TIGAR Enhanced Free Ca 2+ Concentration in Hepatocellular Carcinoma Cells to Accelerate the Sustained Proliferation and Drug Resistance

Background: To study the role of TP53-induced glycolysis and apoptosis regulator (TIGAR) in hepatocellular carcinoma (HCC) and drug resistance. Methods: HCC cells (HepG2 and SMMC7721) were used in this study. Fura 2-AM was used to assess cytosolic free Ca 2+ concentrations ([Ca 2+ ]i) within the two HCC cell lines. Nimodipine (NMDP), a Ca 2+ antagonist, was used to reduce cytosolic [Ca 2+ ]i level. Proliferation of HCC was measured using cell counting kit-8 (CCK-8). The roles of TIGAR and Ca 2+ in drug resistance of HCC cells were assessed using epirubicin (Epi), 5-uorouracil (5-FU), or cisplatinum (DDP). Results: Knockdown of TIGAR signicantly suppressed cell viability, reduced [Ca 2+ ]i, restrained protein expression of Ca 2+ -activated cysteine proteinases (Calpain1 and 2), as well as blocked the activation of nuclear factor kappa B (NF-κB) through an increase of cytoplasmic NF-κB and reduction of nuclear NF-κB. However, overexpression of TIGAR (oeTIGAR) resulted in the opposite. Evidence also shows that oeTIGAR suppressed the sensitivity of HCC to Epi, which was retarded by NMDP as an additional treatment. TIGAR interference could enhance the sensitivity of HCCs with high TIGAR expression to drugs. Conclusions: TIGAR promoted HCC progression and induced drug resistance, and the mechanism involved was [Ca 2+ ]i-mediated activation of Calpain 1 and 2 and NF-κB signaling. in HCC cells. Elevated Ca 2+ appears to be the mechanism in which TIGAR promotes proliferation and drug resistance in HCC. Our study suggests that targeting TIGAR is a therapeutic strategy for HCC chemotherapy.


Background
Hepatocellular carcinoma (HCC) is commonly caused by alcohol or viral infections (e.g., Hepatitis B and C virus), and accounts for 2.4% of all malignancies and 9% of cancer-related deaths (1). Patients with HCC usually receive a three-drug chemotherapy protocol consisting of cisplatinum (DDP) and 5uorouracil (5-FU), followed by partial surgical resection. However, 70% of cases recur due to drug resistance (2,3). This necessitates a deeper analysis and better understanding of the molecular processes that favor HCC progression and the development of drug resistance to improve treatment of HCC.
TP53-induced glycolysis and apoptosis regulator (TIGAR) is an oncogene that is associated with progression and drug resistance of various cancers (4)(5)(6). In HCC progression, TIGAR promotes proliferation by inhibiting glycolysis, promoting anti-oxidative activities, reducing ROS as well as retarding ROS-associated DNA damage, apoptosis, and autophagy (7)(8)(9). Moreover, TIGAR also reduces the sensitivity of HCC cells to epirubicin (Epi) (8), suggesting that TIGAR serves as a promoter of drug resistance in HCC. These studies suggest that TIGAR can be a candidate for targeted cancer therapy.
An increase in free Ca 2+ concentrations ([Ca 2+ ]i) acts as a second messenger to stimulate cytosolic events that participate in HCC proliferation, apoptosis, and migration (10,11). Inhibition of Ca 2+ signaling in HCC also suppresses multi-drug resistance by suppressing epithelial-mesenchymal transition, blunting hypoxia-inducible factor1-α signaling as well as attenuating DNA damage repair (12). TIGAR functions similar to fructose-2,6 bisphosphatase, whose activity is enhanced by high [Ca2 + ]i in human broblasts (13). Whether [Ca 2+ ]i plays a role in HCC proliferation and drug resistance remains to be proven.
To explore the role of Ca 2+ in TIGAR's promotion of cancer cell proliferation in HCC in vitro, siRNAmediated TIGAR knockdown (siTIGAR) and lentivirus-mediated TIGAR overexpression (oeTIGAR) were transfected into human HCC cells (HepG2 and SMMC7721). Nimodipine (NMDP) was used as Ca 2+ inhibitor to decrease cytosolic ([Ca 2+ ]i). Calpains, also known as Ca 2+ -activated cysteine proteinases, as well as nuclear factor κB (NF-κB) in the nucleus and cytoplasm were assessed. The sensitivity of HCC cells to Epi were also detected under oeTIGAR transduction or NMDP treatment.

Bioinformatics analysis
To study the expression and association of TIGAR, Calpain 1, and Calpain2 in HCC progression, tumor tissue and surrounding normal tissue were taken from patients with HCC (n = 13). Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to assess mRNA levels of TIGAR, Calpain 1, Calpain 2, and NF-ĸB in hepatic tissues. Pearson's correlation coe cients (r) and linear regression were carried out to analyze expression correlation between TIGAR and Calpain 1, as well as TIGAR and Calpain2.

Isolation of primary HCC cells
Primary HCC cells were separated from tumor tissue according to previously reported methods (14,15).
Resected HCC specimens were sliced into 1 cm 3 sections, which were suspended in RPM11640 medium (Hyclone, Logan, UT, USA) containing 100 U/ml of penicillin and 100 µg/ml of streptomycin (Solarbio, Beijing, China). Tissue sections were digested into single cells by a collagenase/hyaluronidase solution (Thermo Fisher Scienti c, Waltham, MA, USA) followed by 0.25% trypsin-EDTA at 37℃ for 2 h, then ltered using a 120 mesh sieve. The ltrate was centrifuged at 1000r/min for 10 min at 37℃. Primary HCC cells in the precipitate were further puri ed from stromal cells using a magnetic bead isolate system (Beijing Percans Oncology Research Co., Ltd. To study the involvement of Ca 2+ in TIGAR-induced cancer cell proliferation, HepG2 cells were transfected with TIGAR and then exposed to 10 µM of NMDP (Shanghai yuanye Bio-technology Co., Ltd, access No. cells with oeTIGAR were treated with 10 µM of NMDP plus either Epi 2.5 µg/mL, 5-FU 5 µg/mL, or DDP 1mg/L. Epi, 5-FU, and DDP were all purchased from Selleck (https://www.selleck.cn/) with catalog numbersS1223, S1209, and S1166, respectively.

Groups
The cell experiments were divided into four parts. Part 1 examined the effects of siTIGAR on HepG2 and SMMC7721 cells. The two cell lines were transfected with siTIGAR, cultured for 72 h, then harvested at 0, 24, 48, and 72 h after treatment. The proliferation of cells at each time point was tested by CCK-8 assay.
Furthermore, the rescue assays of siTIGAR in the events involved in HCC were administrated by treatment with oeTIGAR in HepG2 cells, and the above indices were detected. In part 4, 10 primary HCC cell lines were divided into low (A1-A5) and High (B1-B5) groups, according to TIGAR mRNA levels assessed by RT-PCR. All cell lines were treated with anti-HCC drugs (Epi, 5-Fu, and DDP) with or without NMDP, and CCK-8 was used to assess proliferation of primary HCC at 48 h. In addition, we assessed the effects of siTIGAR on drug resistance of two higher TIGAR expression cell lines (B4 and B5 cells) against NMDP (10 µM) plus 5-FU or DDP. Each cell line was divided into six groups: siNC, siTIGAR, siNC + drug, siTIGAR + drug, siNC + NMDP + drug, and siTIGAR + NMDP + drug. The cell samples were harvested at 48 after treatment. The proliferation of cells at each time point was tested by CCK-8 assay, and the protein levels of PCNA and H3 were detected using western blot at 48 h after treatment.

Statistical analysis
Data were calculated using Graphpad Prism 6 (GraphPad Software Inc., USA) and presented as mean ± SEM. Difference effect between groups was determined using one-way ANOVA with Tukey-Kramer method, and P value < 0.05 being signi cant. Correlations between TIGAR and Calpain1, as well as TIGAR and Calpain2 in HCC tumor samples were assessed by Pearson r analysis.

Expression of TIGAR, Calpain 1, and Calpain 2 in HCC
Expression and association of TIGAR, Calpain 1, and Calpain 2 in hepatic tissues of HCC patients were assessed. Figure 1A-D indicated that TIGAR, Calpain 1, Calpain 2, and NF-ĸB were signi cantly enhanced in HCC (P < 0.01). Furthermore, Figure 1E & F show that the correlation between TIGAR and both Calpains were signi cantly positive.
siTIGAR inhibited HCC cell proliferation, down-regulated Ca 2+ signaling, and prevented NF-ĸB activation As shown in Figure 2, siTIGAR remarkably reduced HCC cell proliferation ( Figure 2A&B oeTIGAR enhanced drug resistance of HCC cells via the Ca 2+ pathway To study whether TIGAR acted on [Ca 2+ ]i to induce drug resistance in HCC cells, oeTIGAR-transfected HepG2 cells were exposed to NMDP + anti-HCC drugs (Epi, 5-FU, or DDP). The carcinogenesis of HepG2 cells were assessed by proliferation assay. Figure 5A-C indicated that anti-HCC drugs reduced the proliferation of HepG2 cells with or without oeTIGAR (All P < 0.01). HepG2 proliferation was signi cantly enhanced in oeTIGAR + drug group in comparison to Vector + drug group, but was remarkably reduced in oeTIGAR + drug + NMDP group, in comparison to oeTIGAR + drug group. Moreover, the protein level of PCNA at 48 h after treatment showed the same trends with cell proliferation, as shown in Figure 5D-F.
Given the role of oeTIGAR in regulating Ca 2+ ( Figure 4C), our data suggested that oeTIGAR evoked the Ca 2+ pathway to promote drug resistance against HCC medications.

Effect of TIGAR on primary HCC cells with drugs and NMDP
From the RT-PCR assay of TIGAR mRNA levels in 10 primary HCC cell lines, we con rmed A1-A5 as low TIGAR expression group (n = 5) and B1-B5 as high TIGAR expression group (n = 5) ( Figure 6A). Figure  6B-D indicated the inhibited effect of anti-HCC drugs on proliferation of B1-B5 cell lines was signi cantly reduced in comparison to A1-A5 cell lines; however, they were obviously enhanced with additional NMDP treatment. Moreover, the proliferation of primary HCC cell lines B4 and B5 were reduced by NMDP and anti-HCC drugs, and the inhibited effect of anti-HCC drugs was further aggravated by TIGAR interference ( Figure 6E). The protein levels of PCNA in B4 and B5 cells showed same trend with the proliferation ( Figure 6F&G).

Discussion
TIGAR is a proliferation inducer and anti-apoptosis regulator in HCC (7), and can function as fructose-2,6 bisphosphatase to contribute to energy metabolism in tumors. Evidence suggest that the blockade of the intracellular Ca 2+ release pathway favored apoptosis of HCC cells, based on a study on human broblasts that indicated that enhanced [Ca 2+ ]i is associated with the increased levels of fructose 2,6bisphosphate (13).Thus, in this study, we investigated whether [Ca 2+ ]i also played a role in HCC progression, in addition to TIGAR.
Calpains are a class of Ca 2+ -dependent cysteine proteases and exist primarily in two characterized forms (Calpain 1 and 2, also known as µ-and m-calpain, respectively). Calpain is activated and associated with the retarding of extrinsic apoptotic signaling in hepatitis C virus-related HCC (17). In this study, we con rmed the increased expression of TIGAR, Calpain 1, and Calpain 2 in HCC progression (7,18), and reported the positive correlations between TIGAR and Calpains1 and 2 for the rst time ( Figure 1).
We also investigated whether Ca 2+ -mediated Calpain was involved in TIGAR-induced HCC progression. Our data showed that siTIGAR suppressed HCC proliferation, down-regulated anti-apoptotic protein Bcl2, and up-regulated pro-apoptotic proteins c-caspase 3 and Bax. However, oeTIGAR led to the opposite, con rming the role of TIGAR inhibition in regulating HCC cell death (7,8). These indicate that TIGAR plays an important role in HCC, and suppressing TIGAR expression could be a probable target to treat HCC. More importantly, TIGAR enhanced [Ca 2+ ]i and elevated levels of Calpain 1 and Calpain 2, while NMDP exposure remarkably reduced TIGAR's effects ( Figure 4). These data showed that [Ca 2+ ]i-mediated Calpain signaling was the primary mechanism by which TIGAR exerts its effects on HCC cells.
Activation of NF-κB frequently occurs in tumors, especially in those with drug or radiotherapy resistance (19)(20)(21). The dimeric NF-κB complexes are localized to the cytosol through interaction with Inhibitor of NF-κB (IκBα). Decreased IκBα expression and activity favors the release of NF-κB dimers, which are subjected to nuclear translocation and result in subsequent activation of NF-κB target genes. Evidence suggests that TIGAR inhibits NF-κB activation in murine adipocyte cells by suppressing the activity of IKKβ (adaptor protein for IκBα degradation) (22). In bladder cancer cells, elevated Ca 2+ is essential for NF-κB activation (23). In this study, we showed that TIGAR-activated NF-κB depended on the elevated [Ca 2+ ]i levels in HCC cells (Figure 4).
Inhibition of Ca 2+ entry SOCE (a key pathway for extracellular Ca 2+ in ux) enhanced sensitivity of HCC cells to chemotherapy (24). Thus, we studied whether Ca 2+ was involved in TIGAR-induced drug resistance. TIGAR confers resistance to Epi, as evidenced by enhanced proliferation of HCC in the oeTIGAR + Epi group when compared to Vector + Epi, which is consistent with a previous study (8). However, the anti-cancer effect of Epi in oeTIGAR + Epi was strongly augmented with additional NMDP treatment, substantiating that TIGAR enhanced Ca 2+ levels to induce drug resistance in HCC cells.
Moreover, Figure 5 shows that primary HCC with high TIGAR expression was less sensitive to Epi, and abrogation of the Ca 2+ pathway using NMDP potentiated the death-inducing ability of Epi, which further veri ed that the resistance of primary HCC to Epi-induced killing was attributed to TIGAR-mediated Ca 2+ overload. Furthermore, we obtained similar results using 5-FU and DDP ( Figure 5 & 6), suggesting that TIGAR-mediated enhancement of [Ca 2+ ]i was involved in drug resistance in HCC.

Conclusions
In summary, TIGAR evoked Ca 2+ -dependent Calpain and activated NF-ĸB in HCC cells. Elevated Ca 2+ appears to be the mechanism in which TIGAR promotes proliferation and drug resistance in HCC. Our study suggests that targeting TIGAR is a therapeutic strategy for HCC chemotherapy.

Consent for publication
Written informed consent for publication was obtained from all participants.

Availability of supporting data
The datasets used or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare no competing interests.

Funding
This study was supported by the National Natural Science Foundation of China (81602613), and the grant from Suzhou Introduced Team of Clinical Medical Experts (SZYJTD201803).
Authors' contributions H.Z., Z.Y.Y. and W.C. conceived and designed the research. J.M.X., Y.Q.S. and X.Y.F. performed experiments, analyzed, and interpreted data. Z.Y.F., W.L. and X.M.M. analyzed data. D.K.G. and P.D. interpreted data. W.C. wrote the paper. All authors edited the paper for submission.

Figure 1
Expression of TIGAR and its correlation to Calpain 1 and Calpain 2 in HCC. Rescue assays of TIGAR in the events involved in HCC.