CRNDE is highly expressed in human HCC and positively correlated with tumor size, pathological stage, and poor survival
CRNDE was upregulated in 240 paired in-house HCC specimens via qRT-PCR, compared with adjacent non-cancerous tissue, CRNDE was significantly upregulated (68%) in HCC tumor tissues (cohort 1, Fig. 1A). Similarly, CRNDE expression in HCC tumor tissue specimens was markedly higher relative to normal counterparts (cohort 1, Fig. 1B). These results were consistent with gene expression data from other publicly available datasets comprising several human hepatoma samples (cohort 2, The Cancer Genome Atlas data set; cohort 3, The Oncomine database from Wurmbach Liver Statistics), both showing high expression of CRNDE in HCC tumor tissues (Fig. 1C, D). Overall survival analysis of HCC patients further supported an association of high expression of CRNDE with shorter survival compared with low CRNDE expression (Fig. 1E). To further assess the significance of CRNDE expression in HCC, clinicopathological features were examined. Notably, CRNDE was increased at advanced pathological stage (cohort 1, Fig. 1F; p = 0.0019), larger tumors (cohort 1, Fig. 1G; p = 0.0032), virus infection (cohort 1, Additional file 1: Fig. S1A, HCV: p = 0.0124, HBV + HCV: p = 0.0008), and cirrhosis (cohort 1, Additional file 1: Fig. S1B, p = 0.0470). Our collective results support an oncogenic role of CRNDE in the biological progression of HCC.
CRNDE promotes HCC tumorigenesis and sorafenib resistance
We subsequently examined CRNDE expression in HCC cell lines. To this end, Hep3B and SkHep1 cells highly expressing CRNDE were selected for shRNA-based knockdown analysis (Fig. 2A) and CRNDE expression was induced in Huh7 and J7 cells expressing low levels of CRNDE via plasmid transfection. Knockdown of CRNDE significantly suppressed proliferation of both Hep3B and SkHep1 cells (Additional file 2: Fig. S2A, B). Conversely, CRNDE overexpression promoted Huh7 and J7 cell proliferation relative to cells expressing pcDNA3-control (Additional file 2: Fig. S2C, D). Simultaneously, in vivo experiments revealed that the final tumor volume was significantly greater in the CRNDE overexpression than the pcDNA3-control cell group (Additional file 2: Fig. S2E, F). Moreover, survival benefits were reduced under conditions of CRNDE overexpression, supporting an oncogenic role in HCC (Additional file 2: Fig. S2G). Additionally, CRNDE clearly suppressed migration and invasion of Hep3B and SkHep1 (Additional file 2: Fig. S2H, I, L, M). Conversely, migration and invasion abilities were enhanced in Huh7 and J7 cells overexpressing CRNDE (Additional file 2: Fig. S2J, K, N, O). In vivo suggested that the number of metastatic nodules in lungs was reduced in mice injected with CRNDE-silenced Hep3B (Additional file 2: Fig. S2P) and increased in livers of mice administered Huh7 overexpressing CRNDE (Additional file 2: Fig. S2Q). Silencing of CRNDE in Hep3B and SkHep1 cells promoted sensitization to sorafenib relative to the control shLuc group (Additional file 2: Fig. S2R, S) while CRNDE overexpression in Huh7 and J7 cells induced greater resistance to sorafenib relative to the corresponding pcDNA3-control groups (Additional file 2: Fig. S2T, U). These data are consistent with previous findings [11, 22, 23] that CRNDE is not only highly expressed but also markedly promotes tumorigenesis and sorafenib resistance in HCC specimens.
CRNDE is positively correlated with upregulation of EGFR, thereby increasing proliferation and sorafenib resistance of HCC
Recent findings support an oncogenic role of CRNDE in glioma, whereby its upregulation is positively correlated with activation of EGFR signaling [24]. In our experiments, EGFR downstream signaling molecules (such as p-EGFR and p-STAT3) were reduced upon CRNDE silencing in Hep3B and SkHep1 cells (Figs. 2B, Additional file 3: Fig. S3A), and conversely, enhanced upon CRNDE overexpression in Huh7 and J7 cells (Figs. 2C, Additional file 3: Fig. S3B). Notably, suppression of CRNDE in HCC cells markedly reduced EGFR at both mRNA and protein levels while its overexpression exerted the opposite effect (Fig. 2B–E, Additional file 3: Fig. S3A–D). qRT-PCR analysis was further conducted to confirm the effects of CRNDE silencing and overexpression (Additional file 3: Fig. S3E–H). Compared with the pcDNA3-control group, higher green fluorescence intensity of EGFR was detected in the CRNDE overexpressing group via immunofluorescence staining (Additional file 3: Fig. S3I). Compared with adjacent non-cancerous tissue (N), CRNDE was significantly upregulated in HCC tumor tissues (T) (cohort 1, Fig. 2F). To validate upregulation of EGFR in HCC specimens, EGFR expression patterns were examined in 240 paired HCC specimens. Our qRT-PCR findings revealed significant expression of EGFR in HCC tumor tissues (T) (Fig. 2G). Additionally, the available online dataset suggested that the EGFR expression was no different between normal (N) and tumor (T) in cohort 2 and cohort 3 (Additional file 4: Fig. S3J and S3K). However, EGFR expression in HCC tumor tissue specimens was markedly higher relative to normal counterparts in our 240 paired HCC specimens (Fig. 2G), and EGFR was significantly increased at advanced pathological stage (cohort 1, Additional file 4: Fig. S3L; p = 0.0319), larger tumors (cohort 1, Additional file 4: Fig. S3M; p = 0.0346), and virus infection (cohort 1, Additional file 4: Fig. S3N; HBV: p = 0.0118, HCV: p < 0.0001, HBV + HCV: p < 0.0001). The Spearman correlation coefficient analysis confirmed a significant positive association of CRNDE with EGFR (Fig. 2H, Spearman r: 0.4239, p < 0.0001). Simultaneous in vivo experiments disclosed that both the number of Ki67-positive cells and EGFR expression were increased in CRNDE overexpression relative to pcDNA3-control-transfected J7 cells, as determined via IHC staining (Fig. 2I). We further assessed whether EGFR affects HCC cell proliferation and sorafenib resistance under CRNDE overexpression conditions. In both pcDNA3-control and CRNDE overexpressing Huh7 and J7 cell lines, knockdown of EGFR led to a significant reduction in cell proliferation (Fig. 2J, Additional file 4: Fig. S3O) and suppression of CRNDE-induced sorafenib resistance (Fig. 2K, Additional file 4: Fig. S3P). Western blot experiments were conducted to ascertain siRNA-mediated inhibition of EGFR protein in pcDNA3-control and CRNDE overexpressing Huh7 and J7 cells (Fig. 2L, Additional file 4: Fig. S3Q). Our results indicate that CRNDE-mediated enhancement of EGFR expression is involved in proliferation and sorafenib resistance of HCC cells.
CRNDE upregulates EGFR through mediating chromatin relaxation in HCC
To establish the mechanisms by which CRNDE promotes EGFR mRNA transcription, the promoter assay was performed. The activity of the region 600 bp upstream of the promoter (Fig. 3A) was significantly reduced upon CRNDE silencing in Hep3B and SkHep1 cells (Fig. 3B, Additional file 5: Fig. S4A), and conversely, enhanced with CRNDE overexpression in Huh7 and J7 cells (Fig. 3C, Additional file 5: Fig. S4B). Subcellular fractionation analysis using GAPDH and U1 as cytoplasmic and nuclear localization markers, respectively, showed that CRNDE primarily localizes in the nucleus of HCC cells (Additional file 5: Fig. S4C, D). Acetylated H3K27 and H3K9 are associated with alterations in transcription regulation [25, 26]. High H3K27Ac expression at the endogenous EGFR promoter was predicted using the UCSC Genome Browser (https://genome.ucsc.edu/). To further ascertain whether CRNDE is involved in the regulation of EGFR gene expression via chromatin relaxation, we examined the status of representative histone modifications. Acetylated H3K9 and H3K27 signals were reduced upon CRNDE silencing in Hep3B and SkHep1 cells (Fig. 3D, Additional file 5: Fig. S4E). Conversely, CRNDE overexpression enhanced acetylated H3K9 and H3K27 signals relative to control Huh7 and J7 cells (Fig. 3E, Additional file 5: Fig. S4F). Recent studies have shown that H3K9 and H3K27 are acetylated by p300, a member of the histone acetyltransferase family [27]. Accordingly, we investigated whether p300 is involved in the mediation of EGFR expression by CRNDE. Interestingly, knockdown of p300 inhibited EGFR protein and mRNA expression under CRNDE overexpression conditions in Huh7 cells (Fig. 3F, G) but had no effect in the corresponding pcDNA3-control group. Additionally, suppression of p300 reduced EGFR promoter activity in CRNDE overexpressing HCC cells (Fig. 3H, Additional file 5: Fig. S4G). To determine whether EGFR is transcriptionally activated by chromatin relaxation via p300 acetyltransferase activity mediated by CRNDE, the effect of C646 (a p300 acetyltransferase inhibitor [28, 29]) on EGFR expression was evaluated under CRNDE overexpression conditions. EGFR, H3K9Ac, and H3K27Ac protein signals were significantly induced in CRNDE overexpressing relative to the control cell groups. Notably, treatment with C646 led to a marked reduction in expression levels of these proteins (Fig. 3I, Additional file 5: Fig. S4H). Furthermore, C646 suppressed EGFR mRNA levels to a significant extent in Huh7 and J7 cells overexpressing CRNDE (Fig. 3J, Additional file 5: Fig S4I). In the luciferase reporter assay, treatment with C646 reduced EGFR promoter activity compared to that in DMSO under CRNDE overexpression (Fig. 3K, Additional file 5: Fig. S4J). These results suggest that CRNDE induces EGFR upregulation by increasing histone acetylation through the regulation of p300.
YY1 promotes HCC tumorigenesis via induction of EGFR transcription activity under conditions of CRNDE overexpression
We identified a series of transcription factors with binding sequences in the 600-bp region upstream of the promoter via the PROMO database (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3) prediction, including C/EBPβ [30], c-myc [31], STAT3 [32], and YY1 [33], which could also interact with p300. To establish whether these transcription factors are involved in the regulation of EGFR in response to CRNDE, specific shRNAs were used for their knockdown in Huh7 cells overexpressing CRNDE. Interestingly, only the knockdown of YY1 inhibited the CRNDE-induced increase in EGFR mRNA (data not shown). Consistently, CRNDE-induced EGFR protein was inhibited upon YY1 silencing (Fig. 4A, B; Additional file 6: Fig. S5A, B), along with EGFR transcription activity (Fig. 4C, Additional file 6: Fig. S5C). The PROMO database predicts that the 600-bp region upstream of the promoter potentially contains two YY1 binding sites. Next, we designed variants of the YY1 binding sites in the EGFR promoter via site-directed mutagenesis (SDM), designated M1, M2, and M3 (Fig. 4D). EGFR promoter activity was inhibited in M2 and M3 variants under conditions of CRNDE overexpression (Fig. 4E, Additional file 6: Fig. S5D), supporting the involvement of YY1 in the transcriptional activation of EGFR by CRNDE. The ChIP assay was conducted to confirm YY1 binding to the promoter region of EGFR, ranging from 464 to 476 bp upstream of the transcription start site. A 249-bp PCR product containing the YY1-binding site on the EGFR promoter was obtained. The locations and sequences of PCR primers are depicted in Fig. 4F. YY1 binding was reduced upon CRNDE silencing in Hep3B and SkHep1 cells (Fig. 4G, H; Additional file 6: Fig. S5G). GADPH was used as a negative control (Additional file 6: Fig. S5E). Conversely, CRNDE overexpression in Huh7 and J7 (Fig. 4I, J; Additional file 6: Fig. S5H; GADPH as a negative control, Additional file 6: Fig. S5F) cells enhanced YY1 binding to the EGFR promoter region. Based on these findings, we conclude that EGFR transcriptional activity is induced by enhancing specific YY1 binding to its promoter region following CRNDE expression.
CRNDE regulates H3K9Ac/p300/YY1 complex binding on the EGFR promoter
p300 can act as both a coactivator and corepressor through interactions with YY1 and modulation of YY1-mediated transcriptional regulation [33]. Here, we further examined the hypothesis that p300 associates with YY1 as a transcriptional complex that regulates EGFR under conditions of CRNDE overexpression. Data from the co-immunoprecipitation (co-IP) assay revealed decreased association of p300 with YY1 following CRNDE silencing in Hep3B (Fig. 5A). Conversely, CRNDE overexpression in Huh7 promoted the binding of p300 and YY1 (Fig. 5B). The RNA immunoprecipitation (RIP) assay confirmed the association of p300 with CRNDE in HCC (Fig. 5C, D; Additional file 7: Figs. S6A, B). These results suggest that p300 is not only associated with CRNDE in HCC but also further stimulates associations with YY1 under conditions of CRNDE overexpression. Furthermore, we observed decreased binding of p300/YY1 to the EGFR promoter under CRNDE silencing in the re-ChIP assay (Fig. 5E, Additional file 7: Fig. S6C; negative control GADPH, Additional file 7: Fig. S6D, E). Conversely, CRNDE overexpression led to elevated binding to the EGFR promoter (Fig. 5F, Additional file 7: Fig. S6F; negative control GADPH, Additional file 7: Fig. S6G, H). To further determine whether CRNDE influences p300/YY1 binding to the EGFR promoter through chromatin relaxation, histone markers were assayed. Initially, H3K9Ac binding to the EGFR promoter was markedly reduced upon CRNDE silencing (Fig. 5G, Additional file 7: Fig. S6I; negative control GADPH, Additional file 7: Fig. S6J, K) while CRNDE overexpression led to increased binding to the EGFR promoter (Fig. 5H, Additional file 6: Fig. S5L; negative control GADPH, Additional file 7: Fig. S6M, N). Data from the further re-ChIP analysis revealed reduced H3K9Ac/YY1 binding to the EGFR promoter upon CRNDE silencing (Fig. 5I, Additional file 7: Fig. S6O; negative control GADPH, Additional file 7: Fig. S6P, Q). Conversely, overexpression of CRNDE dramatically stimulated these interactions (Fig. 5J, Additional file 7: Fig. S6R; negative control GADPH, Additional file 7: Fig. S6S, T). However, no changes in the binding ability of H3K27Ac/YY1 to the EGFR promoter under conditions of CRNDE expression were observed in the re-ChIP assay (data not shown). Finally, interactions of both H3K9Ac and YY1/H3K9Ac to the EGFR promoter were enhanced under CRNDE overexpression, as determined from ChIP and re-ChIP analyses, and reduced following C646 treatment (Fig. 5K, L; negative control GADPH, Additional file 7: Fig. S6U, V). The collective results suggest that CRNDE mediates both chromatin relaxation and H3K9Ac/p300/YY1 complex binding at the EGFR promoter region to regulate EGFR transcriptional activity.
CRNDE promotes CDX tumor growth in mice via p300/YY1/H3K9Ac regulation of EGFR
In HCC specimens, p300 enhances proliferation via epigenetic regulation of gene transcription [34,35,36]. Here, we assessed whether the promontory effects of p300 on HCC cell proliferation are related to CRNDE. Proliferation was significantly reduced in CRNDE overexpressing Huh7 and J7 cells and conversely, increased in pcDNA3-control cells with knockdown of p300 (Fig. 6A, B). Additionally, both migration and invasion abilities were increased in Huh7 cells with CRNDE overexpression and inhibited followed by knockdown of p300 or YY1 (Additional file 8: Fig. S7A, B). Quantification of Huh7 cell numbers using the transwell assay is presented in the right panel (Additional file 8: Fig. S7C, D). The collective results suggest that CRNDE influences migration, invasion, and proliferation of HCC cells through p300 and YY1-mediated. Recent studies have shown that C646 effectively reduces the proliferation of HCC cells [35, 37, 38]. CRNDE overexpressing Huh7 and J7 cells exposed to C646 for 72 h displayed decreased viability in a dose-dependent manner compared to the pcNDA3-control groups (Fig. 6C, D). We further examined the potential therapeutic effect of C646 on HCC xenografts in nude mice. To this end, two groups of mice were subcutaneously injected with J7 cells (transfected with pcDNA3-control or CRNDE overexpressing constructs). CRNDE overexpressing J7 cells induced a significant increase in tumor sizes examined on day 5 following injection. A representative tumor sample is depicted in Fig. 6E. Final tumor sizes were considerably greater in the CRNDE overexpressing than pcDNA3-control cell-injected groups (Fig. 6F). C646 treatment for 21 days induced the strongest inhibition of tumor growth, resulting in markedly smaller tumor sizes and weights in CRNDE overexpressing cancer cell line-derived xenograft (CDX) mice (Fig. 6G, H). As expected, the survival curve for the CRNDE overexpression group treated with C646 at the sub-pharmacologic dose was longer than that for untreated mice (Fig. 6I). Additionally, EGFR mRNA levels were significantly increased upon CRNDE overexpression and conversely, decreased following treatment with C646 (Fig. 6J). Consistently, IHC analysis revealed marked inhibition of EGFR protein expression in cells treated with C646 for 21 days compared to untreated cells overexpressing CRNDE (Fig. 6K). The collective results support the utility of the p300 inhibitor in suppressing tumor growth under conditions of CRNDE expression.
CRNDE enhances sorafenib resistance through upregulation of EGFR with p300 in HCC
The influence of p300 inhibitors on sorafenib resistance in HCC has not been extensively investigated to date. Based on the finding that C646 is effective against HCC in vitro and in vivo (Fig. 6), we further examined whether C646 could reduce cell viability in vitro upon co-treatment with sorafenib in CRNDE overexpressing Huh7 and J7 cells. Notably, sorafenib resistance was reduced in C646-treated CRNDE overexpressing Huh7 and J7 cells in a dose-dependent manner (Fig. 7A, Additional file 9: Fig. S8A). To assess the effects of C646 on sorafenib resistance of CRNDE overexpressing HCC cells in vivo, a mouse xenograft model was used. CRNDE overexpressing Huh7 xenografts displayed elevated tumor sizes compared to those expressing pcDNA3-control (Fig. 7B, C). Simultaneously, sorafenib resistance was enhanced under CRNDE overexpression, with no significant decrease in tumor size following sorafenib treatment relative to the pcNDA3-control (Fig. 7B, C). Only the pcNDA3-control group showed a marked reduction in tumor size following treatment with sorafenib (Fig. 7B, C). Under conditions of CRNDE overexpression, combined treatment with C646 and sorafenib for 21 days dramatically reduced resistance compared to treatment with sorafenib only (Fig. 7B–D). However, in the presence of C646 only, HCC tumor size was equivalent to that recorded following treatment with a combination of C646 and sorafenib under conditions of CRNDE overexpression (Fig. 7B, C). In terms of survival curves, pcDNA3-control mice died within 43 days, with a median survival of 27 days, while mice in the CRNDE overexpression group died within 10 days, with a median survival of 10 days (Fig. 7D, E). Treatment of pcDNA3-control mice with either sorafenib or C646 led to increased survival times (Fig. 7D). Sorafenib treatment under CRNDE overexpression conditions increased survival, with a median survival time of 30 days (Fig. 7E). Notably, C646-treated or sorafenib and C646-treated mice displayed significantly longer survival times compared to non-treated or sorafenib only groups (Fig. 7E). EGFR mRNA and protein expression were significantly induced upon CRNDE overexpression and reduced by both C646 alone and in combination with sorafenib (Fig. 7F, G). These results support a critical role of CRNDE in sorafenib resistance and suggest that the p300 inhibitor C646 exerts inhibitory and additive effects on sorafenib resistance to prolong overall survival through downregulation of EGFR expression.