CCN2–MAPK–Id-1 loop feedback amplification is involved in maintaining stemness in oxaliplatin-resistant hepatocellular carcinoma
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Hepatocellular carcinoma (HCC) is the second leading cause of cancer death worldwide. Chemotherapy is an alternative treatment for advanced HCCs, but chemo-resistance prevents cancer therapies from achieving stable and complete responses. Understanding the underlying mechanisms in chemo-resistance is critical to improve the efficacy of HCC.
The expression levels of Id-1 and CCN2 were detected in large cohorts of HCCs, and functional analyses of Id-1 and CCN2 were performed both in vitro and in vivo. cDNA microarrays were performed to evaluate the alterations of expression profiling of HCC cells with overexpression of CCN2. Finally, the role of downstream signaling of MAPK/Id-1 signaling pathway in oxaliplatin resistance were also explored.
The increased expression of Id-1 and CCN2 were closely related to oxaliplatin resistance in HCC. Upregulation of CCN2 and Id-1 was independently associated with shorter survival and increased recurrence in HCC patients, and significantly enhanced oxaliplatin resistance and promoted lung metastasis in vivo, whereas knock-down of their expression significantly reversed the chemo-resistance and inhibited HCC cell stemness. cDNA microarrays and PCR revealed that Id-1 and MAPK pathway were the downstream signaling of CCN2. CCN2 significantly enhanced oxaliplatin resistance by activating the MAPK/Id-1 signaling pathway, and Id-1 could upregulate CCN2 in a positive feedback manner.
CCN2/MAPK/Id-1 loop feedback amplification is involved in oxaliplatin resistance, and the combination of oxaliplatin with inhibitor of CCN2 or MAPK signaling could provide a promising approach to ameliorating oxaliplatin resistance in HCC.
KeywordsHepatocellular carcinoma Oxaliplatin CCN2 Id-1 MAPK signaling
Connective tissue growth factor
Inhibitor of DNA binding protein 1
Gene Expression Omnibus
Epithelial cell adhesion molecule
Epithelial mesenchymal transition
Transcatheter arterial chemoembolization
Cumulative recurrence rates
Hepatitis B virus
HBV surface antigen
Kyoto Encyclopedia of Genes and Genomes
Cancer stem cells
Hepatocellular carcinoma (HCC), which accounts for over 80% of primary liver cancers occurring worldwide, is the second leading cause of cancer death worldwide, with more than 50% of the total number of cases and deaths in China . Despite progress in early detection and treatment of HCC, more than 70% of patients are at an advanced stage at diagnosis and are not suitable for curative therapies. Therefore, platinum-based local or systemic chemotherapy is an alternative treatment. Unfortunately, primary and acquired resistance to chemotherapy is common with HCC, which represents a major challenge in the treatment of advanced HCC . Accordingly, the molecular characterization of chemo-resistant HCC is critical for further improving the efficacy of chemotherapy. In our previous cDNA microarray study of oxaliplatin-resistant HCCs, we have found significant upregulations of 267 genes including CCN2 and Id-1 (inhibitor of DNA binding protein-1) , while the accurate role and relationship of which in oxaliplatin resistance is still not yet clear in HCC.
The CCN family is a six-member family of cysteine-rich regulatory proteins in humans that share a multi-modular structure with an N-terminal secretory signal domain followed by four conserved domains . Because they possess four functional domains, CCN proteins do not behave like traditional growth factors or cytokines and do not appear to have a unique receptor to which they bind with high affinity to induce signal transduction . CCN2/Connective tissue growth factor (CTGF), one of the secreted factors upregulated in oxaliplatin-resistant HCC, is involved in proliferation, chemotaxis, adhesion, migration, and cell fate in different cell types and tissues . However, evidences that suggest the role and mechanism of CCN2 in malignant tumors are still vague [7, 8].
Id-1 plays important roles in blocking cell differentiation and stimulating cell proliferation by mimicking the activities of other oncogenes, and inhibiting the tumor suppressor activities by targeting the proteins harboring the basic helix–loop–helix (HLH) motif . Due to its role in cell differentiation, Id-1 has also been implicated in the biology of cancer stem cells . Matsuda et al.  reported that increased Id-1 expression in HCC plays an important role in hepatocarcinogenesis and serves as a useful marker for risk prediction of occurrence. Sharma et al.  found that Id-1 has a tumor promotion role in the metabolic reprogramming of cancer cells including aerobic glycolysis and glutaminolysis. Further, the low postnatal expression of Id-1 and its high expression in cancer stem cells mark them as attractive targets for anti-cancer therapy. However, the role of Id-1 in HCC and the regulatory mechanisms it shares with CCN2 remain unclear, especially in HCC with oxaliplatin resistance.
In the present study, we determined that increased Id-1 and CCN2 expression were closely correlated with oxaliplatin resistance in HCC, verified that malignancy and poor prognosis were associated with Id-1 in human HCC, and explored the negative roles of Id-1 and the regulatory mechanisms of CCN2 in HCC. Finally, we demonstrated enhanced oxaliplatin resistance could be reversed by CCN2–MAPK–Id-1 signaling loop inhibition.
Patients and follow-up
A total of 268 patients who underwent curative liver resection for HCC between January 2004 and December 2006 at the Zhongshan Hospital Fudan University were enrolled in this study. Among which, the training set contained 64 cases (including HCC tissues and the paired non-tumor liver tissues from 48 patients were used for real-time PCR and that from 16 patients for Western blot), another set of 184 patients was used for validation (the validation set). Curative resection was defined as the complete resection of tumor nodules, leaving the tumor margins free of cancer upon histologic examination. The histopathologic diagnosis was confirmed by two independent experienced pathologists. Patients were followed after surgical treatment until December 2013, and the median follow-up was 63 months (range 0–110 months). Details of the follow-up procedures were previously described . The clinicopathologic characteristics of HCC patients in the validation set were provided in Supplementary Tables 1 and 2.
Animal model and treatment procedures
MHCC97H-CCN2-sh cells, Hep3B-CCN2 cells, MHCC97H-Id1-sh cells, MHCC97H-Id1 cells, MHCC97H mock cells, and their associated control cells were implanted subcutaneously into the upper left flank region of mice to establish subcutaneous xenografts. The synergistic effects of the combination therapy of oxaliplatin and sorafenib were evaluated. Twenty-four nude mice bearing subcutaneous xenografts were randomly divided into the control, oxaliplatin, sorafenib, and oxaliplatin + sorafenib groups (n = 6 per group). The treatments included a tail vein injection of oxaliplatin (10 mg/kg/week), a 0.2 mL oral dose of sorafenib (30 mg/kg/week). Tumor weights were evaluated in 4 weeks after the treatments. The subcutaneous implantation models were also established in 12 C57 mice using Hep1-6 cell, to carry out the same above evaluations. Intraperitoneal injection of pentobarbital (5 mg/kg) combined with cervical spondylolisthesis was used for the killing of mice after the study.
cDNA microarray analysis
cDNA microarrays were performed using the Human OneArray® (Phalanx Biotech Group, San Diego, CA) to evaluate the alterations of expression profiling. Total RNA was extracted from Hep3B-CCN2 and Hep3B-vector cells and the isolations and microarray analyses were performed in triplicate according to the manufacturer’s instructions. All data were uploaded to the Gene Expression Omnibus (GEO accession number 124529).
Vector construction, transfection, and lentivirus transduction
The human full-length Id-1 (NM_002165) and CCN2 (NM_001901.2) cDNA were obtained from GeneCards (Shanghai, China) and cloned into the pCDH lentiviral expression vector (System Biosciences, CA, USA). Using the In-Fusion® HD Cloning Kit (Takara, Tokyo, Japan), the amplified fragment was inserted into the pCDH plasmid (between XbaI and EcoRI sites). Lentiviral shRNA expression plasmids PLVT, PLKO.1, and the target sequences were listed in Supplementary Table 7.
Statistical analyses were performed using SPSS 15.0 for Windows (SPSS, Inc., Chicago, IL). A p value < 0.05 was considered statistically significant.
Increased expression levels of Id-1 and CCN2 were closely related to oxaliplatin resistance in HCC
Increased Id-1 and CCN2 expression trends are positively associated with HCC malignancy and poor prognosis
To illustrate the clinical role of Id-1 and CCN2 in HCC, 184 patients in the validation cohort were sorted according to the Id-1 and CCN2 expression levels (Supplementary Fig. 3A, B). The patients in the Id-1high group had significantly lower overall survival (OS) and higher cumulative recurrence rates (CCR) compared with those in the Id-1low group (Fig. 2d). The patients in the CCN2high group also had significantly lower OS and higher CCR compared with those in the CCN2low group (Fig. 2e). We then classified the patients into three subgroups according to CCN2 and Id-1 expression levels. Group I had low expression levels of both CCN2 and Id-1, group II had high expression of either CCN2 or Id-1, and Group III had high expression of both CCN2 and Id-1. The patients in group I had the best prognosis, their OS rate was significantly higher than that of the patients in groups II and III, and their CCR was significantly lower (Fig. 2f).
Cox regression analysis revealed that increased Id-1 expression was only significantly correlated with tumor differentiation. No significant association was found between Id-1 expression and the other clinico-pathological characteristics including age, gender, hepatitis B virus (HBV) surface antigen (HBsAg) status, cirrhosis, serum alpha-fetoprotein (AFP) and alanine aminotransferase (ALT) levels, tumor dimension, tumor number, vascular invasion, or tumor encapsulation (Supplementary Table 1). Cox regression analysis also revealed that increased CCN2 expression was significantly correlated with tumor number and tumor differentiation. However, no significant association was found between CCN2 expression level and the other clinical and pathological characteristics (Supplementary Table 2).
A univariate analysis revealed that tumor size, tumor number, vascular invasion, Id-1 and CCN2 expression were significantly associated with the post-operative OS and CCR of HCC patients. However, no prognostic significance was found for the other characteristics including age, gender, HBsAg, HCV-Ab, liver cirrhosis, AFP, ALT, tumor encapsulation or differentiation (Supplementary Table 3). The multivariate Cox proportional hazards model revealed that tumor size, number of tumor nodules, and Id-1 and CCN2 expression were also independent prognostic indicators for OS and CCR of HCC patients; however, vascular invasion was an independent prognostic indicator for OS in HCC patients (Supplementary Table 4).
To confirm the prognostic value of Id-1 and CCN2 for HCC, we detected its protein levels in frozen tissue samples from HCC patients by immunoblotting, and found a significantly increased Id-1 and CCN2 protein levels in HCC tissues with early recurrence compared with the others. In addition, increased Id-1 and CCN2 expression was found to be associated with poor differentiation of HCC (Supplementary Fig. 4).
Upregulation of Id-1 and CCN2 are related to enhanced stemness of HCC cells
To evaluate the role of CCN2 in HCC, we stably silenced and restored CCN2 expression in HCC cells. Among of the three CCN2-shRNA, CCN2-sh1 was found to exert the most efficient interference of CCN2 compared with MHCC-97H-mock cells, and restoration of CCN2 expression could rescue the altered expression of CCN2 (Fig. 3c; Supplementary Fig. 8). The downregulation of CCN2 significantly inhibited the oxaliplatin resistance which could be reversed when CCN2 expression was restored. The downregulation of CCN2 expression significantly impaired the invasiveness, migration, proliferation and sphere formation abilities compared with the controls. Furthermore, after rescuing CCN2 expression, the impaired abilities were significantly restored (Supplementary Fig. 9). The in vivo subcutaneous tumor growth capacity of MHCC-97H cells transfected with CCN2 shRNA in nude mouse models was significantly diminished, whereas it is significantly enhanced when the CCN2 was rescued (Fig. 3d).
Overexpression of CCN2 induced significant changes in gene expression profiles of HCC cells
Interestingly, spp1, which encodes OPN, a secreted phosphoprotein that plays a crucial role in HCC metastasis [14, 15, 16, 17], was also significantly upregulated in HCC with CCN2 overexpression, whereas CDH1, which encodes E-cadherin and mediates the epithelial phenotype in tumor cells , was significantly downregulated. Many other significantly altered genes that play important roles in tumor progression were also identified to be closely related to CCN2 (all data were uploaded into GEO).
CCN2 significantly activates the MAPK/Erk/Id-1 signaling pathway and Id-1-positive feedback amplified the expression of CCN2
In the case of construction of oxaliplatin-resistant HCC cell lines, firstly, we treated the oxaliplatin-resistant MHCC-97H cells with different concentrations of oxaliplatin, and found that the expression levels of CCN2, Id-1, LRP6, p-Erk, and E-cadherin varied in a dose-dependent manner (Supplementary Fig. 10A). And, the variations in CCN2, Id-1, and p-Erk expression levels also occurred in a time-dependent pattern with a significant difference in the maximum time point during the oxaliplatin treatment (Supplementary Fig. 10B).
We re-analyzed the difference between oxaliplatin-resistant and wild-type HCC, and the activation of the MAPK/Erk signaling cascade was demonstrated to be concomitant with the upregulation and phosphorylation of p-C-RAF, p-MEK, and p-Erk1/2, which eventually led to the upregulation of Id-1 (Fig. 5e). When sorafenib or U0126 was added to the oxaliplatin-resistant HCC cells, the MAPK/Erk signaling pathway was impaired and Id-1 expression was downregulated (Fig. 5f). On the other hand, when the CCN2 expression in oxaliplatin-resistant HCC cells was silenced using specific shRNA, the MAPK/Erk signaling and Id-1 expression were obviously inhibited (Fig. 5g). Furthermore, Id-1 overexpression or interference induced an obvious variation of CCN2 protein level in the same trend in HCC cells (Fig. 3a).
Oxaliplatin combined with CCN2/MAPK/Erk signaling inhibition results in improved treatment effects in HCC
Furthermore, in the subcutaneous xenograft models established in nude mice using MHCC-97H cells, the combination of sorafenib with oxaliplatin induced a more significant inhibitory effect on in vivo tumor growth compared with the oxaliplatin alone (Fig. 6c). In the subcutaneous xenograft models of C57BL/6 mice established with Hep1-6 cells, oxaliplatin did not exhibit an inhibitory effect on tumor growth, but when in combination with sorafenib, oxaliplatin exhibited a significant inhibitory effect on in vivo growth of HCC (Fig. 6d).
Previously, our research group studied various HCC therapies, including curative and palliative treatments, and found that EMT occurred after treatment with oxaliplatin  and that the downregulation of CSCs and inhibition of stemness occur in the sensitization of HCC to oxaliplatin . We then constructed oxaliplatin-resistant HCC models and found increased expression of both CCN2 and Id-1 in the oxaliplatin-resistant HCC using cDNA microarrays . In the present study, we showed that the expression of Id-1 and CCN2 were closely correlated with malignancy in HCC patients, especially in the patients resistant to TACE. The adverse role of Id-1 and CCN2, and the mutual regulatory role were also explored in HCC patients and validated in HCC with primary and acquired oxaliplatin resistance.
Id-1 plays an important role in a number of cellular processes, including cellular development, senescence, differentiation, angiogenesis, and migration [28, 29]. The ability of Id-1 to drive self-renewal was first established in neural stem cells . However, Id-1 is also significantly associated with breast , pancreas , cervical , ovarian , and colorectal cancers in humans . In HCC, Id-1 expression is related to HCC dedifferentiation , and might serve as a potential prognostic marker for HBV-related HCC . Recently, Sharma et al.  found that Id-1 promoted metabolic reprogramming in HCC cells. In the current study, we found that Id-1 was closely related to oxaliplatin resistance in mouse models of HCC. Increased Id-1 expression was likewise demonstrated in HCC and oxaliplatin-resistant HCC, and was significantly correlated with tumor malignancy and poor prognosis.
Members of the CCN family of secreted proteins typically interact with various cytokines to elicit cell proliferation, adhesion, invasion, migration, embryonic development, angiogenesis, wound healing, fibrosis, or inflammation. In HCC, we proved that CCN2 expression is an independent factor associated with shorter OS and prolonged CCR, and upregulation of CCN2 significantly enhanced oxaliplatin resistance. Hou et al.  showed that CCN2 is related to the formation of bone metastases in HCC, whereas other studies have indicated that CCN2 could serve as a potential prognostic biomarker  and that the inhibition of CCN2 blocks the progression of HCC . Previously, we found a positive relationship between Id-1 and CCN2 expression in HCC by cDNA microarray , which was confirmed in the present study. Exploration of the forward regulatory mechanism of CCN2 and Id-1 using cDNA microarrays revealed that Id-1 and the MAPK signaling pathway were downstream of CCN2 signaling. Hence, Id-1 could partially upregulate CCN2 in a positive feedback manner. We likewise demonstrated that MAPK/Erk/Id-1 signaling was one of the most important autocrine signaling pathways regulated by CCN2 in oxaliplatin-resistant models and that the mechanism is involved in stemness maintenance and oxaliplatin resistance in HCC.
The MAPK/Erk signaling pathway plays a central role in HCC progression and is crucial for HCC proliferation . Many studies have implicated the MAPK/Erk pathway in both the development and progression of HCC and indicated that activation of the MAPK/Erk pathway correlates with a poor prognosis in human HCC . In the present study, we demonstrated that the activation of MAPK/Erk signaling and the upregulation of Id-1 were related to oxaliplatin resistance and enhanced stemness in HCC and that oxaliplatin treatment significantly activated MAPK/Erk signaling and upregulated Id-1 expression in a time- and dose-dependent manner. Further, when we silenced endogenous CCN2 expression or treated HCC cell lines with a combination of sorafenib or U0126 and oxaliplatin, MAPK/Erk signaling was impaired and Id-1 expression was downregulated. Hence, we believe that Id-1 could be an effector molecule of CCN2 and that the upregulation of CCN2 and Id-1 could serve as molecular markers of chemo-resistance. Additionally, the use of additive or synergistic therapies in the subgroup of HCC with highly activated CCN2/MAPK/Erk/Id-1 signaling could enhance current knowledge of chemotherapy and aid in developing individual treatments.
In summary, our study demonstrated that Id-1 and CCN2 were frequently upregulated in HCC patients and served as an independent prognostic factor associated with malignancy. The upregulation of Id-1 involved changes in the upregulation of CCN2 in HCC after oxaliplatin treatments and was accompanied by the activation of the MAPK/Erk signaling pathway. Thus, the combination of oxaliplatin with strategies designed to inhibit CCN2/MAPK/Erk signaling could provide a promising approach to ameliorating HCC progression, especially oxaliplatin resistance.
From our experimental results and our review of the literature, we propose the following conclusions: (1) Id-1 and CCN2 in HCC are associated with a malignant phenotype and poor prognosis in HCC; (2) enhanced stemness is profoundly influenced by the expression levels of Id-1 and CCN2; (3) CCN2 significantly activates the MAPK/Erk/Id-1 signaling pathway and Id-1 positive feedback amplified the expression of CCN2; (4) oxaliplatin combined with CCN2/MAPK/Erk signaling inhibition results in improved treatment effects in HCC.
XL, YB, SSJ, FAJ, FC, XLX, GS, MZ, PBN, and QAJ contributed to the study design, analysis, and interpretation of data. QAJ conceived the study, XL, YB, and XLX performed the majority of the experiments. SSJ and MZ participated in statistical analysis. FAJ, FJ, GS and PBN participated in the establishment of the nude mouse model. QAJ drafted and prepared the manuscript. All authors approved the final manuscript.
This research project was mainly supported by the National Natural Science Foundation of China (81502694). This research project was also partly supported by the Fundamental Research Funds for the Central Universities (1191329835), Postdoctoral Science Foundation of China (2015M570330), and Key projects of Ningxia Natural Science Foundation (NZ15130). The funders had no role in the study design, data collection and interpretation, or decision to submit the work for publication.
Compliance with ethical standards
Conflict of interest
Xia Liao, Yang Bu, Shanshan Jiang, Fan Chang, Fengan Jia, Xuelian Xiao, Ge Song, Mei Zhang, Pengbo Ning, Qingan Jia declare that they have no competing interests.
Ethics approval and consent to participate
Ethical approval was obtained from the Research Ethics Committee of Fudan University and written consent was obtained from each patient. Animal protocols were approved by the ethics committee on Experimental Animals of Xi’an Jiaotong University.
Consent for publication
Availability of data and material
All data generated or analyzed during this study are included in this published article. The datasets used and/or analyzed and materials developed during the current study are available from the corresponding author by reasonable request.
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