MicroRNA-375 Suppresses the Growth and Invasion of Fibrolamellar Carcinoma

Abstract

Background & Aims Fibrolamellar carcinoma (FLC) is a rare liver cancer that primarily affects adolescents and young adults. It is characterized by a heterozygous approximately 400-kb deletion on chromosome 19 that results in a unique fusion between DnaJ heat shock protein family member B1 (DNAJB1) and the alpha catalytic subunit of protein kinase A (PRKACA). The role of microRNAs (miRNAs) in FLC remains unclear. We identified dysregulated miRNAs in FLC and investigated whether dysregulation of 1 key miRNA contributes to FLC pathogenesis. Methods We analyzed small RNA sequencing (smRNA-seq) data from The Cancer Genome Atlas to identify dysregulated miRNAs in primary FLC tumors and validated the findings in 3 independent FLC cohorts. smRNA-seq also was performed on a FLC patient-derived xenograft model as well as purified cell populations of the liver to determine whether key miRNA changes were tumor cell–intrinsic. We then used clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (Cas9) technology and transposon-mediated gene transfer in mice to determine if the presence of DNAJB1-PRKACA is sufficient to suppress miR-375 expression. Finally, we established a new FLC cell line and performed colony formation and scratch wound assays to determine the functional consequences of miR-375 overexpression. Results We identified miR-375 as the most dysregulated miRNA in primary FLC tumors (27-fold down-regulation; P = .009). miR-375 expression also was decreased significantly in a FLC patient-derived xenograft model compared to 4 different cell populations of the liver. Introduction of DNAJB1-PRKACA by clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 engineering and transposon-mediated somatic gene transfer in mice was sufficient to induce significant loss of miR-375 expression ( P < .05). Overexpression of miR-375 in FLC cells inhibited Hippo signaling pathway proteins, including yes-associated protein 1 and connective tissue growth factor, and suppressed cell proliferation and migration ( P < .05). Conclusions We identified miR-375 as the most down-regulated miRNA in FLC tumors and showed that overexpression of miR-375 mitigated tumor cell growth and invasive potential. These findings open a potentially new molecular therapeutic approach. Further studies are necessary to determine how DNAJB1-PRKACA suppresses miR-375 expression and whether miR-375 has additional important targets in this tumor. Transcript profiling: GEO accession numbers: GSE114974 and GSE125602.

Single-Cell RNA Sequencing Identifies Yes-Associated Protein 1–Dependent Hepatic Mesothelial Progenitors in Fibrolamellar Carcinoma

“…Introducing miR-375 mimics into prFLC cells significantly suppressed YAP1 expression, reduced the expression of YAP1 target genes, and inhibited growth and migration of the cancer cells. 24 Finally, in the present study, similar responses were achieved by treating prFLC cells with verteporfin, a well-accepted pharmacologic inhibitor of YAP1. 25 YAP1 is known to maintain the proliferative and migratory capabilities of multipotent progenitors and, thus, antagonizes the terminal epithelial differentiation of such cells during development.…”

“…These partially reprogrammed FLC cells maintain expression of the FLC signature DNAJB1-PRKACA fusion ( Figure 3D). As previously reported, such prFLC cells express YAP1 and YAP1 target genes, 24 and thus prFLC cell line and the YAP1 inhibitor drug verteporfin 25 were used to test whether inhibiting YAP1 alters tumor-associated activities (Figure 3, E and F). Compared with vehicle, verteporfin treatment significantly reduced the rate of cell growth, colony formation, and migratory capacity of prFLC cells ( Figure 3F).…”

MicroRNAs Regulating Hippo-YAP Signaling in Liver Cancer

“…In PLC/PRF/5 and MHCC-97L HCC cell lines, ectopic expression of miR-375 downregulated the expression level of YAP protein and CTGF mRNA, which can be transcribed by activated YAP [ 57 ]. Recently, Dinh and Jewell et al [ 58 ] found that miR-375 is the most downregulated miRNA in primary fibrolamellar carcinoma (pFLC), a rare liver cancer that primarily affects adolescents and young adults, compared with nonmalignant human livers. The loss of miR-375 was induced by the presence of the DNAJB1-PRKACA fusion gene, a hallmark of FLC, although it is yet unknown how DNAJB1-PRKACA inhibits miR-375 expression [ 58 ].…”

“…Recently, Dinh and Jewell et al [ 58 ] found that miR-375 is the most downregulated miRNA in primary fibrolamellar carcinoma (pFLC), a rare liver cancer that primarily affects adolescents and young adults, compared with nonmalignant human livers. The loss of miR-375 was induced by the presence of the DNAJB1-PRKACA fusion gene, a hallmark of FLC, although it is yet unknown how DNAJB1-PRKACA inhibits miR-375 expression [ 58 ]. As in HCC cells, the overexpression of miR-375 in FLC cells inhibited YAP and CTGF, mitigating the proliferative and migratory ability of tumor cells [ 58 ].…”

<p>Curcumin Modifies Epithelial–Mesenchymal Transition in Colorectal Cancer Through Regulation of miR-200c/EPM5</p>

“…6,8 For instance, miRNAs from the miR-200 family repress EMT by directly targeting and downregulating ZEB1/ZEB1/ SNAIL/SLUG via miR-200-binding sites located within their 3′UTRs, resulting in enhanced E-cadherin expression and inhibition of cancer cell migration and motility. [12][13][14] On the contrary, theses EMT-TFs bind directly to the common promoter regions of the miR-200c family and cause transcriptional repression. 15 Therefore, the balance between miR-200c and EMT-TFs determines, at least in part, the status of epithelial or mesenchymal phenotype.…”