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Down-regulation of collagen hydroxylation in colorectal liver metastasis

ABSTRACT: Collagen, a major component of the extracellular matrix, is significantly upregulated in colorectal liver metastasis (CRLM) compared to liver tissue. Expression levels of specific collagen types in CRLM resemble those in colorectal cancer (CRC) and colon tissue. We investigated whether the collagen hydroxylation pattern from the primary tumor also migrates with the metastatic tumor. The degree of collagen alpha-1(I) hydroxylation in colon, CRC, liver, and CRLM tissue in the same individuals (n=14) was studied with mass spectrometry. The degree of hydroxylation was investigated in 36 collagen alpha-1(I) peptides, which covered 54% of the triple helical region. The average degree of hydroxylation in liver tissue was similar to that in colon tissue. The overall degree of hydroxylation was significantly lower (9% +/- 14%) in CRC tissue and also significantly lower (12% +/-22%) in CRLM tissue compared to colon or liver tissue. Still, in previous research of liver and CRLM enzymes involved in collagen hydroxylation were found to be upregulated, whereas P4HB (4-prolyl hydroxylase beta unit) was downregulated. Furthermore, 11 peptides are present with a specific number of hydroxylations that are significantly different between CRLM and liver tissue; these peptides could be candidates for the detection of CRLM. For one of these eleven peptides, a matching naturally occurring peptide in urine has been identified as being significantly different between patients suffering from CRLM and healthy controls. We conclude that the degree of hydroxylation in CRC and CRLM tumor tissue is in general significantly downregulated in comparison to healthy colon and liver tissue. The hydroxylation pattern in CRLM resemble partly the pattern in liver, primary colorectal cancer and colon.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Colon

DISEASE(S): Colon Cancer

SUBMITTER: Nick van Huizen

LAB HEAD: T.M. Luider

PROVIDER: PXD015015 | Pride | 2020-09-16

REPOSITORIES: Pride

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Project description:The liver is the most common site for colorectal cancer (CRC) metastasis and new tissue culture models are needed to study hepatic metastasis from CRC as none of the current models mimic the biological, biochemical and structural characteristics of the metastatic microenvironment. Decellularization provides a novel approach for the study of cancer extracellular matrix (ECM) as decellularized scaffolds retained their tissue-specific features and biological properties. In the present study we created a 3D model of CRC and matched CRC liver metastasis (CRLM) using patient-derived decellularized ECM scaffolds seeded with HT-29 CRC cell line. Here we show increased HT-29 cell proliferation and migration capability when cultured in cancer-derived scaffolds compared to same-patient healthy colon and liver tissues. CRLM scaffolds also induced activation of epithelial-mesenchymal transition (EMT) with cancer cells showing loss of E-cadherin expression and increased Vimentin expression. EMT was confirmed by gene expression profiling, with the most represented biological processes in CRLM-seeded scaffolds involving demethylation, deacethylation, cellular response to stress metabolic processes, response to oxygen level and to starvation. The complex 3D culture environment reduced the HT-29 cell response to anti-CRC drug 5-FU when used at standard IC50 determined in 2D cultures. In conclusion, our 3D culture system with patient-derived tissue-specific decellularized ECM better recapitulates the metastatic microenvironment compared to conventional 2D culture conditions and represents a relevant approach for the study of liver CRC metastasis formation and progression. Transcriptomic analysis was performed comparing the global gene expression profiles of HT29 cells grown on CRLM and HL scaffolds. The transcriptomic profile of each 3D subtype was compared with HT29 cells grown in plastic. Hierarchical clustering analysis revealed that the gene expression signature of recellularized CRLM scaffolds was more comparable to recellularized HL scaffolds, than to HT29 cells grown in 2D. Gene set enrichment analysis (GSEA) of the differently expressed genes (DEG) up-regulated in recellularized CRLM scaffolds compared to HT29 grown in 2D revealed that genes involved in demethylation, deacethylation and metabolic process belong to the most enriched biological pathways in repopulated-CRLM scaffolds. Comparing the DEG between recellularized CRLM scaffolds vs HT29 grown in 2D with a series of a-priori defined gene-set, called Hallmark, we obtained concordant results with the “HYPOXIA” and “EPITHELIAL_MESENCHYMAL_TRANSITION” pathway, enriched in recellularized CRLM, supporting the idea that 3D culture models are the ability to re-create a complex environment in terms of oxygen tension, nutrients, and metabolic gradient, similarly to the in vivo environment.

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Down-regulation of collagen hydroxylation in colorectal liver metastasis

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