Comparative analysis of 14-3-3 isoform expression and epigenetic alterations in colorectal cancer

Abstract

BackgroundThe 14-3-3 family is a group of intracellular proteins found in all eukaryotic organisms. Humans have seven isoforms that serve as scaffolds to promote interactions of regulatory phospho-proteins involved in many vital cellular processes and previous studies have shown that disturbances in native 14-3-3 levels can contribute significantly to the development of various cancers.MethodsDNA and RNA was extracted from frozen tissue samples collected by the Human Cooperative Tissue Network. RNA samples were reverse transcribed and subjected to qRT-PCR analysis using fluorescently labelled probes. Genomic DNA was treated with bisulfite and cloned into bacterial vectors for subsequent high-resolution sequencing. Mammalian NIH3T3 cells were transformed with 14-3-3 eta and Ras expression vectors synthesized from cDNA. Colonies were counted and transforming capability assessed after 21 days of growth. Cell lysates were analyzed by western blot to verify protein expression.ResultsHere we examined normal and cancerous 14-3-3 expression levels of all seven isoforms in a cohort of sporadic colorectal adenocarcinomas and in a group of tumors and their matched normals using qRT-PCR analysis. We found a statistically significant decrease in the levels of 14-3-3 sigma, eta, and zeta observed among adenocarcinomas compared to normal tissue. A parallel analysis of microarray data from the TCGA dataset confirmed that expression of sigma and eta were down-regulated in colon tumors. To explore the mechanisms behind 14-3-3 expression changes, we examined the methylation status of the sigma, eta, and zeta gene promoters in selected samples. Our data identified novel CpG methylation sites in the eta promoter consistent with epigenetic silencing of both 14-3-3 sigma and eta isoforms during colon tumorigenesis. Because epigenetic silencing is the hallmark of a tumor suppressor we tested eta in focus formation assays and found that it is capable of suppressing ras-induced transformation of NIH3T3 cells.ConclusionTo our knowledge, this is the first study to identify the 14-3-3 eta gene as a tumor suppressor and that its expression is suppressed in colon tumors by DNA hypermethylation. These data suggest a link between 14-3-3 expression levels and the development of colon cancers.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1856-y) contains supplementary material, which is available to authorized users.

Serum Extracellular Vesicle Stratifin Is a Biomarker of Perineural Invasion in Patients With Colorectal Cancer and Predicts Worse Prognosis

“…Although SFN plays important role in various cancers, its role in CRC is still obscure. To date, only a few studies have characterized the expression levels of SFN in CRC (57-59), although it has been reported that SFN mRNA expression is significantly decreased in CRC tissue (59). However, in our study, we discovered that SFN expression was obviously upregulated in serum-derived EVs of CRC patients with PNI compared to those without PNI.…”

NOTUM Is Involved in the Progression of Colorectal Cancer

“…Integrin linked kinase (ILK), actin cytoskeleton, RhoGDI, sirtuin, and 14-3-3-mediated signaling were activated, while actin-based mobility by Rho, leukocyte extravasation, RhoA, sumoylation, and Rho family GTPase signaling were deactivated in the secretome of SW620 ( Figure 1B). This indicates that the canonical pathways involved in metastatic features of CRC (19) were changed. We plotted a virtual 2-D protein map by using the fold change of protein expression in SW480 and SW620 against the total ion intensity ( Figure 1C).…”

Differential dependence on microbiota of IL‐23/IL‐22‐dependent gene expression between the small‐ and large‐intestinal epithelia

“…IL‐22 is reported to promote the progression of colon cancer (McCuaig et al, 2020; Perez et al, 2020; Wang et al, 2017). Meanwhile, several recent reports have suggested that Stratifin/14‐3‐3σ functions as a tumor suppressor in colorectal cancer (Winter et al, 2021; Young et al, 2015). These previous and current findings suggest that the downregulation of Stratifin/14‐3‐3σ by the IL‐23/IL‐22 axis may be one of the causes of colon cancer progression.…”