DEPDC1 promotes cell proliferation and suppresses sensitivity to chemotherapy in human hepatocellular carcinoma

Hepatocellular carcinoma, one of the most prevalent human malignancies, ranks the third most leading cause of tumor-related mortality worldwide, with approximately 600,000 deaths occurring annually [1–3]. It is estimated by American Cancer Research Society that more than 40,000 HCC cases were newly diagnosed and approximately 28,000 patients died of this cancer in the United States in 2017 [4]. It is widely acknowledged that drug resistance and distant metastasis are partially responsible for the high mortality of HCC patients [5–7]. Although significant progress has already been achieved in the diagnosis and treatment, the long-term prognosis is still rather poor. Hence, it is of great significance to develop novel and efficient therapeutic strategies. It is recognized that gene therapy may be a promising candidate therapy against HCC.

DEP domain containing 1 (DEPDC1), which is located at 1p31.3, is a highly conserved gene among many species ranging from Caenorhabditis elegans to human [8,9]. It is well documented that DEPDC1 encodes a highly conserved 92-kDa protein, which plays crucial roles in many biological processes, including cell proliferation, cell cycle progression, cell apoptosis and signaling transduction [10–12]. Recent studies have reported that DEPDC1 is implicated in diverse types of human cancers, such as bladder cancer [13], prostate cancer [14], nasopharyngeal carcinoma [15], lung adenocarcinoma [16] and glioma [17]. Nonetheless, the biological role of DEPDC1 in HCC remains largely unknown.

In the present study, we found that DEPDC1 was significantly up-regulated in HCC tissues compared with adjacent normal tissues. In addition, increased DEPDC1 expression was associated with poor prognosis of patients. Functional investigations demonstrated that DEPDC1 facilitated HCC cell proliferation and suppressed chemotherapy sensitivity. Furthermore, mechanistic studies revealed that c-Jun N-terminal kinase (JNK) signaling pathway mediated the oncogenic function of DEPDC1 in HCC. Collectively, this study may provide some evidence for DEPDC1 as a candidate therapeutic target against HCC.

Tumor tissues and adjacent non-cancerous tissues were collected from 60 HCC patients who received surgical resection at Sichuan Academy of Medical Sciences and Sichuan Provincial People’s Hospital (Chengdu, China) between April 2010 and November 2016. Clinicopathological parameters of HCC patients were listed in Table 1. Overall survival time was defined as the interval between the date of primary surgery treatment and the date of death or last follow-up. All the patients gave their written consents. The present study was approved by the Ethics Committee of Xi’an Jiaotong University (Xi’an, China).

Four human HCC cell lines (HepG2, SK-Hep1, Huh7 and Huh6) and normal human liver L02 cell line were purchased from Chinese Academy of Sciences (Shanghai, China). All the cells were cultured in DMEM medium (Gibco, Grand Island, NY, U.S.A.) containing 10% fetal bovine serum (FBS) in 5% CO₂ humidified atmosphere at 37°C.

Cell transfection was performed using Lipofectamine 2000 according to the manufacturer’s protocols (Invitrogen, Carlsbad, CA, U.S.A.). DEPDC1 overexpression and knockdown studies were conducted using pcDNA3.1 and pLKO.1 (GenePharma Co. Ltd., Suzhou, China), respectively. Short hair RNA (shRNA) specially targeting DEPDC1 was designed by GenePharma and its sequence was as followed: 5′-GAACTATCAAGAGTAGTTCGT-3′. Transfection efficiency was examined after 48-h incubation.

Total RNA was extracted using RNeasy Kit (Qiagen, Valencia, CA) following the manufacture’s protocol. cDNA was synthesized by PrimeScript RT Master Mix (Takara, Dalian, China) based on the manufacturer’s instructions. The qRT-PCR analysis was carried out under an ABI Prism 7500 Fast Real-time PCR system using a SYBR Premix Ex Taq (Takara). GAPDH was used as an internal control. The sequences of primers were as followed: DEPDC1, forward 5′-ACGAAGGTATCCAGAATTG-3′and reverse 5′-AGATAATACCCAGTGAGGGA-3′; GAPDH, forward 5′-CTCCTCCACCTTTTGACGCTG-3′and reverse 5′-TCCTCTTGTGCTCTTGCTGG-3′. Relative DEPDC1 expression levels were calculated using 2^−△△C_t method.

Cell viability was measured using cell counting kit-8 (CCK8; Dojindo, Kumamoto, Japan). Cells (∼1 × 10³ per well) were seeded into 96-well plates and incubated for different time periods. Afterwards, 11 μl of CCK8 was added and incubated for another 4 h. The absorbance was determined using microplate reader (BioRad, Hercules, CA, U.S.A.) at 450 nm. The chemoresistance of HCC cells to cisplatin was examined by CCK8. Various concentrations of cisplatin were supplemented into 96-well plate and incubated for 12 h. Then, CCK8 was added and incubated for another 4 h. The absorbance was measured to evaluate the chemoresistance to cisplatin. All the experiments were conducted in triplicates.

Colony formation assays were conducted to evaluate clonogenic capabilities of HCC cells. In brief, cells (∼1 × 10³ cells) were plated into six-well plates. The cells were cultured for 14 days to form colonies. Then the colonies were fixed by 4% paraformaldehyde for 20 min, followed by being stained with 0.2% Crystal Violet. Visible colonies were then photographed and counted under an invert light microscope (Nikon, Tokyo, Japan).

Cell protein was extracted using RIPA lysis buffer (Takara) containing 1 nM PMSF (Invitrogen, Carlsbad, CA, U.S.A.) according to the manufacturer’s instructions. Then, 20 μg protein per well was electrophoresed in 10% SDS-PAGE gels and transferred onto PVDF membrane (Millipore, Bedford, MA, U.S.A.) and incubated with different primary antibodies, including anti-DEPDC1 (ab197246), anti-GAPDH (ab181603), anti-JNK (ab176645) and anti-p-JNK (ab107407) at 4°C overnight. The membrane was then incubated with HRP-labeled secondary at temperature for 2 h. The bands were detected using an ECL Western blotting kit (Invitrogen) and analyzed on a Gel Doc XR System (Bio-Rad Laboratories, Hercules, CA, U.S.A.).

Cell apoptosis was evaluated using Annexin-V FITC/PI Apoptosis Detection Kit (BD Bioscience, CA, U.S.A.) according to the manufacturer’s protocol. Cells were collected by low-speed centrifugation and rinsed twice with ice-cold PBS. Subsequently, cells were incubated with 300 μl of binding buffer. Afterward, cells were treated with 5 μl of PI and 5 μl of Annexin V-FITC (Invitrogen) at 4°C for 20 min in the dark. Then, cell apoptosis was detected by flow cytometry (Becton Dickinson).

This animal protocol was approved by Animal Care and Use Committee of Sichuan Academy of Medical Sciences and Sichuan Provincial People’s Hospital (Chengdu, China). Ten 4-week-old male nude mice were purchased from the Model Animal Research Institute of Nanjing University (Nanjing, China). Xenograft models were established to evaluate the effects of DEPDC1 depletion on tumor growth in vivo. Briefly, Huh6 cells (5 × 10⁶) treated with pLKO.1 and pLKO.1-DEPDC1 were subcutaneously injected into the flanks of the mice. Tumor size was measured using slide caliper every 3 days, and tumor volumes were calculated according to the formula:volume (mm³) = [width² (mm²) × length (mm)]/2. At day 21 post-inoculation, all the nude mice were killed, and the tumors were harvested and weighted.

Paraffin-embedded tissues were sectioned at 4.5-μm thickness. After being dewaxed and hydrated, sections were incubated with 3% H₂O₂ for 30 min to block the endogenous peroxidase (POD) activity. Following antigen recovery by repeated cooling and heating, 5% bovine serum albumin (BSA) was applied to block non-specific binding. The sections were then incubated with primary antibodies overnight at 4°C. Anti-DEPDC1 (ab197246) and anti-Ki67 (ab92742) were purchased from Abcam and used at a dilution of 1:500. After being rinsed with PBS three times for 5 min each, slices were treated with biotinylated secondary antibody for 1 h, followed by incubation with streptavidin-horseradish peroxidase (HRP) for 20 min. Diaminobenzidine substrate was used to visualize ZNF322-positive cells. Slides were then observed under a microscope (Nikon).

TUNEL analysis was conducted to monitor the apoptosis in the collected tumors using the In Situ Cell Death Detection Kit (Roche Diagnostic, Basel, Switzerland) following the manufacturer’s instructions. In brief, sample slides were incubated with 50 μl of TUNEL reaction mixture for 1 h at 37°C in the dark. After being rinsed with PBS three times, cells were then labeled with TdT labeling reaction mix. Positively stained cells were subsequently observed and photographed under an EVOS FL microscope (Thermo Fisher Scientific, Waltham, MA, U.S.A.).

All the values were expressed as mean ± SD. SPSS18.0 software was applied to conduct statistical analysis. Differences between three or more groups were compared using analysis of variance (ANOVA) followed by Dunnett’s multiple comparison. Differences between two groups were compared using Student’s t test. The log-rank and Kaplan–Meier survival analysis were used to determine the correlation between DEPDC1 expression and overall survival. Fisher’s exact test was performed to evaluate the relationship between DEPDC1 expression and clinicopathological characteristics of patients. P < 0.05 was considered statistically significant.

Even though DEPDC1 has been identified as a crucial regulator in multiple types of human neoplasms, its role in HCC remains poorly understood. To characterize the biological role of DEPDC1 in HCC, we first determined its mRNA expression levels in 60 pairs of tumorous tissues and adjacent normal liver tissues by qRT-PCR analysis. As presented in Figure 1A, HCC tissues exhibited higher DEPDC1 mRNA expression levels than matched para-cancerous tissues. Besides, Western blotting analysis and IHC staining showed that DEPDC1 protein expression was significantly up-regulated in cancerous tissues compared with corresponding normal tissues (Figure 1B and C). To assess the relationship between DEPDC1 expression and overall survival of HCC patients, tumorous tissues were classified into high DEPDC1 expression group and low DEPDC1 expression group based on the average value of its mRNA expression levels. The log-rank test and Kaplan–Meier survival analysis demonstrated that patients with high DEPDC1 expression experienced a significantly shorter overall survival (Figure 1D). Furthermore, Fisher’s exact test showed that high DEPD1 expression was associated with larger tumor size and advanced TNM stage (Table 1). Consistently, we found that DEPDC1 was significantly up-regulated in HCC cell lines (HepG2, Huh7, SK-Hep1 and Huh6) in comparison with normal human liver L02 cells (Figure 1E). Collectively, our data indicate that increased DEPDC1 expression correlates with poor prognosis of HCC patients.

In view of the findings mentioned above, we speculated that DEPDC1 was involved in human hepatocellular carcinogenesis and progression. To further investigate the biological role of DEPDC1 in HCC, we performed overexpression and knockdown studies in HepG2 cells (lowest endogenous DEPDC1 expression) and Huh6 cells (highest endogenous DEPDC1 expression), respectively. Transfection efficiency was determined by Western blotting (Figure 2A). As evident from CCK8 assays, DEPDC1 overexpression promoted Hep2G cell viability compared with control group, whereas DEPDC1 ablation inhibited Huh6 cell viability (Figure 2B). As shown in Figure 2C, DEPDC1 overexpression enhanced clonogenic ability of HepG2 cells in comparison with control group, while DEPDC1 depletion suppressed clonogenic capability of Huh6 cells. As displayed in Figure 2D, DEPDC1 overexpression repressed HepG2 cell apoptosis compared with control group, whereas DEPDC1 knockdown accelerated Huh6 cell apoptosis. Taken together, these results suggest that DEPDC1 promotes HCC cell proliferation and survival.

To validate the effects of DEPDC1 in vivo, we established murine xenograft models by subcutaneous injection of shRNA-DEPDC1-treated Huh6 cells in the flank. As shown in Figure 3A, DEPDC1 knockdown significantly inhibited tumor growth in vivo. Besides, IHC staining demonstrated that less Ki67-positive cells were observed in the tumors collected from the shRNA-DEPDC1 group compared with control group (Figure 3B). Furthermore, TUNEL analysis revealed that DEPDC1 knockdown promoted cell apoptosis in the tumor compared with negative control group (Figure 3C). To sum up, our results suggest that DEPDC1 depletion inhibits tumor growth in vivo.

Previous studies have demonstrated that JNK signaling pathway plays critical roles in cancer cell proliferation, growth and motility. To clarify the potential molecular mechanisms underlying HCC, we evaluated the effects of DEPDC1 overexpression or ablation on JNK signaling pathway. As presented in Figure 4A, DEPDC1 overexpression promoted the phosphorylation of JNK compared with control group, whereas DEPDC1 knockdown suppressed the phosphorylation of JNK. Furthermore, Western blotting analysis showed that usage of SP600125, a specific JNK inhibitor, reversed the promoting effects of DEPDC1 overexpression on the phosphorylation of JNK (Figure 4B). Besides, we found that JNK specific inhibitor SP600125 also reversed the inhibitory effect of DEPDC1 overexpression on HepG2 cell apoptosis (Figure 4C). Taken together, these findings indicate that DEPDC1 promotes activation of JNK signaling pathway in HCC cells.

To verify the potential molecular mechanisms by which DEPDC1 promotes HCC viability and survival, we performed subsequent studies. As displayed in Figure 5A, DEPDC1 overexpression enhanced viability of cisplatin-treated HepG2 cells compared with control group, whereas DEPDC1 depletion inhibited viability of cisplatin-treated Huh6 cells. Furthermore, FCM analysis demonstrated that DEPDC1 overexpression inhibited apoptosis of cisplatin-treated HepG2 cells, whereas DEPDC1 depletion promoted apoptosis of cisplatin-treated Huh6 cells (Figure 5B). Besides, we found that JNK specific inhibitor SP600125 suppressed viability and survival of cisplatin-treated Huh6 cells compared with control group, suggesting that SP600125 possesses similar effect on HCC cell viability and survival to DEPDC1 ablation (Figure 5C). Collectively, these results indicate that DEPDC1 ablation enhances sensitivity of HCC cells to chemotherapy through JNK pathway.

HCC, one of the most lethal malignancies, has imposed great pressure on public health and posed enormous threats to human life worldwide [18,19]. It is widely acknowledged that dys-regulation of tumor-related signaling pathways is implicated in human carcinogenesis and development [20–24]. Furthermore, targeting dysregulated cancer-related signal pathways is considered as a promising therapeutic strategy for HCC patients. Even through great advances have already been achieved in the therapy of HCC, the long-term prognosis remains rather discouraging. Hence, novel and efficient therapeutic strategies are in urgent demand. Accumulating evidence has demonstrated that aberrant expression of crucial tumor-related genes contributes to tumorigenesis and malignant progression of human HCC. Wang et al. [25] demonstrated that FAM83D was up-regulated and promoted cell proliferation through activating MEK/ERK signaling pathway in HCC. Chen et al. [26] found that IQGAP1 was overexpressed and promoted cell proliferation by activating Akt signaling in HCC. Fang et al. [27] reported that FBI-1 promoted cell proliferation and enhanced chemotherapy resistance in HCC. Dai et al. [28] revealed that GOLPH3 promoted cell aggressiveness via activating NF-κB pathway in HCC. Despite the fact that a great deal of studies have focused on the ectopic expression of cancer-related genes up to now, much attention should be paid to identifying more genes which are implicated in the initiation and development of HCC.

Past studies showed that DEPDC1 was implicated in multiple types of human neoplasms. Harada and co-workers [13] demonstrated that DEPDC1 contributed to bladder cancer oncogenesis. Huang and co-workers [14] reported that DEPDC1 promoted prostate cancer cell proliferation and tumor growth via activation of E2F signaling pathway. Feng et al. [15] revealed that DEPDC1 facilitated nasopharyngeal carcinoma cell cycle progression and motility. Wang and co-workers [17] found that increased DEPDC1 expression was associated with poor prognosis of patients with lung adenocarcinoma. However, the biological role of DEPDC1 in HCC remains poorly understood. Hence, it may be worthy to characterize the role of DEPDC1 in HCC.

To investigate the role of DEPDC1 in HCC, we first analyzed the expression of DEPDC1 in tumorous tissues and matched adjacent non-cancerous tissues and found that DEPDC1 was highly expressed in HCC tissues. In addition, we noticed that increased DEPDC1 expression predicts poor prognosis of patients. Functional studies showed that DEPDC1 overexpression facilitated HCC cell proliferation, inhibited cell apoptosis and chemotherapy sensitivity, whereas DEPDC1 depletion suppressed cell proliferation, promoted cell apoptosis and chemotherapy sensitivity. Emerging evidence has demonstrated that abnormal control of cancer-related signaling transduction pathways contributes to HCC initiation and malignant development [29–31]. It is well documented that activation of JNK signaling pathway contributes to carcinogenesis and tumor progression of various types of human malignancies [32–35]. In the present study, mechanistic investigations identified that DEPDC1 overexpression promoted HCC cell viability and chemotherapy resistance by activating JNK signaling pathway, while DEPDC1 knocked suppressed cell viability and chemotherapy resistance by down-regulating JNK pathway; meanwhile, JNK specific inhibitor SP600125 was observed to have similar effects to DEPDC1 depletion, indicating that JNK pathway mediated the oncogenic effects of DEPDC1 in HCC. DEPDC1-induced JNK pathway activation may be attributed to its recruiting phosphorylases to facilitate phosphorylation of JNK1/2 proteins [36]. There is no doubt that some limitations exist in the current studies. In our further work, we will conduct corresponding studies to explore the effects of DEPDC1 on HCC cell motility and metastasis to better characterize its biological functions.

In conclusion, the present study for the first time demonstrated that DEPDC1 was significantly up-regulated in HCC tissues compared with matched non-cancerous tissues and that high DEPDC1 expression correlated with poor prognosis of patients. Furthermore, our findings indicate that DEPDC1 promotes HCC cell proliferation and suppresses chemotherapy sensitivity through activating JNK signaling pathway. Therefore, our study provides some new insights into understanding the molecular mechanisms underlying hepatocellular carcinogenesis, implying that DEPDC1 may be used as a promising candidate therapeutic target against HCC.

Yue Wu, Chao Zhou and Pu Wang conceived this study, performed statistical analysis and prepared this manuscript. Mengtian Tu, Yi Huang and Fei Xiong conducted experimental manipulation and participated in statistical analysis. All the authors approved this manuscript submission.

This study was supported by National Key Specialty Construction of Clinical Pharmacy [30305030698] and Research Fund for the Doctoral Program of Sichuan Academy of Medical Sciences and Sichuan Provincial People’s Hospital [30305030843P].

The authors declare that there are no competing interests associated with the manuscript.

CCK8

cell counting kit-8

DEPDC1

DEP domain containg 1

HCC

hepatocellular carcinoma

IHC

immunohistochemical

JNK

c-Jun N-terminal kinase