Proto-oncogene KRAS, GTPase (KRAS) is one of the most intensively studied oncogenes in cancer research. Although several mouse models allow for regulated expression of mutant KRAS, selective isolation and analysis of transforming or tumor cells that produce the KRAS oncogene remains a challenge. In our study, we present a knock-in model of oncogenic variant KRASG12D that enables the "activation" of KRASG12D expression together with production of red fluorescent protein tdTomato. Both proteins are expressed from the endogenous Kras locus after recombination of a transcriptional stop box in the genomic DNA by the enzyme flippase (Flp). We have demonstrated the functionality of the allele termed RedRas (abbreviated KrasRR) under in vitro conditions with mouse embryonic fibroblasts and organoids and in vivo in the lung and colon epithelium. After recombination with adenoviral vectors carrying the Flp gene, the KrasRR allele itself triggers formation of lung adenomas. In the colon epithelium, it causes the progression of adenomas that are triggered by the loss of tumor suppressor adenomatous polyposis coli (APC). Importantly, cells in which recombination has successfully occurred can be visualized and isolated using the fluorescence emitted by tdTomato. Furthermore, we show that KRASG12D production enables intestinal organoid growth independent of epidermal growth factor (EGF) signaling and that the KRASG12D function is effectively suppressed by specific inhibitor MRTX1133.

• Keywords
• Colon cancer, Gene targeting, Intestinal organoids, KRAS oncogene, Lung cancer, MRTX1133 inhibitor,
• MeSH
• Red Fluorescent Protein * MeSH
• DNA Nucleotidyltransferases genetics   metabolism   MeSH
• Gene Knock-In Techniques MeSH
• Luminescent Proteins * genetics   metabolism   MeSH
• Disease Models, Animal MeSH
• Mice, Transgenic MeSH
• Mice MeSH
• Lung Neoplasms genetics   pathology   MeSH
• Proto-Oncogene Proteins p21(ras) * genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Red Fluorescent Protein * MeSH
• DNA Nucleotidyltransferases MeSH
• Hras protein, mouse MeSH Browser
• Luminescent Proteins * MeSH
• Proto-Oncogene Proteins p21(ras) * MeSH

Decreased human epididymis protein 4 (HE4) plasma levels were reported in cystic fibrosis (CF) patients under CFTR potentiator ivacaftor therapy, which inversely correlated with lung function improvement. In this study, we investigated whether HE4 expression was affected via modulation of CFTR function in CF bronchial epithelial (CFBE) cells in vitro. HE4 protein levels were measured in the supernatants of CFBE 41o- cells expressing F508del-CFTR or wild-type CFTR (wt-CFTR) after administration of lumacaftor/ivacaftor or tezacaftor/ivacaftor, while HE4 expression in CFBE 41o- cells were also analyzed following application of adenylate cyclase activators Forskolin/IBMX or CFTRinh172. The effect of all of these compounds on CFTR function was monitored by the whole-cell patch-clamp technique. Induced HE4 expression was studied with interleukin-6 (IL-6) in F508del-CFTR CFBE 41o- cells under TNF-α stimulation for 1 h up to 1 week in duration. In parallel, plasma HE4 was determined in CF subjects homozygous for p.Phe508del-CFTR mutation receiving lumacaftor/ivacaftor (Orkambi®) therapy. NF-κB-mediated signaling was observed via the nuclear translocation of p65 subunit by fluorescence microscopy together with the analysis of IL-6 expression by an immunoassay. In addition, HE4 expression was examined after NF-κB pathway inhibitor BAY 11-7082 treatment with or without CFTR modulators. CFTR modulators partially restored the activity of F508del-CFTR and reduced HE4 concentration was found in F508del-CFTR CFBE 41o- cells that was close to what we observed in CFBE 41o- cells with wt-CFTR. These data were in agreement with decreased plasma HE4 concentrations in CF patients treated with Orkambi®. Furthermore, CFTR inhibitor induced elevated HE4 levels, while CFTR activator Forskolin/IBMX downregulated HE4 in the cell cultures and these effects were more pronounced in the presence of CFTR modulators. Higher activation level of baseline and TNF-α stimulated NF-κB pathway was detected in F508del-CFTR vs. wt-CFTR CFBE 41o- cells that was substantially reduced by CFTR modulators based on lower p65 nuclear positivity and IL-6 levels. Finally, HE4 expression was upregulated by TNF-α with elevated IL-6, and both protein levels were suppressed by combined administration of NF-κB pathway inhibitor and CFTR modulators in CFBE 41o- cells. In conclusion, CFTR dysfunction contributes to abnormal HE4 expression via NF-κB in CF.

• Keywords
• CFTR modulator, HE4, bronchial epithelial cell, cystic fibrosis, inflammation,
• Publication type
• Journal Article MeSH

Neoplastic growth is frequently associated with genomic DNA methylation that causes transcriptional silencing of tumor suppressor genes. We used a collection of colorectal polyps and carcinomas in combination with bioinformatics analysis of large datasets to study the expression and methylation of Hypermethylated in cancer 1 (HIC1), a tumor suppressor gene inactivated in many neoplasms. In premalignant stages, HIC1 expression was decreased, and the decrease was linked to methylation of a specific region in the HIC1 locus. However, in carcinomas, the HIC1 expression was variable and, in some specimens, comparable to healthy tissue. Importantly, high HIC1 production distinguished a specific type of chemotherapy-responsive tumors.

• Publication type
• Journal Article MeSH