Chemical, Molecular, and Single-nucleus Analysis Reveal Chondroitin Sulfate Proteoglycan Aberrancy in Fibrolamellar Carcinoma

Abstract

Fibrolamellar carcinoma (FLC) is an aggressive liver cancer with no effective therapeutic options. The extracellular environment of FLC tumors is poorly characterized and may contribute to cancer growth and/or metastasis. To bridge this knowledge gap, we assessed pathways relevant to proteoglycans, a major component of the extracellular matrix. We first analyzed gene expression data from FLC and non-malignant liver tissue (n=27) to identify changes in glycosaminoglycan (GAG) biosynthesis pathways and found that genes associated with production of chondroitin sulfate, but not other GAGs, are significantly increased by 8-fold. We then implemented a novel LC-MS/MS based method to quantify the abundance of different types of GAGs in patient tumors (n=16) and found that chondroitin sulfate is significantly more abundant in FLC tumors by 6-fold. Upon further analysis of GAG-associated proteins we found that versican (VCAN) expression is significantly up-regulated at the mRNA and protein levels, the latter of which was validated by immunohistochemistry. Finally, we performed single-cell assay for transposon-accessible chromatin-sequencing on FLC tumors (n=3), which revealed for the first time the different cell types in FLC tumors and also showed that VCAN is likely produced not only from FLC tumor epithelial cells but also activated stellate cells. Our results reveal a pathologic aberrancy in chondroitin (but not heparan) sulfate proteoglycans in FLC and highlight a potential role for activated stellate cells.

DNAJB1-PRKACA fusion protein-regulated LINC00473 promotes tumor growth and alters mitochondrial fitness in fibrolamellar carcinoma

“…Despite the clear upregulation of LINC00473 in FLC revealed by bulk tissue analysis, it is unknown what cell type(s) within the tumor drive this signal. To address this knowledge gap, we first analyzed our recently published single-nucleus ATAC-seq data on one matched set of FLC primary tumor, metastatic tumor, and NML tissue [ 60 ]. By examining open chromatin signal on a genome-wide scale, we could infer active transcription at gene loci with single nucleus resolution.…”

“…snATAC-seq : Single-nucleus ATAC (snATAC) analysis was performed as previously described [ 60 ]. Data generated from our snATAC-seq studies were previously published and are available at the Gene Expression Omnibus through accession number GSE202315.…”

DNAJB1-PRKACA fusion protein-regulated LINC00473 promotes tumor growth and alters mitochondrial fitness in fibrolamellar carcinoma

“…Despite the clear upregulation of LINC00473 in FLC revealed by bulk tissue analysis, it is not known what cell type(s) within the tumor drive this signal. To address this knowledge gap, we first analyzed our recently published single-nucleus ATAC-seq data on one matched set of FLC primary tumor, metastatic tumor and NML tissue 60 . By examining open chromatin signal on a genome-wide scale, we were able to infer active transcription at gene loci with single nucleus resolution.…”