. However, when comparing central areas of primary colorectal carcinomas and corresponding metastases, we again found the same differentiated epithelial growth patterns. These characteristic phenotypic changes were associated with distinct expression patterns of beta-catenin, the main oncogenic protein in colorectal carcinomas, and E-cadherin. Nuclear beta-catenin was found in dedifferentiated mesenchyme-like tumor cells at the invasive front, but strikingly, as in central areas of the primary tumors, was localized to the membrane and cytoplasm in polarized epithelial tumor cells in the metastases. This expression pattern was accompanied by changes in E-cadherin expression and proliferative activity. On the basis of these data, we postulate that an important driving force for progression of well-differentiated colorectal carcinomas is the specific environment, initiating two transient phenotypic transition processes by modulating intracellular beta-catenin distribution in tumor cells.

Tip60 is a cell-type-specific transcriptional regulator.

Tip60 was originally identified as cellular HIV-Tat interacting protein and has been shown to augment Tat-dependent transcription. It has also been shown to interact with various cellular transcription factors and to belong to the nuclear histone acetyltransferase (HAT) family. To further elucidate the function of Tip60 and its HAT domain in transcription regulation, we compared Tip60 activity in HeLa and Jurkat T lymphoma cells. Here we show that Tip60 augments the HIV-1 Tat activity at the HIV-LTR promoter in HeLa but inhibits it in Jurkat cells. Moreover, we isolated two new variants of the Tip60 protein (Tip60Delta1, Tip60Delta2) from Jurkat cells. The Tip60Delta2 variant lacks the entire HAT domain but modulates HIV-1 Tat activity like full-length Tip60. In addition, Tip60 and the transcriptional repressor ZEB (zinc finger E box binding protein) interact specifically in the yeast two-hybrid system and additively inhibit the CD4 enhancer/promoter activity in Jurkat cells. Thus, Tip60 may function as corepressor of the ZEB protein. In summary, these data show that Tip60 functions as a cell-type-specific transcriptional regulator and that the HAT domain is not required for either transcriptional activation or inhibition. This indicates that Tip60 may function by recruiting additional cell-type-specific cofactors.

Negative regulation of CD4 expression in T cells by the transcriptional repressor ZEB.

ZEB, an E-box binding transcriptional repressor, is an important regulator of T cell and muscle development. Targeted disruption of ZEB in mice resulted in a strong reduction of thymocytes and the few T cells that reached the mature stage were predominantly CD4(+). CD4 expression during the various stages of T cell differentiation is controlled at the transcriptional level by a complex array of regulatory elements in the CD4 gene locus, consisting of at least three enhancers, one promoter and one silencer. Here we present evidence that CD4 gene expression is negatively regulated by ZEB. We show that ZEB binds to the 5'E-box in the CD4-3 element of the proximal CD4 enhancer in competition with the transcriptional activators E12 and HEB, thereby reducing CD4 expression on CD4 single-positive but not CD4/CD8 double-positive T cells. The conversion of the CD4 proximal enhancer into a potential silencer element by the transcriptional repressor ZEB offers an additional concept of CD4 gene regulation in T cells.

beta-catenin regulates the expression of the matrix metalloproteinase-7 in human colorectal cancer.

Most colorectal cancers have loss of function mutations in the adenomatosis polyposis coli (APC) tumor suppressor gene. This leads to accumulation of beta-catenin, which together with the DNA binding protein TCF-4 functions as a transcriptional activator. Recently defined target genes are c-myc and cyclin D1, linking the APC gene defect to the capacity for autonomous proliferation of colon tumors. Here we report the identification of the matrix metalloproteinase MMP-7 as another target gene of beta-catenin/TCF-4. MMP-7 is overexpressed in 80% of human colorectal cancers and known to be an important factor for early tumor growth, with a potential function also for later progression steps, like invasion and metastasis. Our results explain the high percentage of MMP-7 overexpression in colon tumors. Moreover they indicate that defects in the APC tumor suppressor gene may also have an influence on later steps of colon tumor progression.

Multiple NF-ATc isoforms with individual transcriptional properties are synthesized in T lymphocytes.

The transcription factor NF-ATc that controls gene expression in T lymphocytes and embryonic cardiac cells is expressed in three prominent isoforms. This is due to alternative splice/polyadenylation events that lead to the predominant synthesis of two long isoforms in naive T cells and a shorter NF-ATc isoform in effector T cells. Whereas the previously described isoform NF-ATc/A contains a relatively short C terminus, the longer isoforms, B and C, span extra C-terminal peptides of 128 and 246 aa, respectively. We show here that in addition to the strong N-terminal trans-activation domain, TAD-A, which is common to all three NF-ATc isoforms, NF-ATc/C contains a second trans-activation domain, TAD-B, in its C-terminal peptide. Various stimuli of T cells that induce the activity of TAD-A also enhance the activity of TAD-B, but, unlike TAD-A, TAD-B remains unphosphorylated by protein from 12-O-tetradecanoyl 12-phorbol 13-acetate-stimulated T cells. The shorter C-terminal peptide of isoform NF-ATc/B exerts a suppressive transcriptional effect. These properties of NF-ATc/B and -C might be of importance for gene regulation in naive T lymphocytes in which NF-ATc/B and -C are predominantly synthesized.