Loss of lncRNA LINC01056 leads to sorafenib resistance in HCC

Abstract

Background and aims: Sorafenib is a major nonsurgical option for patients with advanced hepatocellular carcinoma (HCC); however, its clinical efficacy is largely undermined by the acquisition of resistance. The aim of this study was to identify the key lncRNA involved in the regulation of the sorafenib response in HCC.

Materials and methods: A clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) single-guide RNA (sgRNA) synergistic activation mediator (SAM)-pooled lncRNA library was applied to screen for the key lncRNA regulated by sorafenib treatment. The role of the identified lncRNA in mediating the sorafenib response in HCC was examined in vitro and in vivo. The underlying mechanism was delineated by proteomic analysis. The clinical significance of the expression of the identified lncRNA was evaluated by multiplex immunostaining on a human HCC microtissue array.

Results: CRISPR/Cas9 lncRNA library screening revealed that Linc01056 was among the most downregulated lncRNAs in sorafenib-resistant HCC cells. Knockdown of Linc01056 reduced the sensitivity of HCC cells to sorafenib, suppressing apoptosis in vitro and promoting tumour growth in mice in vivo. Proteomic analysis revealed that Linc01056 knockdown in sorafenib-treated HCC cells induced genes related to fatty acid oxidation (FAO) while repressing glycolysis-associated genes, leading to a metabolic switch favouring higher intracellular energy production. FAO inhibition in HCC cells with Linc01056 knockdown significantly restored sensitivity to sorafenib. Mechanistically, we determined that PPARα is the critical molecule governing the metabolic switch upon Linc01056 knockdown in HCC cells and indeed, PPARα inhibition restored the sorafenib response in HCC cells in vitro and HCC tumours in vivo. Clinically, Linc01056 expression predicted optimal overall and progression-free survival outcomes in HCC patients and predicted a better sorafenib response. Linc01056 expression indicated a low FAO level in HCC.

Conclusion: Our study identified Linc01056 as a critical epigenetic regulator and potential therapeutic target in the regulation of the sorafenib response in HCC.

Keywords: CRISPR/Cas9 screens; Fatty acid oxidation; Hepatocellular carcinoma; LINC01056; PPARα; Sorafenib.

Fig. 1

CRISPRa screens identified Linc01056 as a regulator of sorafenib response in HCC cells. (a) Flowchart of CRISPRa screening on MHCC97L cells. (b) 7-day treatment of sorafenib significantly suppressed the proliferation ability of MHCC97L cells. (c) Violin plot of the normalized read count of the sequencing result. The average count of the sorafenib-treated group was slightly higher. (d) Volcano plot of the changes of expression of lncRNAs upon 7-day exposure of sorafenib in HCC cells. (e) Linc01056 was one of most downregulated lncRNA upon acquisition of sorafenib resistance in HCCs. The acquired sorafenib resistance MHCC97L cells were obtained by prolonged seven-day 5 µM sorafenib treatment. (f) In sorafenib-resistant HCC tumour cells, the expression of Linc01056 was potently suppressed. (g) HCC cells were exposed to 1-, 7- and 12-day sorafenib at the dose of 10µM, it was observed that 1-day treatment of sorafenib induced Linc01056 expression, while long-term treatment of sorafenib suppressed Linc01056 expression. (h) Cell viability of MHCC97L were measured against sorafenib treatment for wild-type or 1-day or 7-day treated cells. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 2

Linc01056 was required for sorafenib response in HCC. (a) Knockdown of Linc01056 in MHCC97L and PLC/PRF/5 cells by shRNA interference. (b) Knockdown of Linc01056 increased cell viability in sorafenib-treated HCC cells. (c) Rescue of Linc01056 potentiated the resistant HCC cells to sorafenib treatment. (d) Knockdown of Linc01056 improved colonic formation of HCC cells in the presence of sorafenib. (e) Knockdown of Linc01056 reduced sorafenib-induced apoptosis in HCC cells. Stable knockdown of Linc01056 (f) accelerated in vivo tumour growth and (g) end-point tumour size, (h) reduced expression of cell apoptosis marker cleaved caspase-3 and (i) promoted the cell proliferation marker Ki67. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3

Linc01056 knockdown induced metabolic shift towards fatty acid oxidation. (a). Proteomic analysis on sorafenib-treated MHCC97L cells with or without Linc01056 knockdown. Pathway enrichment on differential gene expression showed that (b) increased genes enriched in pathways related to fatty acid oxidation, while c) reduced genes enriched in pathways related to glycolysis/gluconeogenesis. (d) Changes in expression of FAO-related proteins upon Linc01056 knockdown. (e) GSEA analysis showed enrichment of genes related to FAO. (f) Linc01056 knockdown maintained cellular ATP level upon sorafenib treatment in HCC cells. Knockdown of Linc01056 (g) increased the OCR and (h) decreased the ECAR in HCC cells in the presence of sorafenib. Linc01056 knockdown (i) increased mitochondrial ROS, and (j) maintained the basal respiratory and (k) maximal respiratory capacity in sorafenib-treated HCC cells. (l) Linc01056 increased fatty acid storage in sorafenib-treated HCC cells.(m) Linc01056 knockdown increased content of C16 intermediates of fatty acid in sorafenib-treated HCC cells. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4

FAO inhibition sensitised Linc01056-knockdowned HCC cells upon sorafenib treatment. (a) Knockdown of Linc01056 resulted in an increase in fatty acid uptake in HCC cells, which was further augmented under sorafenib treatment. FAO suppression by etomoxir (b) increased cytotoxicity of sorafenib, (c) suppressed colonic capacity, and (d) induced apoptosis in HCC cells with Linc01056 knockdown. FAO suppression by etomoxir (e & f) reduced in vivo tumour growth and (g & h) end-point tumour size, (i) increased expression of cell apoptosis marker cleaved caspase-3 and reduced the cell proliferation marker Ki67 in sorafenib-treated in vivo HCC tumours without Linc01056 knockdown. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 5

Linc01056 interfered PPARα transcription activity-associated FAO activation. (a) Linc01056 knockdown induced binding of PPARα on to the promoter region of FAO-related genes. (b) Linc01056 knockdown did not change the protein expression of PPARα in HCC cells. (c) Linc01056 located in the cytoplasm of MHCC97L cells with or without sorafenib treatment. (d) Linc01056 specifically bound to PPARα but not PPARγ or FOXO1. (e) PPARα specifically bound to Linc01056 but not other lncRNA like MALAT1. Suppression of PPARα activity by GW6471 (f) reversed the increase of OCR and (g) decrease of ECAR in Linc01056-knockdown HCC cells upon sorafenib treatment. GW6471 inhibited (h) production of mitochondrial ROS, (i) intracellular ATP, (j) cell viability, (k) colonic capacity, and (l) apoptosis of Linc01056-knockdown HCC cells upon sorafenib treatment. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 6

Clinicopathological significance of Linc01056 in HCC. (a) representative image of multiplex staining on tissue microarray of human HCC samples. (b) Expression of Linc01056 was significantly downregulated in HCC compared to adjacent liver. High expression of Linc01056 predicted the good prognosis of (c) overall survival and (d) progression-free survival of HCC patients. (e) Expression of Linc01056 was negatively correlated with FAO marker CPT1 in human HCC samples. (f) Expression of Linc01056 was positively correlated with the cytoplasmic localization of PPARα. (g) Expression of Linc01056 was positively correlated with the sorafenib sensitivity marker p-Erk. *p < 0.05, ***p < 0.001