Glycolysis-related metabolites and genes were highly expressed in HCC cases with low miR-192 expression
To investigate the metabolic feature in HCCs with miR-192 silencing, we used HCC Cohort 1 with available metabolome and transcriptome data (Fig. 1a). An integration analysis of miR-192 with the global metabolome in tumor tissues from 22 HCC patients showed that 17 metabolites were significantly correlated with miR-192 (|r-value| >0.4, Fig. 1b, Supplementary Table S3). Among them, 7 metabolites presented |r-value| >0.5 and three of them were glycolysis-related metabolites, i.e., G6P, F6P, and NADPH. Meanwhile, 652 genes were significantly correlated with miR-192 with |r-value| >0.4, revealed by an integration analysis of miR-192 with mRNA transcriptome in tumor tissues from 176 HCC patients (Fig. 1c). KEGG pathway analysis using these genes displayed 13 enriched metabolomic features (p<0.001), among which three of them related to glycolysis and its related pathway. These results suggest an altered glycolytic feature in HCC cases with low miR-192 expression.
Available glycolysis-related metabolites and genes in our profiling data (Fig. 1d) were then compared between HCCs with high miR-192 levels (termed HCC192High) and HCCs with low miR-192 levels (termed HCC192Low), based on the miR-192 median cut-off in HCC tumors. Levels of G6P, F6P, and NADPH were significantly higher in tumors from HCC192Low patients than HCC192High patients, while as a control there was no difference in their non-tumor tissues (Fig. 1e, Supplementary Fig. S1a). Consistently, many genes coding key enzymes in glycolysis such as GLUT1, HK2, PFKFB3, PFKP, and PKM2 were significantly upregulated in HCC192Low tumors compared to HCC192High tumors (Fig. 1e) but showed negligible alteration in their non-tumor tissues (Supplementary Fig. S1b). MCT1 was used as a negative control due to its main role in lactate import, but not in glycolysis[23, 24]. All of these demonstrate a hyperglycolytic metabolic feature in HCC cases with low miR-192 level.
We further investigated the hyperglycolytic feature in CSC+ HCC cases, i.e., cases with the top quartile expression of CSC biomarker as previously defined . The hierarchical clustering analysis with glycolytic genes in Cohort 1 revealed two HCC subgroups presenting different expression levels of glycolytic genes. Consistently in HCC subgroup with high expression levels of glycolysis-related genes, miR-192 level was low while various groups of CSC+ HCC cases were enriched (Fig. 1d). Statistical analysis also presented that glycolytic genes expressed significantly higher levels in various groups of CSC+ HCCs than CSC- HCCs, while this effect was not noticed from comparisons of their non-tumor tissues (Supplementary Fig. S1c-d). Comparable data were observed in Cohort 2 with 372 HCC patients that significantly high levels of glycolytic genes presented in HCCs192Low and in multiple groups of CSC+HCCs (Supplementary Fig. S1e). Together, these data indicated that the hyperglycolytic feature presented in various groups of CSC+HCCs with low level of miR-192.
HCC cells with miR-192 loss were hyperglycolytic.
We next investigated roles of miR-192 in regulating glycolysis. Lentivirus miRZip-192 was used to reduce miR-192 activity in Huh7 cells (Supplementary Fig. S2a), as we have done previously. Huh7 cells with suppressed miR-192 by miRZip-192 exhibited the overall increased expression of glycolytic genes (Supplementary Fig. S2a), a distinctly increased ECAR and a reduced OCR (Fig. 1g). The extracellular acid produced by cells is derived from lactate produced by glycolysis and CO2 produced during respiration. OCR is mainly representing mitochondrial respiration. Therefore, the increased ECAR in Huh7 cells with suppressed miR-192 was mainly due to lactate produced from glycolysis but not CO2 from mitochondrial respiration. Consistently, overexpressed miR-192 in HLF and HLE cells generally reduced the expression levels of glycolytic genes (Supplementary Fig. S2b) and lowered the extracellular acid production from glycolysis as shown by a reduced ECAR but an increased OCR (Fig. 1h). These results demonstrate an important role of miR-192 in operating a Warburg-like effect in HCC cells.
To better elucidate the role of miR-192 in regulating glycolysis, we established two miR-192 knock-out (termed 192KO) human HCC cell lines and miR-192 was undetected in HLE-192KO and HLF-192KO cells (Fig. 2a). As a control, the expression of miR-194, 109 nucleotides away from mir-192, was not affected. As we expected, HLF-192KO cells displayed significantly increased CSC features, such as increased populations of CD44+, CD24+ and EpCAM+ CSCs (Fig. 2b, Supplementary Fig. S2c); increased mRNA levels of multiple CSC biomarkers and reduced expression of a differentiation-related gene CYP1A2 (Supplementary Fig. S2d); and enlarged and more spheroid formation (Supplementary Fig. S2e). HLE-192KO cells displayed the increased CSC features at a moderate level (Fig. 2b, Supplementary Fig. S2c-d). Consistently, these two 192KO lines also showed the hyperglycolytic features. Many key enzyme proteins in glycolysis pathway were more highly expressed in 192KO cells than in wild-type cells (Fig. 2c). Both 192KO lines also exhibited increased ECARs but decreased OCRs (Fig. 2d), indicating that miR-192 loss largely increased the glycolysis-related extracellular acidification.
Furthermore, overexpressed miR-192 in HLF-192KO cells significantly reduced the CSC features and the lactate accumulation in culture medium by miR-192 loss (Fig. 2e). As a control, intracellular lactate remained no change. Meanwhile, following culture time, lactate accumulation in the medium was gradually increased and significantly higher in both HLF-192KO cells in HLE-192KO cells compared with that in their corresponding wild-type cells, which was lowered by overexpressed miR-192 (Fig. 2f). It could also be visualized via the orange/yellow medium of 192KO cells (referring to a lower pH value) vs. the pink medium of wild-type cells at 72 hours after seeding. Consistent data were obtained in detection of metabolites using non-targeted metabolomics in HLF cells with different expression of miR-192-5p in both internal cells and culture medium (Supplementary Fig S2f). Together, miR-192 loss in HCC cells led to a hyperglycolytic feature.
HCC cells with miR-192 loss had high glucose consumption
We further examined that glucose consumption among HCC cells with different levels of miR-192 and between HCC cells and their surrounding cells in the liver. As shown in Fig. 3a, both HLF-192KO and HLE-192KO cells exhibited significantly higher glucose consumption than HCC cells overexpressing miR-192. In Huh7 cells, suppressing miR-192 by miRZip-192 increased their glucose usage (Supplementary Fig. S3a). Consistently, 192KO HCC cells were more sensitive after exposure to 2-DG, a glucose analog, as shown by the significantly reduced cell viability compared to HCC cells with miR-192 expression (Fig. 3b). Comparable data were obtained in HuH7 cells (Supplementary Fig. S3b).
In co-culture systems of HCC cells with environmental non-HCC cells including LX2, HL7702, and THP1, we further compared the glucose uptake of HCC cells with different miR-192 level and non-HCC cells via 2-NBDG uptake assay. HLF HCC cells infected with pmiR-ctrl/RFP and pmiR-192/RFP lentiviruses were used, which showed that red fluorescent labeling efficiency is nearly 100% (Supplementary Fig. S3c). In this system, with/without co-culturing with other cells, HLF-192KO cells consistently showed higher 2-NBDG uptakes than the wild-type cells (Fig. 3c-d). In contrast, LX2 and HL7702 co-cultured with HLF-192KO cells presented lower 2-NBDG uptakes compared to those co-cultured with HLF-WT cells (Fig. 3d). Moreover, forced-expression of miR-192 in HLF-192KO cells reduced the 2-NBDG uptake in HLF cells but increased the 2-NBDG uptake in LX2 and HL7702 cells in the co-culture system. The 2-NBDG uptake alteration was not noticed in THP1 from our co-culture system (Fig. 3d, Supplementary Fig. S3d). Similar data were noticed in HLE cells as well as in the co-culture assay of HLE with LX2 and HL7702 cells (Supplementary Fig. S3e-f). These results demonstrate that HCC cells with loss of miR-192 actively utilize glucose from their environment to ensure a hyperglycolysis status.
Three important glycolytic regulators were miR-192 bona fide targets and also contributed to stemness features of HCC cells
To investigate the molecular mechanisms of miR-192 regulating glycolysis flow, we assessed genes negatively correlated with miR-192 in 176 HCC cases (r<-0.3) and significantly up-regulated in HCC192Low tumors than in HCC192High tumors (log2fold >0.2, p<0.01). Among these 554 genes, two main gene-groups were noticed. One group contained genes related to cell migration as we reported before. The other group included eight glycolysis-related genes (Fig. 4a) and three of them (GLUT1, HK2, and PKM2) were reported as targets of c-Myc, a key regulator in glycolysis[25, 26].
TargetScan program and manual miRNA target prediction identified four of these glycolytic genes containing miR-192 binding sites in their 3’UTR and/or coding regions (Fig. 4a). They were PFKFB3, GLUT1, MCT4, and MYC. In HLF and HLE cells, overexpressed miR-192 reduced the protein levels of Pfkfb3, Glut1, and c-Myc, but not that of Mct4 (Fig. 4b). Further, the predicted miR-192 binding regions in these three genes were cloned into a luciferase reporter and forced expression of miR-192 reduced the luciferase activities when the wild-type sequences for PFKFB3 and GLUT1 as well as the #2 binding site of MYC were present (Fig. 4c). These effects were significantly reduced when the corresponding miR-192 binding sites were mutated. Moreover, silencing Pfkfb3, Glut1, or c-Myc using two different siRNAs reduced ECAR in both HLF-WT and HLF-192KO cells (Fig. 4d, Supplementary Fig. S4a). In HLF-192KO cells, silencing Pfkfb3, Glut1 or c-Myc notably reduced the ECAR rate to a level similar to that of HLF-WT cells with silencing of these genes. Comparable data were also noticed in HCC patients from Cohorts 1 and 2 that PFKFB3, GLUT1, and MYC presented higher levels in HCC192Low tumors compared to HCC192High tumors (Supplementary Fig. S4b-c). These results indicate that Pfkfb3, Glut1, and c-Myc are miR-192 targets and involved in the hyperglycolysis caused by miR-192 loss.
Pfkfb3, Glut1, and c-Myc were reported to maintain stemness features in cancers at certain levels [27-30]. Consistently, si-PFKFB3, si-GLUT1 or double knockdown led to reduced levels of four CSC biomarkers, i.e., CD44, CD24, EpCAM and CD90, as determined by RT-qPCR (Fig. 4e). FACS analysis also showed that si-PFKFB3 and GLUT1 reduced the populations of CD44+ and CD24+ CSCs (Fig. 4f). Meanwhile, si-MYC seemed to only reduce CD44+ CSCs moderately, but not CD24+ CSC populations (Supplementary Fig. S4d). Together, these data demonstrate that three glycolytic regulators, Pfkfb3, Glut1 and c-Myc were bona fide targets of miR-192, and they contributed to both hyper-glycolysis and CSC features of HCCs caused by loss of miR-192 to different extents.
c-Myc suppressed miR-192 transcription, ensuring a positive feedback of high c-Myc/low miR-192 in hyperglycolytic CSC+HCCs
In our previous miRNA profiles of tumors and non-tumors from a hydrodynamic injection HCC FVB mouse model, miR-192 expression was significantly reduced in c-Myc-induced HCCs (Fig. 5a). Further, in a hydrodynamic injection HCC ICR mouse model, miR-192 level was also reduced >100 times in c-Myc induced HCCs but not much reduced in Ras-induced HCCs than that in the corresponding non-HCC liver tissues. In four different HCC cell lines, si-MYC led to an increased expression of miR-192, while forced expression of c-Myc reduced the level of miR-192 (Fig. 5b). These indicate that c-Myc might regulate miR-192 transcription.
Consistently, among four different lengths of mir-192 promoter regions, the -266 nt to +186 nt region showed the strongest promoter activity (Fig. 5c) and the mir-192 promoter activity (-266 nt to +186 nt) was reduced by exogenous c-Myc, while enhanced by si-MYC (Fig. 5d). Wild-type p53 could bind to the mir-192 promoter region and induce its expression[13, 31]. Consistently, in HepG2 cells with wild-type TP53, the expression of miR-192 was induced by p53 via exposure to Nutlin-3a (an MDM2 antagonist to stabilize p53) and reduced by silencing of p53 (Fig. 5e, Supplementary Fig. S5a-b). Meanwhile, over-expressed c-Myc significantly suppressed miR-192 expression in HCC cells with either activated p53 or silenced p53, indicating that c-Myc-mediated miR-192 down-regulation was independent on p53.
Comparable data were noticed in HCC patients. In both HCC cohorts, hierarchical clustering analysis revealed two subgroups with distinct c-Myc activation status based on 76 c-Myc target genes from the online Human MYC Targets Profiler (Supplementary Fig. S5c-d). In Cohort 1, miR-192 expression in the c-Myc activation subgroup was significantly lower than that in c-Myc non-activation subgroup (Fig. 5f). In Cohort 2, mir-192 expression was always significantly lower in each c-Myc activation subgroup than in the corresponding non-activation subgroup, which was independent on statuses of TP53 and mir-192 promoter methylation (Fig. 5f. In addition, different groups of CSC+HCCs consistently presented a low level of miR-192, a high level of c-Myc activation and high frequency of MYC amplification (Supplementary Fig. S6). Together, c-Myc suppressed miR-192 transcription, which led to a positive feedback of high c-Myc/low miR-192 in CSC+HCC cells with glycolytic feature.
Overproduced lactate from CSC+HCCs activated the Erk pathway in HCC environmental cells, and this effect further increased HCC cell stemness and malignancy features
As the end product of glycolysis, the continuously produced lactate from hyperglycolytic miR-192-loss HCC cells might affect their environment and contribute to HCC malignancy. The transport of lactate across the plasma membrane is mainly catalyzed by MCT1 and MCT4, with MCT1 typically involved in the import while MCT4 involved in export of lactate[23, 24]. In HCC patients, the expression ratio of MCT1 vs. MCT4 presented no difference between tumor and non-tumor tissues of Cohort 1 but was significantly higher in non-tumor tissues than tumor tissues of Cohort 2 (Supplementary Fig. S7a), indicating the possibility of lactate uptake by tumor environmental cells. Lee et al reported that lactate could stabilize the NDRG3, which in turn activated the Erk pathway to promote cell malignancy. Consistently, lactate treatment stimulated Erk phosphorylation noticeably in HCC environmental cells, i.e., LX2 and THP1 and HL7702 cells (Fig. 6a).
In a chamber co-culture system, pErk level was increased in LX2 and THP1 cells when they were co-cultured with HLF-192KO cells compared to when co-culturing with HLF-WT (Fig. 6b). Moreover, pErk was further reduced in LX2 and THP1 when exposed to HLF-192KO cells with miR-192 overexpression (Fig. 6b). Similar results were observed from co-cultured LX2 and THP1 cells with HLE cells (Supplementary Fig. S7b). However, pErk did not seem to be altered in HL7702 cells in this co-culture system. Thus, HCC cells with miR-192 loss could actively affect certain group of cells in their environment via an increased lactate production and secretion.
Fig. 6c-e showed that the lactate-induced pErk in HCC environmental cells partially relied on NDRG3 and MCT1. LX2 and THP1 cells expressed relatively high levels of MCT1 and NDRG3 (Fig. 6c). Silencing NDRG3 or MCT1 in LX2 and THP1 cells reduced the level of lactate-induced pErk (Fig. 6d-e). Low expression levels of MCT1 and NDRG3 in HL7702 cells were consistent with its minor response to lactate (data not shown).
We then explored the effects of an altered lactate/MCT1/NDRG3/pErk axis in LX2 or THP1 cells on the malignancy features of HCC cells. HLF-192KO/RFP cells were co-cultured with LX2 cells pre-transfected with si-Ctrl, or si-NDRG3, or si-MCT1 (termed LX2si-Ctrl, LX2si-NDRG3, and LX2si-MCT1, respectively). Wound-healing assay under red fluorescence showed that cell migration of HLF-192KO cells was slower in co-culture with LX2si-NDRG3 or LX2si-MCT1 than in co-culture with LX2si-Ctrl (Fig. 7a). Consistent data were observed in HLF-192KO cells co-cultured with THP1 (Fig. 7a). Moreover, spheroid assays of HLF-192KO cells were performed with different conditioned medium. The number of spheroids of HLF-192KO cells was significantly lower under exposure to conditioned medium from co-culture of HLF-192KO with LX2si-NDRG3 or LX2si-MCT1 than from co-culture of HLF-192KO with LX2si-Ctrl (Fig. 7b). CD44+ and CD24+ HLF-192KO populations were also significantly reduced when they were co-cultured with LX2si-NDRG3 or LX2si-MCT1 (Fig. 7c). As a control, HLF-192KO with overexpressed miR-192 did not exhibit much alteration on migration, spheroid formation and CSC populations when co-culturing with different LX2 cells (Fig. 7a-c). Therefore, in co-culture system, blocking a lactate/Erk pathway in HCC environmental cells suppressed the malignancy and stemness features of HCC cells.
In both HCC cohorts, patients were divided into four groups based on miR-192 expression in their tumor (HCC192Low and HCC192High, medium cut-off) and levels of NDRG3 and MCT1 in non-tumors (NTHigh_NDRG3 or MCT1 and NT Low_NDRG3 and MCT1, medium cut-offs). There was no expressional difference of NDRG3 or MCT1 in non-tumors between HCC192Low and HCC192High patients (Fig. 7d, Supplementary Fig. S7C). In Cohort 1, patients with HCC192High NTLow_NDRG3 and MCT1 had the best prognosis, as shown by a prolonged time to recurrence and overall survival. In HCC192Low subgroup, patients with NTHigh_NDRG3 or MCT1 had worse prognosis compared to patients with NTLow_NDRG3 or MCT1 (Fig. 7e). Similar but less significant data were obtained from Cohort 2, which might be due to the limited number of patients (n=49) with available non-tumor mRNA reading (Supplementary Fig. S7d). GSEA analysis in HCC192Low patients revealed that several stem cell related gene-sets were enriched in patients with NTHigh_NDRG3 or MCT1 those with NTLow_NDRG3 or MCT1 (Supplementary Fig. S8). Together, when HCC cells with miR-192 loss surrounded by environmental cells with high MCT1 or NDRG3 expression, they presented a highly malignant feature.