Protocol to utilize fresh uncultured human lung tumor cells for personalized functional diagnostics

Peer reviewe

Keratins regulate colonic epithelial cell differentiation through the Notch1 signalling pathway

Keratins (K) are intermediate filament proteins important in stress protection and mechanical support of epithelial tissues. K8, K18 and K19 are the main colonic keratins, and K8-knockout (K8(-/-)) mice display a keratin dose-dependent hyperproliferation of colonic crypts and a colitis-phenotype. However, the impact of the loss of K8 on intestinal cell differentiation has so far been unknown. Here we show that K8 regulates Notch1 signalling activity and differentiation in the epithelium of the large intestine. Proximity ligation and immunoprecipitation assays demonstrate that K8 and Notch1 co-localize and interact in cell cultures, and in vivo in the colonic epithelial cells. K8 with its heteropolymeric partner K18 enhance Notch1 protein levels and activity in a dose dependent manner. The levels of the full-length Notch1 receptor (FLN), the Notch1 intracellular domain (NICD) and expression of Notch1 downstream target genes are reduced in the absence of K8, and the K8-dependent loss of Notch1 activity can be rescued with re-expression of K8/K18 in K8-knockout CRISPR/Cas9 Caco-2 cells protein levels. In vivo, K8 deletion with subsequent Notch1 downregulation leads to a shift in differentiation towards a goblet cell and enteroendocrine phenotype from an enterocyte cell fate. Furthermore, the K8(-/-) colonic hyperproliferation results from an increased number of transit amplifying progenitor cells in these mice. K8/K18 thus interact with Notch1 and regulate Notch1 signalling activity during differentiation of the colonic epithelium

The role of keratins in proliferation, differentiation and hypoxia signaling in colonic epithelia

Keratin (K) intermediate filament proteins are important in stress protection and in providing mechanical support to colonic epithelial cells. Deletion of K8 in mice (K8-/-) leads to colonic epithelial hyperproliferation, mistargeted membrane proteins, diarrhea and a Th2-type colitis. However, the reasons for hyperproliferation in K8-/- mice are not fully understood. In addition, it is not known if keratins modulate colonic epithelial cell differentiation, or if the colonic epithelial oxygen balance is affected by the deletion of keratins. In the studies included in this thesis, it was hypothezised that keratins are important in these cellular processes. Proliferation of colonic epithelial cells is followed by differentiation, which is highly regulated by Notch signaling. However, the regulation of Notch signaling in the colonic epithelium is not fully understood. Our aim was to investigate if keratins have an impact on epithelial cell differentiation and Notch signaling. By immunoprecipitation and proximity ligation assays, we identified K8/K18 as an interaction partner with Notch. Notch protein levels and target gene transcription were decreased in K8-/- mice and Caco-2 cells. However, the K8 dependent Notch phenotype could be rescued by reinsertion of K8/K18 in vitro. Furthermore, overexpression of a mutated form of K8 where serine 74 has been changed to alanine resulting in inactivation of phosphorylation on this site, increased Notch1 protein levels. Despite this, K8Ser74Ala could not rescue the expression of the Notch1 target gene Hey1 in K8-/- Crispr/Cas9 Caco-2 cells. Therefore, keratin phosphorylation could play a role in the link between K8 and Notch. In vivo, K8-/- mice exhibit an unbalanced colonic epithelial cell differentiation, with a shift towards the goblet cell fate, a phenotype that can be explained by a decrease in Notch signaling. K8-/- mice do not develop spontaneous tumors but express many hallmarks of cancer in their colonic epithelium. These include hyperproliferation and colonic inflammation, which both can be regulated by STAT3 signaling. In the second study, our aim was to investigate if the lack of keratins predisposes mice to colorectal cancer (CRC). K8-/- mice were dramatically more susceptible to induced CRC in a genetic and a chemical cancer model, compared to K8+/+ mice. This could be explained by an increase in the activity of STAT3 and levels of IL-22 after K8 deletion. Furthermore, investigation of proteins upstream of IL-22 revealed that K8 co-immunoprecipitaes pro-caspase 1 of the inflammasome and could thereby modulate cell inflammation and proliferation signaling in colonic epithelium. Hypoxia signaling pathways are active in the colonic epithelium and are important in the development of cancer. Using microarray analysis, several genes associated to hypoxia were altered in K8-/- colonic epithelial cells. As a result, our aim was to investigate if keratins participate in the hypoxia signaling pathway and if keratins are altered during acute hypoxia in colon epithelium. Decreased levels of hypoxia inducible transcription factor 1 alpha (Hif-1α) could be detected in K8-/- mice together with decreased activity of several Hif-1α target genes, such as VEGF-A2 and Bcl2. Deletion of K8 with Crispr/Cas9 from Caco-2 cells resulted in increased Hif-1α protein levels in acute hypoxia, with a robust increase in Hif-1α translocation to the nucleus of K8-/- cells compared to K8+/+ cells. This proposes that keratins participate in the regulation of the Hif-1α signaling pathway. Additionally, K19 and K20 were upregulated in acute colonic hypoxia in mice, suggesting that hypoxia is an additional stress where keratin levels are increased. In summary, keratins are novel regulators of colonic epithelial cell proliferation, differentiation, hypoxia mechanisms and can modify cell signaling proteins in epithelia

Increased number of proliferating cells in K8^+/- but unaltered anoikis of colonic crypts.

<p>A. and B. Mice were injected with BrdU (Bromodeoxyuridine, 5-bromo-2-deoxyuridine) i.p. 4 hours before sacrifice and proliferative cells were stained with anti-BrdU and quantified. Proliferating cells were significantly increased in both K8^<b>+/-</b> and K8^<b>-/-</b> mice DC (distal colon; A. b, c) and PC (proximal colon; A. e, f) compared to K8^<b>+/+</b> mice (A. a, d). Scale (a, d) = 50 μm. Brackets indicate the proliferative cell zone at the bottom of the crypt. B. The fraction of proliferative cells was quantified by counting the number of proliferative cells in relation to the total number of cells in the colonic crypts. Increased number of BrdU positive cells directly indicates the hyperproliferation in K8^<b>+/-</b> and K8^<b>-/-</b> colon. *** p<0.001. C. The level of apoptosis was assessed from K8^<b>+/-</b> and K8^<b>-/-</b> colon lysates by immunoblotting for cleaved caspase 7 (cCasp7) directly after excising from the mouse or after 1 hour of incubation at 37°C in cell culture medium. Hsc70 and caspase 7 (Casp7) were used as loading controls.</p

K8^+/- mice have decreased levels of colonic keratins and K8 mRNA.

<p>A. Total lysates of colon from K8^<b>+/+</b>, K8^<b>+/-</b> and K8^<b>-/-</b> mice (n = 3) were analyzed for the amount of K8, K18, K19 and K20 using SDS-PAGE and western blotting. Equal amount of protein was loaded to each well and Hsc70 was used as loading control. B. Keratin protein expression was quantified using ImageJ software. C. RT-PCR was performed to measure keratin mRNA levels in total RNA isolated from colon scrapings. Target genes were amplified using specific primers and KAPA probe Fast ABI Prism qPCR mix. Gene expression levels were normalized to the housekeeping gene β-actin. * = P < 0.05. Results are given as means ± SEM.</p

K8^+/- mice have increased crypt length.

<p>A. The K8^<b>+/-</b> baseline morphology was studied by H&E staining of the distal colon (DC) and proximal colon (PC) and the crypt lengths were measured (black arrows). B. Crypt lengths in PC and DC were measured from digital photographs of H&E stained colon sections (n = 3) taken with Zeiss Axiovert 200M microscope and processed using ImageJ software. The crypt lengths are expressed as mean length ± SD, calculated based on crypts from 3 animals / genotype.</p

Summary of the K8^+/- colonic phenotype.

<p>ND, not determined.</p><p>Summary of the K8^+/- colonic phenotype.</p