The human vault RNA enhances tumorigenesis and chemoresistance through the lysosome in hepatocellular carcinoma

Abstract

The small non-coding VTRNA1-1 (vault RNA 1-1) is known to confer resistance to apoptosis in several malignant cell lines and to also modulate the macroautophagic/autophagic flux in hepatocytes, thus highlighting its pro-survival role. Here we describe a new function of VTRNA1-1 in regulating in vitro and in vivo tumor cell proliferation, tumorigenesis and chemoresistance. Knockout (KO) of VTRNA1-1 in human hepatocellular carcinoma cells reduced nuclear localization of TFEB (transcription factor EB), leading to a downregulation of the coordinated lysosomal expression and regulation (CLEAR) network genes and lysosomal compartment dysfunction. We demonstrate further that impaired lysosome function due to loss of VTRNA1-1 potentiates the anticancer effect of conventional chemotherapeutic drugs. Finally, loss of VTRNA1-1 reduced drug lysosomotropism allowing higher intracellular compound availability and thereby significantly reducing tumor cell proliferation in vitro and in vivo. These findings reveal a so far unknown role of VTRNA1-1 in the intracellular catabolic compartment and describe its contribution to lysosome-mediated chemotherapy resistance.Abbreviations: ATP6V0D2: ATPase H+ transporting V0 subunit d2; BafA: bafilomycin A1; CLEAR: coordinated lysosomal expression and regulation; CQ: chloroquine; DMSO: dimethyl sulfoxide; GST-BHMT: glutathionine S-transferase N-terminal to betaine-homocysteine S-methyltransferase; HCC: hepatocellular carcinoma; LAMP1: lysosomal associated membrane protein 1; LLOMe: L-leucyl-L-leucine methyl ester; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MAPK: mitogen-activated protein kinase; MITF: melanocyte inducing transcription factor; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ncRNA: non-coding RNA; RNP: ribonucleoprotein; SF: sorafenib; SQSTM1/p62: sequestosome 1; STS: staurosporine; tdRs: tRNA-derived RNAs; TFE3: transcription factor binding to IGHM enhancer 3; TFEB: transcription factor EB; vtRNA: vault RNA transcript.

Keywords: Chemoresistance; lysosome; non-coding RNA; tumorigenesis; vault RNA; vtRNA1-1.

Figure 1.

VTRNA1-1/vtRNA1-1 plays a crucial role in tumor cell proliferation and tumorigenesis. (A) Total RNA was extracted from Huh-7 cells and analyzed by northern blotting to confirm successful VTRNA1-1 complementation in Huh-7 KO cells. RNA5-8S5 (RNA, 5.8S ribosomal 5) serves as internal loading control. (B) Mean ± SD relative proliferation of Huh-7 WT, KO and complementation cells was measured with the MTT assay. Values were normalized to day 1, n = 3. (C) Three-day cell proliferation (cells/ml) by automated cell counter. Values were normalized to day 1, n = 5 ± SD. (D) Clonogenic assay: data are expressed as the mean values of three independent experiments ± SD. (E) Tumor volume ± SEM of Huh-7 subcutaneous xenografts mouse model (WT n = 4 and KO n = 5). (F) Huh-7 subcutaneous xenografts immunohistochemistry for MKI67. Counterstain: hematoxylin. Scale bar: 50 μm. The graphs show the mean percentage ± SD of MKI67-positive cell/area per tumor/mouse (five random fields were used for the analysis per tumor/mouse). P values < 0.05 were considered statistically significant and are indicated as follows: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.

Figure 2.

Defective lysosome function impairs autophagy-mediated clearance in VTRNA1-1 KO cells. (A) IC₅₀ values of Huh-7 WT and KO cells after 24 h of staurosporine treatment. (B) Representative immunoblots for PARP1 (FL, full-length) and ACTB/β-actin in Huh-7 and HeLa cells after Staurosporine IC₃₀ treatment (24 h, 5 μM). HeLa cells were used as control for apoptosis induction. (C) Representative immunoblots for LAMP1, SQSTM1/p62, LC3B-I, LC3B-II (o. e., overexposed) and ACTB/β-actin in Huh-7 cells under complete culture medium (10% FBS – Ctrl) and treatment with chloroquine (CQ – 20 μM, 4 h) or bafilomycin A₁ (BafA, 100 nM, 4 h). (D) Top panel, mean ± SD of lysosomal pH values measured by flow cytometry in Huh-7 pre-incubated in culture medium supplemented with FITC-dextran (0.1 mg/mL, 72 h) followed starvation (0.1% FBS) for 24 h and high starvation (HS, with low glucose, without amino acids and FBS) for 6 h. n = 3. Bottom panel, illustrative histograms from flow cytometry showing the shift of FITC-dextran emission wavelength. (E) Representative immunoblots for GST-BHMT of total lysate obtained from Huh-7 cells transiently transfected with the GST-BHMT construct and either maintained in complete culture media or starving media supplemented with leupeptin and E64d and without essential amino acids and FBS for 6 h. Red Ponceau staining served as loading control. P values < 0.05 were considered statistically significant and are indicated as follows: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.

Figure 3.

VTRNA1-1/vtRNA1-1 plays a key role in TFEB nuclear translocation and TFEB-driven CLEAR network genes expression. (A) Representative immunoblots for p-MTOR, total MTOR and GAPDH in Huh-7 WT and VTRNA1-1 KO cells under normal culture conditions (10% FBS). (B) Left, representative immunoblots for p-MAPK1/ERK2-MAPK3/ERK1, total MAPK1/3 and ACTB/β-actin in Huh-7 WT, VTRNA1-1 KO and complementation cells under normal culture conditions (10% FBS). Right, quantification of p-MAPK1/3 obtained by normalizing the p-MAPK1/3 and total MAPK1/3 levels by ACTB/β-actin followed by the ratio of p-MAPK1/3 over total MAPK1/3. Values are expressed as the mean values of three (for WT and KO) or two (for complementation) independent experiments ± SD normalized to WT level. (C) Left, representative nuclear/cytoplasm fractionation immunoblots for TFEB, TUBA/α-tubulin, histone H3 in Huh-7 WT and VTRNA1-1 KO cells under high starvation for 3 h. Right, quantification of TFEB nuclear levels normalized to histone H3. Values are expressed as the mean values of three independent experiments ± SD. (D) Representative nuclear-cytoplasm fractionation immunoblots for TFE3, GAPDH, histone H3 in Huh-7 WT and VTRNA1-1 KO cells under high starvation for 3 h. (E) Real-time qPCR data (mean ± SEM) of TFEB, LAMP1, CTSD, CLCN7 and ATP6V0D2 mRNA levels in Huh-7 VTRNA1-1 KO cells grown under complete medium (10% FBS – Ctrl) and starving culture conditions (0.1% FBS) for 24 h, normalized to Huh-7 WT (n = 3) and VTRNA1-1 KO-derived tumors normalized to WT-transplanted ones (WT n = 4, KO n = 9). (F) Real-time qPCR mean ± SEM data of SQSTM1/p62 in Huh-7 KO cells and derived tumors normalized to Huh-7 WT and WT-transplanted tumors respectively. P values < 0.05 were considered statistically significant and are indicated as follows: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.

Figure 4.

Removal of VTRNA1-1 potentiates the cytotoxicity of sorafenib in vitro and in vivo. (A) Mean ± SD relative viability of Huh-7 WT, KO and complementation cells, after sorafenib IC₃₀ treatment (24 h, 14 μM) measured with the MTT assay. Values were normalized to untreated cells, n = 3. (B) Representative immunoblots for p-MAPK1/3, total MAPK1/3 and GAPDH in Huh-7 WT and VTRNA1-1 KO cells in presence or absence of sorafenib (5 μM) as indicated. (C) Tumor volume of Huh-7-transplanted xenografts mice treated with vehicle and sorafenib (SF – 100 mg/kg – daily oral gavage) for 14 days (SF n = 6/group; vehicle WT n = 4, KO n = 5). P values < 0.05 were considered statistically significant and are indicated as follows: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.

Figure 5.

Model for lysosome and autophagy mediated clearance regulation. In VTRNA1-1/vtRNA1-1 WT cells low MAPK1/3 phosphorylation level allows TFEB nuclear translocation leading to CLEAR network genes expression and ensuring intracellular catabolic compartment stability and function (left). Reduced VTRNA1-1 levels (right) lead to the hyperphosphorylation of MAPK1/3 and TFEB, and consequently to its reduced nuclear translocation, leading to a downregulation of the CLEAR network genes and lysosomal compartment dysfunction.