TRPM2 regulates cell cycle through the Ca²⁺-CaM-CaMKII signaling pathway to promote HCC

Background:

HCC is one of the most common causes of cancer-related deaths. Transient receptor potential melastatin 2 (TRPM2), a Ca²⁺-permeable cation channel, was reported to be involved in carcinogenesis and tumor growth recently. However, whether TRPM2 is involved in the pathogenesis and progression of HCC remains unclear. Herein, we systematically elucidated the functional role of TRPM2 in HCC cell cycle regulation and proliferation.

Approach and Results:

We determine TRPM2 expression to be strongly upregulated in the tumor tissues of HCC patients and associated with a negative prognosis. TRPM2 is highly expressed in HCC cell lines Huh-7 and HepG2 cells, rather than in normal hepatocytes. Inhibition or silencing of TRPM2, or inhibition of the downstream Ca²⁺-CaM-CaMKII signaling pathway, significantly suppressed the proliferation of Huh-7 and HepG2 cells by arresting the cell cycle at the G1/S phase, accompanied with reduced expression of G1/S checkpoint proteins. Importantly, inhibition or depletion of TRPM2 remarkably slowed down the growth of patient-derived xenografts and Huh-7 xenografts in mice.

Conclusion:

Our results indicate that TRPM2 promotes HCC cell proliferation via activating the Ca²⁺-CaM-CaMKII signaling pathway to induce the expression of the key G1/S regulatory proteins and accelerate the cell cycle. This study provides compelling evidence of TRPM2 involvement in a previously unrecognized mechanism that drives HCC progression and demonstrates that TRPM2 is a potential target for HCC treatment.

INTRODUCTION

HCC is one of the most common, aggressive, and deadly cancers and and ranks third globally in cancer-related deaths.1 Despite advanced and improvements in clinical treatments, the 5-year survival rate of HCC patients remains low (~12%).2 Obtaining a more comprehensive understanding of the cellular and molecular mechanisms responsible for HCC tumorigenesis and progression is therefore of paramount importance for identifying therapeutic targets.

Increasing evidence highlights that oxidative stress and the loss of ionic homeostasis play critical roles in the initiation and progression of HCC.3 For example, abnormal increases in the production of reactive oxygen species (ROS) cause oncogenic genetic alterations in hepatocytes and thus promote the expansion of malignant cells.4 In addition, aberrant changes in the homeostasis of intracellular ions in HCC cells,3 particularly intracellular Ca²⁺ as a well-recognized and ubiquitous second messenger, are considered to be important factors in liver cell injury, uncontrolled cell proliferation, and drug resistance for HCC.5,6 Ion channels, which can simultaneously respond to oxidative stress and regulate intracellular ion homeostasis, have therefore become foci in the molecular mechanisms of HCC pathogenesis and may also serve as potential targets for pharmacotherapies of HCC.7

Transient receptor potential melastatin 2 (TRPM2) is a Ca²⁺-permeable cation channel which could be activated in response to oxidative stress8 and plays an important role in regulating intracellular Ca²⁺ homeostasis.9,10 Recent studies have documented TRPM2 expression in multiple cancers and suggested linked oncogenic or tumor-suppressive roles, depending upon the cancer type. In neuroblastomas, TRPM2 is noted as highly expressed where its activation is seen to be linked both to cellular proliferation increase and a reduction in sensitivity to chemotherapy.11,12 Links to cellular proliferation are noted in other cancers where TRPM2 is specifically connected to gastric cancer chemotherapy resistance13 and noted as a potential target for the selective treatment of prostate cancer.14 However, the mechanisms by which TRPM2 regulates the function of cancer cells remain elusive. TRPM2 has been demonstrated to function as an oxidative stress-sensitive Ca²⁺-permeable channel on the cell surface of hepatocytes and plays an important role in mediating acetaminophen-induced liver injury by causing intracellular Ca²⁺ overload.9 Statistical analysis of the International Cancer Genome Consortium (ICGC) dataset has revealed the association of higher TRPM2 expression with increased risk of HCC previously.15 However, how TRPM2 specifically contributes to HCC pathogenesis remains unclear.

In this study, we showed that TRPM2 is highly upregulated in human HCC tissues and exhibits a strong negative correlation with disease prognosis. The pharmacological or genetic intervention of TRPM2 is shown to inhibit HCC cell proliferation by arresting the cell cycle in vitro and suppress HCC growth in vivo. More specifically, we reveal that TRPM2 mediates HCC pathogenesis through the Ca²⁺-CaM-CaMKII signaling pathway to regulate the G1/S phase of the cell cycle. Collectively, these findings provide compelling evidence that TRPM2 plays a pivotal role in promoting the progression of HCC. Thus, TRPM2 may be a potential target for developing HCC therapeutics.

METHODS

Patients and tissue specimens

A total of 104 pairs of HCC tissue samples were collected from HCC patients who had undergone surgical resections between August 2014 and November 2018 at the Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital of Zhejiang University. Eighty-seven pairs of paraffin-embedded HCC tumor, nontumor, and/or peritumor tissues were used for hematoxylin and eosin and immunohistochemistry. One hundred four pairs of fresh frozen tissue samples were assessed using western blotting. All research was conducted in accordance with both the Declarations of Helsinki and Istanbul. The study was approved by The First Affiliated Hospital of Zhejiang University Ethics Committee and informed consent was obtained in advance from each patient.

Cell culture

Huh-7 were purchased from the National Collection of Authenticated Cell Cultures. HepG2, Hep3B, SNU387, and SNU449 were supplemented from American Type Culture Collection. HCC-LM3 and HL-7702 were obtained from the National Infrastructure of Cell Line Resource. MIHA was kindly provided by Professor Hui Lin, from Sir Run Run Shaw Hospital Zhejiang University School of Medicine. Cells were maintained in DMEM or RPMI-1640 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in a humidified 5% CO₂ incubator at 37°C.

Western blotting

Total proteins were extracted from the HCC patient’s tissue or cultured cells using RIPA lysis buffer. Proteins of 20–40 μg were separated into 8%–16% or 4%–20% SurePAGE. Subsequently, proteins were transferred to polyvinylidene difluoride membranes and blocked with 5% Difco skim milk in TBST, then incubated with the primary antibodies (summary in Table S3, http://links.lww.com/HC9/A210) overnight at 4°C. After washing, membranes were incubated with goat anti-rabbit or goat anti-mouse second antibody for 1 hour. Finally, protein bands were visualized and analyzed using an LI-COR Odyssey Imaging System, and quantified by Image Studio software.

CCK-8 assay

In brief, 0.5–2×10⁴ cells were seeded in 96-well plates and incubated for 12 hours before use. After treatment with different reagents for 24 hours, CCK-8 was used to determine cell viability. The absorbance at 450 nm was measured by a SynergyMx M5 microplate reader with a reference wavelength of 630 nm.

Colony formation assay

A colony formation assay was performed as described previously.16 In brief, 1–2×10⁴ cells were seeded in 6-well plates and incubated overnight for adherence. Cells were then incubated with indicated the drugs for 10–14 days with the medium changed twice a week. Cells were fixed with 4% paraformaldehyde and then stained with 0.1% crystal violet.

Flow cytometry analysis

Cells were fixed in ice-cold 75% ethanol for at least 24 hours at −20°C. After washing, cells were incubated in a propidium iodide staining solution for 30 minutes. The cell distribution was detected and analyzed on a NovoCyte flow cytometer (ACEA Biosciences).

Patient-derived xenograft (PDX) model

The PDX model was established in accordance with the protocols described previously.17 In brief, after being anesthetized with isoflurane through inhalation, fresh HCC patient tissue fragments (~2×2×2 mm³) were subcutaneously implanted into the right flank of BALB/c nude mice (6–8 wk). The established PDX model of passage 3 was used for experiments. When the long diameter of xenograft reached about 0.5 cm, mice were randomly divided into 3 groups, then intraperitoneally injected at doses of 30 mg/kg/d ACA or A10 for 28 days. The long diameter (a) and short diameter (b) of the tumor and the mouse body weight were measured every other day. Tumor volume was calculated according to the formula: V=1/2×a×b ². After 28 days, mice were sacrificed by cervical dislocation and tumors were resected and weighed. All animals were managed according to the Care and Use of Medical Laboratory Animals and all experimental protocols were approved by the Animal Care and Use Committee of The First Affiliated Hospital of Zhejiang University.

Statistical analysis

The data are presented as mean±SD obtained from at least 3 independent experiments. Statistical analyses and plotting were performed using SPSS 18.0 and GraphPad Prism 6.0 software for t tests, 1-way ANOVA followed by the Tukey or Dunnett post hoc tests, or 2-way ANOVA. The correlation between TRPM2 expression and clinicopathological features was examined by the chi-square test. The Kaplan-Meier analysis was used to compare overall survival. A value of p < 0.05 was considered statistically significant.

Other materials and methods are provided in the Supporting Information (http://links.lww.com/HC9/A210).

RESULTS

TRPM2 expression is upregulated in human HCC and exhibits a negative correlation with prognosis

To investigate the relevance of TRPM2 to HCC pathogenesis, we conducted western blotting to examine TRPM2 expression in HCC tissues from a cohort of 104 patients. The mean TRPM2 expression was significantly elevated in tumor tissues when compared with adjacent normal liver tissues of HCC patients and was noted as particularly high in HCC tissues from 88 of the 104 patients (84.4%) (Figure 1A, B). There seemed to be a strong association between upregulated TRPM2 expression and HCC. We also examined the TRPM2 expression by immunohistochemistry (Figure 1C). The TRPM2 expression in HCC tissue were higher compared with those in adjacent normal tissues from 53 of 87 examined patients (60.9%). To evaluate the association between TRPM2 expression and prognosis, we equally divided the cohort of 104 patients into 2 groups based on the TRPM2 expression level (Figure 1D). As shown in Figure 1E, the overall survival of HCC patients in the high TRPM2 group was significantly reduced compared with that of patients in the low TRPM2 group (eg, high: just over 50%, low: just over 70% for 5-y survival). To validate these findings, we further analyzed the prognostic value of TRPM2 in the GSE124535 dataset and The Cancer Genome Atlas (TCGA) database. The TRPM2 mRNA expression level was significantly higher in HCC tissues than in nontumor tissues (Figure S1A, B, http://links.lww.com/HC9/A210). The overall survival rate of patients with high TRPM2 expression was also lower than that of patients with low TRPM2 expression (Figure S1C, http://links.lww.com/HC9/A210). Finally, we explored clear correlations of TRPM2 expression with total bilirubin and tumor number (Table S1, http://links.lww.com/HC9/A210).

FIGURE 1.

TRPM2 expression is upregulated in human HCC and exhibits a negative correlation with prognosis. (A) Representative western blot showing significant upregulated of TRPM2 protein expression in HCC tumors (T) compared with matched adjacent normal (N) tissues. (B) Mean TRPM2 protein expression from 104 HCC patients, showing TRPM2/β-actin in tumor normalized to matched normal tissues; ****p < 0.0001. (C) H&E and TRPM2 staining in HCC tumor, peritumor, and normal tissues. Scale bar, 200 or 50 μm. (D) Representative images showing low and high TRPM2 staining in HCC tissues. (E) High-level TRPM2 expression correlates with poor survival outcome in HCC (high, n=52; low, n=52) cohorts based on survival analysis. Abbreviations: H&E, hematoxylin and eosin; TRPM2, transient receptor potential melastatin 2.

TRPM2 expression in HCC confers a critical mechanism to promote cell proliferation

To understand the role of TRPM2 in HCC pathophysiology, we examined the expression of TRPM2 in HCC cell lines (HCC-LM3, Hep3B, SNU387, SNU449, HepG2, and Huh-7) and normal hepatocytes (MIHA and HL-7702). The TRPM2 expression at both the mRNA and protein levels was significantly higher in Huh-7 and HepG2 cells than in normal hepatocytes and other HCC cells (Figure 2A and Figure S2A, http://links.lww.com/HC9/A210). As shown in whole-cell patch recordings, the intracellular application of ADPR, the TRPM2 channel-specific activator,18 elicited no or negligible ionic currents in HL-7702 or MIHA cells (Figure S2B, C, http://links.lww.com/HC9/A210), but robust current responses in Huh-7 and HepG2 cells. These ADPR-induced currents were abolished by A10, a specific TRPM2 inhibitors synthesized by our laboratory19 (Figure 2B, C). Furthermore, exposure to H₂O₂, which is known as an activator of the TRPM2 channel,18 resulted in a massive Ca²⁺ influx in Huh-7 and HepG2 cells, as documented using live cell calcium imaging. Such Ca²⁺ responses were also inhibited by treatment with A10 or another TRPM2 inhibitor ACA (Figure 2D, E). Taken together, these results provide clear evidence to indicate that the Ca²⁺-permeable TRPM2 channel is highly expressed in Huh-7 and HepG2 cells, and mediates ROS-induced Ca²⁺ signaling.

FIGURE 2.

TRPM2 channel is functionally expressed in HCC cells, inhibition of TRPM2 activity or expression significantly suppresses the proliferation and growth of HCC cells. (A) Western blots showing TRPM2 protein expression was significantly increased in Huh-7 and HepG2 compared with normal hepatocytes. (B and C) Whole-cell patch clamp recordings showing inward currents induced by 500 μM ADPR (left panel) and I–V curves (right panel); 30 μM A10 was used to suppress TRPM2 specific currents. (D and E) Live cell calcium imaging showing that exposure to 3 mM H₂O₂ induced an intracellular Ca²⁺ increase, which could be inhibited by A10 or ACA (30 μM). Left: time course of change in Fluo-4 fluorescence intensity normalized to the baseline (F/F0). Right: mean peak F/F0 values. Cell proliferation activity showed significant suppression in A10 and ACA treatment groups (F) or TRPM2 knockdown (H) measured by CCK-8 assays. *p < 0.05, ***p < 0.001, ****p < 0.0001, A10 or shTRPM2 versus control; *p < 0.05, **p < 0.01, ****p < 0.0001, ACA or shTRPM2-2 versus control. (G and I) Colony formation capacity showed an obvious decrease in A10 treatment group (G) or TRPM2 knockdown group (I). (J and K) Cell proliferation capacity markedly decreased when treated with A10 for 24 hours or TRPM2 knockdown as measured by EdU staining. *p < 0.05; ****p < 0.0001. Abbreviations: ACA, N-(p-amylcinnomoyl) anthralic acid; ADPR, ADP ribose; TRPM2, transient receptor potential melastatin 2.

We next investigated whether TRPM2 plays a role in driving cell growth and colony formation, which are the salient functional properties of cancer cells. In Huh-7 and HepG2 cells, cell growth (Figure 2F) and cell proliferation (Figure 2G) were strongly suppressed by treatment with A10 or ACA. Specific knockdown of TRPM2 with lentivirus-coated shRNA exhibited similar results (Figure 2H, I and Figure S3A, D, http://links.lww.com/HC9/A210). In contrast, such pharmacological and genetic interventions did not affect cell growth and colony formation in normal hepatocyte HL-7702 and MIHA cells (Figure S4A, F, http://links.lww.com/HC9/A210). Furthermore, cell proliferation in both Huh-7 and HepG2 cells, revealed by EdU labeling, was attenuated by treatment with A10 (Figure 2J) or TRPM2 silencing (Figure 2K). These results consistently support the understanding that upregulation of TRPM2 expression promotes HCC cell proliferation.

TRPM2 promotes HCC cell proliferation by regulating the G1/S phase of the cell cycle

To gain insight into the mechanisms by which TRPM2 regulates HCC cell proliferation, we conducted a bioinformatics analysis of gene expressions from the GSE124535 dataset and the TCGA database. We found that significantly differentially expressed genes from the GSE124535 dataset were mostly enriched in cell cycle pathways for HCC patients (Figure 3A). Gene set enrichment analysis using the TCGA database also suggested that cell cycle-associated pathways related to key Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways were significantly upregulated in HCC patients (Figure 3B, C). In particular, the expression levels of key checkpoint regulators of the G1/S phase, including cyclin D1, cyclin D2, cyclin D3, cyclin E1, cyclin E2, cyclin A1, and cyclin A2, were higher in HCC tissues of high TRPM2 expression than that in low TRPM2 expression, or in normal tissues (Figure 3D). Consistent with such findings from bioinformatics analysis, strong immunostaining of Ki-67, a protein marker of cell proliferation, was found in HCC tissues with high TRPM2 expression in our cohort of HCC patients (Figure 3E). Similarly, the expression of proliferating cell nuclear antigen, another key protein marker of cell proliferation, was strongly upregulated in HCC tissues (Figure 3F, G). These results provide further evidence lending strong support to the association of the greater proliferation ability of HCC cells with higher TRPM2 expressions. Moreover, the expression levels of cyclin D1, cyclin E1, and cyclin A2 were remarkably increased in HCC tissues, but with no significant difference in the expression of cyclin B1 (Figure 3F, G). These results indicate that TRPM2 promotes HCC cell proliferation primarily by regulating the G1/S phase of the cell cycle.

FIGURE 3.

TRPM2 expression is positively correlated with the altered cell cycle. (A) Metascape enrichment analysis of the differentially expressed genes from the GSE124535 dataset. The top 20 enriched pathways are shown, and terms of interest are labeled in red. (B and C) Gene Set Enrichment Analysis of differentially expressed genes of the HCC cohort of TCGA database, followed by enrichment analysis of GO and KEGG. (D) Relative mRNA expression levels for cell cycle–related proteins in TCGA database of normal (n=50) and HCC tissues (low TRPM2, n=187; high TRPM2, n=187). (E) H&E staining, TRPM2 and Ki-67 immunostaining of liver sections from HCC patients. Scale bar, 100 μm. (F) Western blots showing the expression of indicated proteins in tumor (T) and matched nontumor (N) tissues from HCC patients. (G) Mean values for indicated proteins. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Abbreviations: GO, Gene Ontology; H&E, hematoxylin and eosin; KEGG, Kyoto Encyclopedia of Genes and Genomes; PCNA, proliferating cell nuclear antigen; TCGA, The Cancer Genome Atlas; TRPM2, transient receptor potential melastatin 2.

We further confirmed the critical role of TRPM2 for HCC cells in regulating the cell cycle by using the TRPM2 inhibitor A10 or TRPM2-shRNA. As expected, both A10 and TRPM2-shRNA treatments increased the proportion of cells in the G1 phase but decreased the proportion of cells in the S phase in Huh-7 and HepG2 cells (Figure 4A–D). We also noted that the proportion of cells in the G2 phase in Huh-7 cells had decreased upon treatment with TRPM2-shRNA (Figure 4C, D). By contrast, there were no significant effects on the cell cycle of HL-7702 and MIHA cells (Figure S5A–D, http://links.lww.com/HC9/A210). To further validate the results, we detected the expression level or activity of cell cycle key proteins. As shown in Figure 4E–H, all key G1/S checkpoint regulators, cyclin D1, p-CDK4 (Thr172), cyclin E1 and p-CDK2 (Thr160), were also downregulated in Huh-7 and HepG2 cells upon treatment with A10 or TRPM2-shRNA. Such pharmacological or genetic interventions gave no significant effect on the protein expression level or kinase activity of cyclin B1, p-CDC2 (Tyr15), JNK, or p-JNK (Thr183/Tyr185) (Figure 4E, H and Figure S6A–D, http://links.lww.com/HC9/A210). Collectively, these results provide strong evidence to support the understanding that TRPM2 promotes HCC cell proliferation by regulating the G1/S phase of the cell cycle. Considering that the Wnt/β-catenin pathway may be involved in the regulation of cyclin D1 expression, we also detected the expression of Key regulatory proteins of the Wnt/β-catenin pathway, such as Wnt3α, β-catenin, β-catenin (Ser37), β-catenin (Thr41/Ser45) and c-myc,20 however, no remarkable changes were observed under the treatment of A10 (Figure S6E, F, http://links.lww.com/HC9/A210). Therefore, we believe that the Wnt/β-catenin pathway might not be affected by the TRPM2-regulated cyclin D1 expression.

FIGURE 4.

Inhibition of TRPM2 activity or expression induces G1/S phase cell cycle arrest in HCC cells. Flow cytometry analysis of the cell cycle distribution of Huh-7 and HepG2 cells under control, treatment with 30 μM A10 (A) or TRPM2-shRNA (C) in the G1, S, and G2 phases (B and D). (E and F) Representative western blots showing the expression of cell cycle key proteins under control, A10 treatment group or TRPM2 knockdown group. (G and H) Mean protein expression levels of indicated proteins as shown in (E) and (F). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Abbreviation: TRPM2, transient receptor potential melastatin 2.

The Ca²⁺-CaM-CaMKII pathway mediates TRPM2-dependent promotion of HCC cell proliferation

The TRPM2 channel is known to regulate cellular proliferation, as well as its other functions, through mediating Ca²⁺ influx to raise the intracellular Ca²⁺ level.11,12,21,22 To confirm whether TRPM2 promotes cell proliferation by regulating Ca²⁺, we used a membrane-permeable intracellular Ca²⁺ chelator BAPTA-AM. As shown in Figure 5A and B and Figure S7A and B (http://links.lww.com/HC9/A210), cell proliferation in both Huh-7 and HepG2 cells was significantly suppressed by treatment with BAPTA-AM. As shown by EdU labeling, the proliferation activity of these cells was also strongly reduced by treatment with BAPTA-AM (Figure 5C, D) where the cell cycle was arrested in the G1/S phase after BAPTA-AM treatment (Figure 5E, F). These results support the understanding that Ca²⁺ plays a key role in the regulation of proliferation as mediated by TRPM2.

FIGURE 5.

Inhibition of TRPM2-mediated Ca²⁺-CaM-CaMKII pathway significantly suppresses cell proliferation and induces G1/S phase cell cycle arrest. (A and B) Colony formation capacity showed significant inhibition under treatment of BAPTA-AM (10 μM) or W-7 (40 μM) and/or combined with A10 (30 μM) for 10–14 days. (C and D) Cell proliferation capacity markedly decreased when treated with BAPTA-AM or W-7 for 24 hours as measured by EdU staining. (E and F) Flow cytometry analysis of the cell cycle distribution under treatment with BAPTA-AM or W-7 for 24 hours in the G1, S, and G2 phases. (G–I) Flow cytometry detected the cell distribution in the G1, S, and G2 phrases (G and H) and EdU labeling analysis of cell proliferation (I), respectively, under KN-93 (5 μM) treatment for 24 hours. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Abbreviation: TRPM2, transient receptor potential melastatin 2.

As a second messenger, Ca²⁺ regulates cell proliferation by activating or inhibiting Ca²⁺-related regulatory proteins.23 Calmodulin (CaM) is the most common Ca²⁺-binding protein, which not only binds to Ca²⁺ but also is able to act as an important Ca²⁺ sensor to monitor intracellular Ca²⁺ dynamics.24 Moreover, some studies have confirmed that an increase in CaM level is one of the most important markers for the rapid proliferation of tumor cells.25,26 Therefore, we hypothesized that TRPM2-mediated increases in intracellular Ca²⁺ levels regulate cell proliferation by engaging CaM. As shown in Figure 5A and B and Figure S7A and B (http://links.lww.com/HC9/A210), the cell proliferation was significantly suppressed upon treatment with W-7, a specific CaM inhibitor, but with no further reduction when adding A10 in combination with W-7 in both Huh-7 and HepG2 cells. Similarly, proliferation activity was strongly reduced by treatment with W-7 (Figure 5C, D), and cell cycle was arrested in the G1/S phase in both HCC cells (Figure 5E, F). Furthermore, the percentage of cells transiting from the G1 to S phase, after being synchronized to the G1/S boundary by double-thymidine block, was decreased by treatment with A10, BAPTA-AM, or W-7 in Huh-7 cells (Figure S7C, http://links.lww.com/HC9/A210). Taken together, these results indicate that the TRPM2-mediated Ca²⁺ signaling plays a crucial role in the promotion of HCC cell proliferation through CaM-dependent regulation of the G1/S phase of the cell cycle.

How exactly does Ca²⁺ and CaM regulate the cell cycle? It is known that Ca²⁺/CaM-dependent protein kinase II (CaMKII) is important in regulating the expression of cell cycle regulatory proteins.27 As confirmed by western blotting, CaMKII activation, occurring through phosphorylation at Thr286, is inhibited by treatment with A10, BAPTA-AM, or W-7 in Huh-7 and HepG2 cells (Figure 6A, C). This indicates that CaMKII is activated through the coordinated actions of TRPM2-mediated Ca²⁺ and CaM. Furthermore, cell cycle arrest in the G1/S phase was induced (Figure 5G, H), and cell proliferation was inhibited (Figure 5I) in both HCC cells upon treatment with KN-93, a competitive inhibitor of CaMKII. These results suggest that the Ca²⁺-CaM-CaMKII signaling pathway mediates TRPM2-dependent regulation of the G1/S phase of cell cycle and promotion of cell proliferation in HCC cells.

FIGURE 6.

Inhibition of the TRPM2 channel and downstream Ca²⁺-CaM-CaMKII pathway significantly suppresses the expression of G1/S phase cell cycle–related proteins. Representative western blots showing the expression of indicated proteins in Huh-7 and HepG2 cells treated with A10 (30 μM), BAPTA-AM (10 μM), or W-7 (40 μM) for 24 hours (A), or treated with KN-93 (5 μM) for 24 hours (B), and mean expression of indicated proteins (C and D). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Abbreviation: TRPM2, transient receptor potential melastatin 2.

There is evidence that the cyclin D/CDK4 and cyclin E/CDK2 complexes play key roles in the regulation of the G1/S phase.28 The expression of cyclin D1 and the activation of CDK4 were significantly inhibited by treatment with A10, BAPTA-AM, W-7, or KN-93 (Figure 6A–D). The formation and activation of the cyclin D/CDK4 complex is also being increasingly shown to result in 2 key outcomes. On the one hand, it can inhibit the expression of the CDK2 inhibitors p21 and p27, thus enhancing the activity of CDK2.29 On the other hand, it can inhibit Rb phosphorylation, thus promoting E2F dissociation from the Rb/E2F complex and entry into the nucleus, which in turn promotes the expression of cyclin E.30–32 In Huh-7 and HepG2 cells treated with A10, BATPA-AM, W-7 or KN-93, with the reduction of cyclin D1 and CDK4, the expression levels of p21 and p27 upregulated, while the expression levels of cyclin E and p-CDK2 (Thr160) downregulated (Figure 6A–D). We also detected the mRNA expression levels of cyclin D1, cyclin E1, p21, and p27, and obtained similar results (Figure S8A, B, http://links.lww.com/HC9/A210). Meanwhile, the phosphorylation levels of Rb at Ser807/811 and Ser795 sites were significantly inhibited (Figure 6A–D). Similar results were observed in Huh-7 and HepG2 cells after treatment with TRPM2-shRNA (Figure S9A, B, http://links.lww.com/HC9/A210). There were no further effects upon treatment with W-7 upon the expression of these proteins in cells that had received prior treatment with TRPM2-shRNA (Figure S9A, B, http://links.lww.com/HC9/A210). These results support the understanding that TRPM2 promotes HCC cell proliferation through activating the Ca²⁺-CaM-CaMKII pathway to regulate the expression of G1/S phase-related proteins.

TRPM2 promotes the growth of PDX and Huh-7 xenografts in mice

To provide more direct evidence of the vital role of TRPM2 in tumorigenesis and the progression of HCC, we constructed PDX in nude mice. Figure 7A shows the model diagram of PDX using tumor tissue from HCC patients. As examined using western blot, the TRPM2 expression was significantly upregulated in tumor tissue of HCC patients in the PDX model (Figure 7B). The growth of PDX was significantly slower upon daily treatment with ACA or A10 through intraperitoneal injection for 28 days (Figure 7C, D), while the body weight of mice exhibited no change (Figure 7G). The tumor weight and tumor growth curve of PDX was significantly decreased by treatment with ACA or A10 (Figure 7E, F). These results indicate that inhibiting TRPM2 significantly suppresses the development of PDX. Similarly, the growth of xenografts constructed by subcutaneous injection of Huh-7 cells in nude mice was reduced by prior treatment of Huh-7 cells with TRPM2-shRNA (Figure 7K, L). The expression level of Ki-67 in both PDX and Huh-7 xenografts was constantly and markedly downregulated by pharmacological inhibition or knockdown of TRPM2 (Figure 7H, M). Moreover, the expression levels of cyclin D1 and cyclin E1 in PDX xenografts were significantly reduced by treatment with A10 (Figure 7I, J). Such results from in vivo models of PDX and Huh-7 xenografts provide direct evidence to support the important role of TRPM2 in driving HCC progression.

FIGURE 7.

Inhibition or knockdown of TRPM2 significantly slows down the growth of PDX and Huh-7 xenografts. (A) Diagram illustrating the PDX model. (B) Western blot analysis of the TRPM2 expression of patient tissue samples used for PDX modeling. (C) Summary of the growth of PDX xenografts under the treatment of control (n=5), ACA (n=5), or A10 (n=5). (D) Size of exfoliated PDX tissues. (E) Mean tumor weights of PDX xenografts. (F) The tumor volume curve of PDX. (G) Body weight-time curve of PDX nude mice. (H) H&E staining, TRPM2 and Ki-67 immunostaining of PDX under the indicated conditions. Scale bar, 100 μm. (I and J) Representative western blots showing the expression of TRPM2, cyclin D1, and cyclin E1 in PDX. (K) Summary of the growth of Huh-7 xenograft model in shNC (n=9) or shTRPM2 (n=9). (L) Mean Huh-7 xenografts weight. (M) H&E staining, TRPM2 and Ki-67 immunostaining of Huh-7 xenografts. Scale bar, 100 μm. *p < 0.05; **p < 0.01; ****p < 0.0001. Abbreviations: ACA, N-(p-amylcinnomoyl) anthralic acid; H&E, hematoxylin and eosin; PDX, patient-derived xenografts; TRPM2, transient receptor potential melastatin 2.

DISCUSSION

Intracellular Ca²⁺ homeostasis regulation is essential for maintaining the physiological functions of cells. Previous studies have implicated Ca²⁺ dyshomeostasis as having a key role in tumorigenesis.5,6 In this study, we reveal that TRPM2-mediated Ca²⁺ influx promotes the cellular proliferation of cancer cells by activating the CaM-CaMKII pathway and upregulating the activities of CDK4 and CDK2 to accelerate the G1/S phase transition (Figure 8).

FIGURE 8.

Model pattern of TRPM2 promoting HCC proliferation by mediating G1/S phase cell cycle regulation. TRPM2 is upregulated in HCC cells and, through the Ca²⁺-CaM-CaMKII signaling pathway, upregulates cyclin D1 expression of and activation of CDK4. This, on the one hand, inhibits p21 and p27 to decrease the CDK2 activity while, on the other hand, stimulating Rb monophosphorylation to promote the expression of cyclin E, which stimulates the formation of the cyclin E/CDK2 complex, resulting in phosphorylation at the Thr160 site and activation of CDK2 and further facilitating the phosphorylation and activation process of Rb. Activation of Rb promotes its dissociation from the Rb/E2F complex and its translocation to the nucleus to then drive the expression of S synthetic-related proteins and thereby enhance G1 to S transition. Abbreviations: PDX, patient-derived xenografts; TRPM2, transient receptor potential melastatin 2.

TRPM2 has been reported to be involved in the regulation of proliferation in a variety of tumor cells such as neuroblastomas,11 gastric cancers,13 and T-cell leukemias.33 In neuroblastomas, TRPM2 overexpression enhances proliferation by modulating HIF-1/2α, mitochondrial function, and mitophagy.11 Whereas in gastric cancer cells, TRPM2 has been noted to mediate cell proliferation in a JNK-dependent manner.13 Conversely, in T-cell leukemia, ionizing radiation stimulated TRPM2-mediated Ca²⁺ entry and CaMKII activation to inhibit the expression of cdc25b and cdc2, thereby inducing G2/M arrest and the inhibition of cell proliferation.33 These findings suggest that the mechanisms by which TRPM2 regulates cell proliferation may be context-dependent or cancer cell-type specific.

Elevated oxidative status has been found in many types of cancer cells, which contributes to carcinogenesis.34,35 Emerging evidence has revealed that oxidative stress-sensitive TRP channels are involved in many cancers. Among such TRP channels, TRPC3, TRPV1, TRPV2, TRPM2, TRPM7, TRPM8, and TRPA1 have been noted as ROS sensors.36–39 For instance, TRPA1 acts in tumorigenesis by promoting oxidative stress tolerance and resistance to ROS-producing chemotherapies36;stimulation of TRPM7 suppresses cancer cell proliferation and metastasis by evoking apoptosis, cell cycle arrest, and ROS elevation40; TRPV2 mediates Ca²⁺ influx to impair glioblastoma stem-like cell proliferation and promotes differentiation41; and downregulating TRPC3 expression leads to a reduction of proliferation by suppressing EGF-induced Ca²⁺ influx induced G2/M cell cycle arrest in ovarian cancer cells.39 Those studies support the understanding that oxidative stress-sensitive TRP channels are often closely involved in abnormal proliferation and alterations of the cell cycle in tumor cells. Recently, TRPM2 was also reported in immune cells to mediate ROS-sensitive Ca²⁺ signaling mechanisms that play crucial roles in several cell functions.42 In this study, we found that TRPM2 regulated cell proliferation and the cell cycle–related factors in HCC by targeting the Ca²⁺-CaM-CaMKII signaling pathway.

Accumulating evidence now indicates that the unlimited malignant proliferation ability of tumor cells is one of the key factors for their malignant progression.43 Intracellular Ca²⁺ plays a central role in this throughout the cancer cell cycle and is especially important to the G1/S phase transition.44 There are many Ca²⁺ signaling proteins, including voltage-operated Ca²⁺ channels, store-operated Ca²⁺ channels, inositol trisphosphate receptors, Ca²⁺-Mg²⁺ ATPase, and Ca²⁺/CaM kinases, that have been identified as potential targets for therapeutic treatment at different stages of HCC or other cancers.45 For example, the TRPV4 channel promotes cell proliferation through its modulation of the ERK signaling pathway in tumor tissues of HCC46. The TRPV4 channel-mediated Ca²⁺ influx was found to be critical for the G1/S phase transition in human colon cancer47. Mitochondrial calcium uniporter–induced mitochondrial Ca²⁺ uptake induces MMP activation and cell motility through the ROS activated JNK pathway, thereby promoting HCC metastasis.48 In this study, we demonstrated that inhibition of the activity or expression of TRPM2 in HCC cells resulted in decreased CaM expression and attenuated CaM KII activation, thereby inhibiting the expression of key proteins regulating the G1/S phase, and thus inducing G1/S phase cell cycle arrest. Other studies have also reported that TRPM2 participates in the regulation of the G2/M phase of the cell cycle, such as where TRPM2 silencing causes G2/M arrest in non–small cell lung cancer by activating the JNK pathway.49 However, in HCC cells, we found that the inhibition or knockdown of TRPM2 neither induced G2/M phase cell cycle arrest nor activated the JNK pathway, suggesting that there may be varied mechanisms by which TRPM2 regulates the cell cycle in different tumors. Such potential differences should be highlighted for further study. Meanwhile, we found that the phosphorylation level of MEK1/2 was remarkably reduced under the treatment of TRPM2 inhibitors or the inhibition of the Ca²⁺-CaM-CaMKII pathway in Huh-7 and HepG2 cells (Figure S10A–D, http://links.lww.com/HC9/A210). A similar result was also reported in a recent study demonstrating that TRPM2 might promote the proliferation of pancreatic cancer by downstream MAPK/MEK pathway activation.50 Since TRPM2-regulated MAPK/MEK pathway may be a general mechanism in TRPM2-mediated cancer cell proliferation, it should be of high priority for future studies to further investigate the underlying mechanism of TRPM2 regulating MAPK/MEK pathway in various cancers. In addition, CaM/CaMKII and cell cycle regulatory proteins are not suitable drug targets because of their important roles in many physiological and pathological processes. Our data suggest that TRPM2 may be developed as a novel therapeutic target for the treatment of HCC.

In summary, our study determined that TRPM2 plays a crucial role in the pathogenesis of HCC by stimulating G1/S phase cell cycle progression and cell proliferation through the Ca²⁺-CaM-CaMKII signaling pathway (Figure 8). Our findings uncover a previously unrecognized mechanism that is important in driving HCC progression. It also suggests that targeting TRPM2 for developing therapeutics to treat HCC is a potential strategy.