During innate immune responses, myeloid differentiation primary response 88 (MyD88) functions as a critical signaling adaptor protein integrating stimuli from toll-like receptors (TLR) and the interleukin-1 receptor (IL-1R) family and translates them into specific cellular outcomes. In B cells, somatic mutations in MyD88 trigger oncogenic NF-κB signaling independent of receptor stimulation, which leads to the development of B-cell malignancies. However, the exact molecular mechanisms and downstream signaling targets remain unresolved. We established an inducible system to introduce MyD88 to lymphoma cell lines and performed transcriptomic analysis (RNA-seq) to identify genes differentially expressed by MyD88 bearing the L265P oncogenic mutation. We show that MyD88L265P activates NF-κB signaling and upregulates genes that might contribute to lymphomagenesis, including CD44, LGALS3 (coding Galectin-3), NFKBIZ (coding IkBƺ), and BATF. Moreover, we demonstrate that CD44 can serve as a marker of the activated B-cell (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) and that CD44 expression is correlated with overall survival in DLBCL patients. Our results shed new light on the downstream outcomes of MyD88L265P oncogenic signaling that might be involved in cellular transformation and provide novel therapeutical targets.

• Keywords
• CD44, DLBCL, Galectin-3, LGALS3, MYD88, NF-kB, lymphoma, oncogenic signaling,
• MeSH
• Adaptor Proteins, Signal Transducing metabolism   MeSH
• Hyaluronan Receptors genetics   metabolism   MeSH
• Lymphoma, Large B-Cell, Diffuse * pathology   MeSH
• Galectin 3 metabolism   MeSH
• Humans MeSH
• Mutation MeSH
• Myeloid Differentiation Factor 88 genetics   metabolism   MeSH
• NF-kappa B * genetics   metabolism   MeSH
• Gene Expression Profiling MeSH
• Basic-Leucine Zipper Transcription Factors genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Adaptor Proteins, Signal Transducing MeSH
• Hyaluronan Receptors MeSH
• BATF protein, human MeSH Browser
• CD44 protein, human MeSH Browser
• Galectin 3 MeSH
• Myeloid Differentiation Factor 88 MeSH
• NF-kappa B * MeSH
• NFKBIZ protein, human MeSH Browser
• Basic-Leucine Zipper Transcription Factors MeSH

BACKGROUND/AIM: Colorectal cancer is currently the third leading cause of cancer-related deaths and recently, alternative splicing has risen as its important regulator and potential treatment target. In the present study, we analyzed gene expression of the MBNL family of regulators of alternative splicing in various stages of colorectal cancer development, together with the MBNL-target splicing events in FOXP1 and EPB41L3 genes and tumor-related CD44 variants. MATERIALS AND METHODS: Samples of tumor tissue and non-malignant mucosa from 108 patients were collected. After RNA isolation and reverse transcription, the relative gene expression of a selected gene panel was tested by quantitative real-time PCR, followed by statistical analysis. RESULTS: MBNL expression was decreased in tumor tissue compared to non-tumor mucosa. In addition, lower expression was observed for the variants of FOXP1 and EPB41L3, while higher expression in tumor tissue was detected both for total CD44 and its cancer-related variants 3 and 6. Transcript levels of the MBNL genes were not found to be related to any of the studied clinicopathological characteristics. Multiple significant associations were identified in the target gene panel, including higher transcript levels of FOXP1 and CD44v3 in patients with distant metastases and connections between recurrence-free survival and altered levels of FOXP1 and CD44v3. CONCLUSION: Our results identified for the first-time deregulation of MBNL genes in colorectal cancer. Down-regulation of their transcripts in tumor tissue compared to matched non-tumor mucosa can lead to transition of alternative splicing patterns towards a less differentiated phenotype, which highlights the importance of alternative splicing regulation for tumor growth and propagation.

• Keywords
• Alternative splicing, MBNL regulatory factors, RNA-binding proteins, colorectal cancer,
• MeSH
• Alternative Splicing MeSH
• Cell Differentiation physiology   MeSH
• Adult MeSH
• Colorectal Neoplasms genetics   metabolism   pathology   MeSH
• Middle Aged MeSH
• Humans MeSH
• RNA-Binding Proteins biosynthesis   genetics   MeSH
• Aged MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• MBNL1 protein, human MeSH Browser
• RNA-Binding Proteins MeSH

Background and aims: The majority of colorectal cancers arise from detectable adenomatous or serrated lesions. Here we demonstrate how deregulated alternative splicing of CD44 gene in diseased colon mucosa results in downregulation of standard isoform of CD44 gene (CD44s) and upregulation of variant isoform CD44v8-10. Our aim is to show that upregulation of CD44v8-10 isoform is a possible marker of precancerous lesion in human colon. Methods: We analysed pairs of fresh biopsy specimen of large intestine in a cohort of 50 patients. We studied and compared alternative splicing profile of CD44 gene in colon polyps and adjoined healthy colon mucosa. We performed end-point and qRT PCR, western blotting, IHC staining and flow cytometry analyses. Results: We detected more than five-fold overexpression of CD44v8-10 isoform and almost twenty-fold downregulation of standard isoform CD44s in colon polyps compared to adjoined healthy tissue with p = 0.018 and p < 0.001 in a cohort of 50 patients. Our results also show that aberrant splicing of CD44 occurs in both biologically distinct subtypes of colorectal adenoma possibly in ESRP-1 specific manner. Conclusion: 92% of the colon polyp positive patients overexpressed CD44v8-10 isoform in their colon polyps while only 36% of them had positive fecal occult blood test which is currently a standard non-invasive screening technique. Impact: We believe that our results are important for further steps leading to application of CD44v8-10 isoform as a biomarker of colorectal precancerosis in non-invasive detection. Early detection of colon precancerosis means successful prevention of colorectal carcinoma.

• Keywords
• CD44 isoforms, RNA splicing, cancer markers, colon polyps, colorectal precancerosis,
• MeSH
• Hyaluronan Receptors genetics   metabolism   MeSH
• Colon metabolism   pathology   MeSH
• Colorectal Neoplasms diagnosis   genetics   metabolism   MeSH
• Humans MeSH
• Biomarkers, Tumor genetics   metabolism   MeSH
• Colonic Polyps metabolism   pathology   MeSH
• Prognosis MeSH
• Protein Isoforms MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Hyaluronan Receptors MeSH
• CD44 protein, human MeSH Browser
• Biomarkers, Tumor MeSH
• Protein Isoforms MeSH