TY - JOUR
AU - Cui, Yejia
AB - Abstract Oxaliplatin (OXA) resistance limits the efficiency of treatment for hepatocellular carcinoma (HCC). Studies have shown that the PDZ-binding kinase (PBK) plays important roles in tumors. However, the role of PBK in HCC is still a problem. In this study, we explored whether PBK is involved in the chemoresistance to OXA in HCC. Expressions of PBK in six HCC cell lines and one human hepatocytes line were determined by real-time quantitative PCR and western blot analysis. SNU-182 and HepG2 cells were chosen to induce OXA resistance. PBK was silenced or overexpressed in OXA-resistant and sensitive cell lines. Then, cell proliferation, migration, and invasion were measured by cholecystokinin-8 assay and Transwell assay, respectively. The Cancer Genome Atlas dataset showed that PBK is highly expressed in HCC and signifies poor prognosis to patient with HCC. Results showed that expression of PBK in HCC cells was significantly higher than that in THLE2 cells, and it was further increased in OXA-resistant HCC cells. Silencing of PBK promoted the sensitivity of drug-resistant HCC cells to OXA. Overexpression of PBK relieved the apoptosis induced by OXA and promoted the migration and invasion of OXA-sensitive HCC cells. Thus, this study revealed that high PBK expression is correlated with OXA resistance in HCC cells, which may provide a promising therapeutic target for treating HCC. hepatocellular carcinoma, chemoresistance, oxaliplatin, PBK/TOPK, metastasis Introduction Hepatocellular carcinoma (HCC) is the fifth most common cancer in terms of incidence and the third in terms of mortality. HCC is the most frequent cause of all liver cancers, accounting for 90% of cases worldwide [1,2]. Recently, the incidence of HCC has increased, mainly in patients with cirrhosis and chronic liver infection caused by hepatitis. HCC is often highly aggressive. Due to the lack of early detection and ineffective chemotherapy, the prognoses of patients with advanced HCC remain as dismal as other cancers, despite multiple treatment options, including liver resection, thermal ablation, trans-arterial chemo or radio-embolization, liver transplant, and systemic chemotherapy [3,4]. Chemotherapy resistance is a significant factor for the recurrence and metastasis of HCC [5–7]. Oxaliplatin (OXA)-based systemic chemotherapy is increasing as the most critical clinical medicine for HCC in advanced stages, being a vital breakthrough for tumor chemotherapy. OXA-based chemotherapy has recently been shown to be effective for the treatment of advanced HCC. However, the development of resistance to treatment severely limits its efficacy [8]. The molecular mechanisms contributing to the chemotherapeutic resistance of HCC are obscure. OXA, a third-generation platinum analog, is a compound with significant anticancer activities against colorectal, breast, gastric, and renal carcinomas and sarcomas [9]. It also has been used in combination with 5-fluorouracil (5-FU) and leucovorin as a first-line chemotherapeutic regimen (FOLFOX4) for advanced HCC [10]. As a bifunctional alkylating agent, OXA can covalently bind with DNA and form platinum–DNA adducts that block DNA replication and transcription [11]. However, ample evidence has shown that the occurrence of chemoresistance is a major limitation to the efficacy of platinum-based therapies in managing HCC [12,13]. Molecular mechanisms involved in OXA resistance of HCC remain poorly defined. PDZ-binding kinase (PBK), also known as lymphokine-activated killer T (T-LAK) cell-originated protein kinase (TOPK), was first cloned from the T-LAK cell subtraction cDNA fragment library [14]. PBK is rarely expressed in normal tissues except for fetal and germ cells but is highly trans-activated in various cancers, making it a promising molecular target for cancer screening and targeted therapy [15,16]. Many studies have indicated that high PBK expression is associated with a more aggressive phenotype in various cancers, including gastric, oral, glioma, lung, colon, cervical, colorectal, breast, prostate, and pancreatic cancers [17–24]. Recent study has also shown that PBK confers cisplatin resistance in high-grade serous ovarian carcinoma [23]. However, its role in HCC drug resistance remains unknown. PBK is a serine/threonine kinase belonging to the mitogen-activated protein kinase (MAPK) kinase (MAPKK) family [25]. MAPKK is the upstream activator of MAPK and can catalyze the phosphorylation of Tyr and Thr residues of MAPK [26]. The MAPK pathway is composed of a cascade of activated serine/threonine protein kinases and plays an essential role in the regulation of the cell cycle and gene expression [27]. PTEN is the first tumor suppressor gene with specific phosphatase activity [28]. It has been shown that PTEN can selectively prevent ERK activation, which is not affected by integrin and growth factor stimulation, thus playing a negative regulatory role in ERK/MAPK signal transduction [29]. Studies have also shown that PTEN can repress Akt phosphorylation through dephosphorylation of PIP3 and the antagonistic oncogene PI3K [30]. It has been revealed that TOPK/PBK can regulate the PI3K/PTEN/AKT pathway in lung cancer [31]. Therefore, we put forward the hypothesis that PBK might be associated with the PTEN/AKT pathway in HCC. In this study, we aimed to illuminate the functions of PBK in the chemoresistance of HCC and further investigate the underlying mechanisms in vitro. Additionally, we tried to elucidate the regulatory mechanism of PBK in HCC. Materials and Methods Data collection The available data on PBK expression of HCC cells and clinical information were obtained from Illumina HiSeq Level 3 isoform quantification files available at the The Cancer Genome Atlas (TCGA) Data Portal website (http://tcga.cancer.gov/dataportal; accessed June 2016). PBK expression in 50 normal samples and 371 primary tumor samples and the survival probability in the groups with high expression of PBK (n=91) and low/medium expression of PBK (n=274) are available at the website (http://ualcan.path.uab.edu/cgi-bin/TCGAExResultNew2.pl?genenam=PBK&ctype=LIHC). Cell culture and treatment Human HCC cell lines (SNU-182, SK-Hep1, Huh7, SMMC-7721, HepG2, and MHCC97H) and human liver THLE2 cells were purchased from iCell Bioscience (Shanghai, China). The cell lines were grown in DMEM (Thermo Fisher Scientific, Shanghai, China) containing 10% fetal bovine serum (FBS; Gibco, Grand Island, USA) and 0.1% antibiotic antimycotic solution, and kept in a 5% CO2 humidified incubator (Thermo Fisher Scientific) at 37°C. To establish stable OXA-resistant HCC models, SNU-182 and HepG2 cell lines were exposed to increasing concentrations of OXA (Sigma-Aldrich, Darmstadt, Germany). Briefly, SNU-182 and HepG2 cells were initially treated with OXA at 0.1 μM for 1 month. Subsequently, the OXA concentration in the culture medium was increased every 2 weeks by 0.1 μM up to a final concentration of 1 μM (until a cell population was selected that demonstrated at least a 3-fold greater IC50 (45 μg/ml) to OXA than the parental cell lines. The established OXA-resistant SNU-182 and HepG2 cells were named SNU-182/OXA-r and HepG2/OXA-r cells, respectively. To eliminate the influence of residual OXA in the culture medium, SNU-182/OXA-r and HepG2/OXA-r cells were cultured in OXA-free DMEM medium for 2 weeks prior to the experiments. To overexpress PBK in SNU-182 and HepG2 cells, coding sequence of PBK was cloned and inserted into the expression plasmid pGL3.0-basic (BioVector, Beijing, China) to produce pGL3.0-PBK. To knock down the expression of PBK and PTEN in SNU-182 and HepG2 cells, cells were transfected with specific siRNAs targeted to PBK and PTEN, which were synthesized by GenePharma (Shanghai, China). When cells reached 65% confluence, pGL3.0-PBK or siRNA (si-PBK: 5ʹ-UAAAAGCACGAUAACCAACAA-3ʹ; si-PTEN: 5ʹ-UUUGUUUCUGCUAACGAUCUC-3ʹ; si-NC: 5ʹ-UUCUCCGAACGUGUCACGUTT-3ʹ) was transfected into cells using Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, USA) following the manufacturer’s instructions. Quantitative reverse transcription-PCR Total RNA was extracted by using the TRIzol reagent (Thermo Fisher Scientific). Subsequently, reverse transcription of the total RNA was performed with a 1-μg RNA template, random primers, and PrimeScript Reverse Transcriptase (Takara, Tokyo, Japan). The PCR primers were designed by the Primer Premier 6.0 software. The primers used were as follows: PBK, 5ʹ-CCAAACATTGTTGGTTATCGTGC-3ʹ (forward) and 5ʹ-GGCTGGCTTTATATCGTTCTTCT-3ʹ (reverse), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5ʹ-TGTTCGTCATGGGTGTGAAC-3ʹ (forward) and 5ʹ-ATGGCATGGACTGTGGTCAT-3ʹ (reverse). Quantitative reverse transcription-PCR (qPCR) was performed by using the SYBR Green qPCR kit (Toyobo, Osaka, Japan) on the 7500 Real-Time PCR System (Applied Biosystems, Foster, USA) as recommended by the manufacturer. The PCR cycling conditions were 1 min at 94°C, 35 cycles of denaturing for 30 s at 94°C, annealing for 2 min at 60°C, and extension for 1 min at 72°C, and 5 min at 72°C. The relative mRNA levels were evaluated using the 2−∆∆Ct method. GAPDH was used as the internal control. Western blot analysis After treatment, cells were lysed in 1× RIPA buffer (400 µl/10-cm dish; Sigma-Aldrich) on ice for 30 min. Protein in the supernatant was collected by centrifugation and its concentration was determined by NanoDrop (Thermo Fisher Scientific) using the BCA Protein Assay kit (Beyotime, Shanghai, China). Then, protein samples were boiled with loading buffer for 5 min. Protein samples (20 μg of protein in each sample) were separated by 10% gradient SDS-PAGE and then transferred to PVDF membranes (Millipore, Billerica, USA) for antibody blotting. The PVDF membranes were blocked with TBST containing 5% skimmed milk at room temperature for 1 h. After washing three times with TBST, the membranes were incubated with anti-PBK (1:800), anti-PTEN (1:400), anti-AKT (1:500), anti-p-AKT (1:400), anti-Bax (1:400), anti-Caspase-3 (1:1500), anti-Bcl-2 (1:1500), and anti-GAPDH (1:2000) primary antibodies (Abcam, Cambridge, UK) at 4°C overnight. Then, the membranes were washed with TBST three times and incubated with HRP-conjugated goat anti-rabbit IgG secondary antibodies (Boster, Wuhan, China) at room temperature for 1 h. Finally, protein bands were visualized using the ECL-Plus reagent (Millipore) and images were acquired with the ChemiDoc™ Imaging System (Bio-Rad, Hercules, USA). The integrated option densities of protein bands were analyzed through Image-Pro Plus software (Media Cybernetics, Rockville, USA) and the relative levels of protein bands were calculated using GAPDH as the loading control. Cell viability assay Cell viability was measured using the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) as recommended by the manufacturer. Briefly, the corresponding cells were seeded into 96-well plates at an initial density of 1 × 104 cells/well 24 h after OXA treatment. Then, 10 μl of cholecystokinin-8 (CCK-8) solution was added into each well of the plate. Each group had three replicates. The plates were incubated for another 2 h at 37°C. Finally, cell viability was determined by measuring the optical density at 450 nm with a Microplate Reader (Thermo Fisher Scientific). Transwell assay For cell migration assay, cells were incubated using 24-well Transwell insert plates (8 μm pore size; Corning Co., Corning, USA). Briefly, cells (1×105) suspended in serum-free medium were plated in the upper chamber and 0.6 ml of medium with 10% FBS was added to the lower chamber. After 24 h of incubation, the migrated cells were fixed in 4% paraformaldehyde, stained by crystal violet (Beyotime), and counted under an Olympus CX41 microscope (Olympus, Tokyo, Japan). For cell invasion assay, the upper chamber of the 24-well Transwell was coated with 100 μl of Matrigel (BD Biosciences, Franklin Lakes, USA) in serum-free culture medium for 4 h at 37°C in advance. The rest of the experiment was the same as the migration experiment. Flow cytometry Cell apoptosis was measured by the flow cytometry assay using the Annexin V-FITC/PI Apoptosis Kit (MultiSciences, Hangzhou, China). The treated cells (5×105) were collected, washed three times with PBS, and transferred to flow tubes after trypsin digestion. Then, cells were incubated with 200 μl of Annexin V-FITC/PI solution for 25 min in the dark at room temperature. Finally, the FITC fluorescence and PI fluorescence were detected on a BD FACS Calibur™ flow cytometer (BD Biosciences). Statistical analysis Each experiment was performed in triplicate and repeated at least three times. Data were expressed as the mean ± standard deviation (SD) of three independent experiments. Statistical analyses were performed using Prism GraphPad software (version 7.0). For comparative analyses, differences between two groups were assessed by two-tailed Student’s t-test and one-way or two-way ANOVA with multiple comparisons tests. A P-value <0.05 was considered statistically significant. Results PBK is highly expressed in HCC To examine the differential expression of PBK in the para-carcinoma specimens and pathological specimens, we analyzed mRNA expression profiles of HCC from the TCGA dataset. The results revealed that the expression of PBK in primary tumors is significantly higher than that in normal tissues, which are available at the website (http://ualcan.path.uab.edu/cgi-bin/TCGAExResultNew2.pl?genenam=PBK&ctype=LIHC) (Fig. 1A). Furthermore, the HCC patients with low expression of PKB had better prognosis than those with high expression of PKB (Fig. 1B). We also used the qPCR assay and western blot analysis to test the expression of PBK in six HCC cell lines and human THLE2 hepatocytes. The results consistently showed that the expression of PBK in HCC cells was significantly higher than that in human liver THLE2 cells. In addition, it was relatively lower in SNU-182 and HepG2 cell lines than in the other four HCC cell lines (Fig. 1C and Supplementary Fig. S1A). These data indicated that PBK is highly expressed in HCC tissues and HCC cell lines. Figure 1. Open in new tabDownload slide PBK was highly expressed in HCC (A) Expression of PBK in HCC is available at the website (http://ualcan.path.uab.edu/cgi-bin/TCGAExResultNew2.pl?genenam=PBK&ctype=LIHC). (B) Effect of PBK expression level on HCC patient prognosis in the TCGA database. (C) Expression of PBK in human liver THLE2 cells and six HCC cell lines detected by qPCR (upper) and western blot analysis (lower). ***P<0.001 vs THLE2 cells. Figure 1. Open in new tabDownload slide PBK was highly expressed in HCC (A) Expression of PBK in HCC is available at the website (http://ualcan.path.uab.edu/cgi-bin/TCGAExResultNew2.pl?genenam=PBK&ctype=LIHC). (B) Effect of PBK expression level on HCC patient prognosis in the TCGA database. (C) Expression of PBK in human liver THLE2 cells and six HCC cell lines detected by qPCR (upper) and western blot analysis (lower). ***P<0.001 vs THLE2 cells. PBK is highly expressed in OXA-resistant HCC cell lines To further study whether PBK is involved in the chemoresistance of HCC, drug resistance of SNU-182 and HepG2 cells was induced by OXA. Then, CCK8 assay was performed. The results showed that the cell viability of OXA-resistant cells was higher than that of wild-type cells (Fig. 2A), and the IC50 value in OXA-resistant cells was significantly higher than that in the wild-type cells (Fig. 2B). Of note, qPCR and western blot analysis results revealed that the expression levels of PBK in OXA-resistant cells were significantly higher compared with those in their parental cells (Fig. 2C,D and Supplementary Fig. S1B). These results suggested that PBK is highly expressed in OXA-resistant HCC cell lines. Figure 2. Open in new tabDownload slide Expression of PBK in OXA-resistant HCC cells (A,B) SNU-182/OXA-s, SNU-182/OXA-r, HepG2/OXA-s, and HepG2/OXA-r cells were treated with different concentrations of OXA, and the half inhibitory concentration (IC50) was determined using CCK-8 assay, respectively. (C,D) PBK expression in OXA-resistant cells and wild-type cells detected by qPCR and western blot analysis. ***P < 0.00.1 vs OXA-s. OXA-s, OXA-sensitive; OXA-r, OXA-resistant. Figure 2. Open in new tabDownload slide Expression of PBK in OXA-resistant HCC cells (A,B) SNU-182/OXA-s, SNU-182/OXA-r, HepG2/OXA-s, and HepG2/OXA-r cells were treated with different concentrations of OXA, and the half inhibitory concentration (IC50) was determined using CCK-8 assay, respectively. (C,D) PBK expression in OXA-resistant cells and wild-type cells detected by qPCR and western blot analysis. ***P < 0.00.1 vs OXA-s. OXA-s, OXA-sensitive; OXA-r, OXA-resistant. Knockdown of PBK increases the sensitivity of OXA-resistant HCC cells by upregulating PTEN To investigate whether PBK affects HCC cell proliferation, migration, invasion, and OXA resistance by regulating PTEN, we knocked down PBK and PTEN in SNU-182/OXA-r and HepG2/OXA-r cells using specific siRNAs. Subsequently, qPCR and western blot analysis were used to test the transfection effects, and results showed that the expression of PBK was significantly downregulated after transfection with si-PBK or si-PBK+si-PTEN (Fig. 3A,B and Supplementary Fig. S1C). In addition, silencing of PBK dramatically upregulated PTEN, and significantly downregulated p-AKT, while silencing of PTEN could markedly prevent the increase of PTEN and the decrease of p-AKT mediated by PBK silencing in SNU-182/OXA-r and HepG2/OXA-r cells; additionally, we discovered that the total AKT had no obvious change after transfection (Fig. 3C and Supplementary Fig. S1C). Furthermore, the immunofluorescence assay proved that knockdown of PBK prominently decreased p-AKT expression, which could also be reversed by PTEN knockdown in SNU-182/OXA-r and HepG2/OXA-r cells (Fig. 3D,E). Figure 3. Open in new tabDownload slide Knockdown of PBK inhibited SNU-182/OXA-r and HepG2/OXA-r cell viability, migration, and invasion by upregulating PTEN (A) qPCR analysis of PBK in SNU-182/OXA-r and HepG2/OXA-r cells after transfection with si-PKB and/or si-PTEN. (B) Western blot analysis of PBK in SNU-182/OXA-r and HepG2/OXA-r cells after transfection with si-PKB and/or si-PTEN. (C) Expression changes of PTEN, AKT, and p-AKT in SNU-182/OXA-r and HepG2/OXA-r cells transfected with si-PKB and/or si-PTEN. (D,E) IF assay was conducted to monitor the expression changes of PBK and p-AKT in SNU-182/OXA-r and HepG2/OXA-r cells transfected with si-PKB and/or si-PTEN. Magnification, 400×. Scale bar=10 μm. (F,G) Cell viability was detected by CCK-8 in SNU-182/OXA-r and HepG2/OXA-r cells under different concentrations of OXA and their IC50 values. (H) Migration of SNU-182/OXA-r and HepG2/OXA-r cells after transfection with si-PKB or/and si-PTEN. (I) Invasion of SNU-182/OXA-r and HepG2/OXA-r cells after transfection with si-PKB and/or si-PTEN. ***P <0.001 vs si-NC; ###P <0.001 vs si-PKB. OXA-r, OXA-resistant. Figure 3. Open in new tabDownload slide Knockdown of PBK inhibited SNU-182/OXA-r and HepG2/OXA-r cell viability, migration, and invasion by upregulating PTEN (A) qPCR analysis of PBK in SNU-182/OXA-r and HepG2/OXA-r cells after transfection with si-PKB and/or si-PTEN. (B) Western blot analysis of PBK in SNU-182/OXA-r and HepG2/OXA-r cells after transfection with si-PKB and/or si-PTEN. (C) Expression changes of PTEN, AKT, and p-AKT in SNU-182/OXA-r and HepG2/OXA-r cells transfected with si-PKB and/or si-PTEN. (D,E) IF assay was conducted to monitor the expression changes of PBK and p-AKT in SNU-182/OXA-r and HepG2/OXA-r cells transfected with si-PKB and/or si-PTEN. Magnification, 400×. Scale bar=10 μm. (F,G) Cell viability was detected by CCK-8 in SNU-182/OXA-r and HepG2/OXA-r cells under different concentrations of OXA and their IC50 values. (H) Migration of SNU-182/OXA-r and HepG2/OXA-r cells after transfection with si-PKB or/and si-PTEN. (I) Invasion of SNU-182/OXA-r and HepG2/OXA-r cells after transfection with si-PKB and/or si-PTEN. ***P <0.001 vs si-NC; ###P <0.001 vs si-PKB. OXA-r, OXA-resistant. Cell viability assay showed that the viability of SNU-182/OXA-r and HepG2/OXA-r cells were gradually decreased with the increase of the OXA concentration, and the IC50 value was notably reduced in the si-PBK group relative to that in the si-NC group, while the reduction of cell viability and IC50 value mediated by PBK knockdown could be dramatically reversed by PTEN knockdown (Fig. 3F,G). The Transwell assay revealed that silencing of PBK significantly inhibited the migration and invasion of SNU-182/OXA-r and HepG2/OXA-r cells, which could also be reversed by PTEN silencing (Fig. 3H,I). Furthermore, cells were stimulated with OXA (10 μg/ml) after transfection, and the flow cytometry assay showed that silencing of PBK significantly promoted OXA-induced cell apoptosis, which could also be reversed by PTEN silencing (Fig. 4A,B). Western blot analysis revealed that the protein levels of Bax and Caspase-3 were upregulated, while the expression of Bcl-2 was downregulated in the si-PBK group relative to that in the si-NC group. PTEN silencing could inhibit the upregulation of Bax and Caspase-3 and the downregulation of Bcl-2 in SNU-182/OXA-r and HepG2/OXA-r cells (Fig. 4C and Supplementary Fig. S1C). These data indicated that knockdown of PBK increased the expression of PTEN, which induced the sensitivity of OXA-resistant HCC cells. Figure 4. Open in new tabDownload slide Knockdown of PBK promoted SNU-182/OXA-r and HepG2/OXA-r cell sensibility to OXA through PTEN SNU-182/OXA-r and HepG2/OXA-r cells were stimulated with OXA (10 μg/ml) after transfection. (A,B) Apoptosis rates of SNU-182/OXA-r and HepG2/OXA-r cells were analyzed by flow cytometry after transfection of si-PKB and/or si-PTEN. (C) The levels of Bax, Caspase-3, and Bcl-2 were detected by western blot analysis. ***P < 0.001 vs si-NC; ###P < 0.001 vs si-PKB. OXA-r, OXA-resistant. Figure 4. Open in new tabDownload slide Knockdown of PBK promoted SNU-182/OXA-r and HepG2/OXA-r cell sensibility to OXA through PTEN SNU-182/OXA-r and HepG2/OXA-r cells were stimulated with OXA (10 μg/ml) after transfection. (A,B) Apoptosis rates of SNU-182/OXA-r and HepG2/OXA-r cells were analyzed by flow cytometry after transfection of si-PKB and/or si-PTEN. (C) The levels of Bax, Caspase-3, and Bcl-2 were detected by western blot analysis. ***P < 0.001 vs si-NC; ###P < 0.001 vs si-PKB. OXA-r, OXA-resistant. Overexpression of PBK promotes proliferation, migration, and invasion in HCC cells To further prove the function of PBK in HCC cell proliferation, migration, and invasion, we overexpressed PBK expression in SNU-182/OXA-s and HepG2/OXA-s cells. qPCR and western blot analysis were used to test expression of PBK. Data showed that expression of PBK was significantly upregulated (Fig. 5A,B and Supplementary Fig. S1D). Furthermore, expression of PTEN was downregulated, and expression of p-AKT was upregulated, while expression of AKT had no obvious change after transfection. CCK8 assay revealed that the cell viability was significantly increased in both SNU-182/OXA-s and HepG2/OXA-s cells after overexpression of PBK (Fig. 5C,D and Supplementary Fig. S1D). Transwell assay showed that overexpression of PBK significantly promoted cell migration and invasion (Fig. 5E,F). These data indicated that overexpressing PBK promoted the proliferation, migration, and invasion of HCC cells. Figure 5. Open in new tabDownload slide Overexpression of PBK promoted SNU-182/OXA-s and HepG2/OXA-s cell viability, migration, and invasion (A) Expression of PBK was determined by the qPCR assay after PBK overexpression in SNU-182/OXA-s and HepG2/OXA-s cells. (B) Western blot analysis of PBK, PTEN, AKT, and p-AKT expressions in SNU-182/OXA-s and HepG2/OXA-s cells transfected with pGL3.0-PBK. (C,D) After PBK overexpression, the viability was assessed by CCK-8 in SNU-182/OXA-s and HepG2/OXA-s cells under different concentrations of OXA and their IC50 values. (E) Migration of SNU-182/OXA-s and HepG2/OXA-s cells transfected with pGL3.0-PBK. (G) Invasion of SNU-182/OXA-s and HepG2/OXA-s cells transfected with pGL3.0-PBK. ***P < 0.05 vs vector. OXA-s, OXA-sensitive. Figure 5. Open in new tabDownload slide Overexpression of PBK promoted SNU-182/OXA-s and HepG2/OXA-s cell viability, migration, and invasion (A) Expression of PBK was determined by the qPCR assay after PBK overexpression in SNU-182/OXA-s and HepG2/OXA-s cells. (B) Western blot analysis of PBK, PTEN, AKT, and p-AKT expressions in SNU-182/OXA-s and HepG2/OXA-s cells transfected with pGL3.0-PBK. (C,D) After PBK overexpression, the viability was assessed by CCK-8 in SNU-182/OXA-s and HepG2/OXA-s cells under different concentrations of OXA and their IC50 values. (E) Migration of SNU-182/OXA-s and HepG2/OXA-s cells transfected with pGL3.0-PBK. (G) Invasion of SNU-182/OXA-s and HepG2/OXA-s cells transfected with pGL3.0-PBK. ***P < 0.05 vs vector. OXA-s, OXA-sensitive. Overexpression of PBK promotes OXA resistance in HCC cells After transfection of pGL3.0-PBK, SNU-182/OXA-s and HepG2/OXA-s cells were stimulated with 10 μg/ml OXA for 24 h. The flow cytometry assay results showed that cell apoptosis was significantly inhibited by overexpression of PBK (Fig. 6A,B). Western blot analysis revealed that protein expressions of Bax and Caspase-3 in the PBK-overexpressed group were lower than those in the vector-control group, while the expression of Bcl-2 was higher than that in the vector-control group (Fig. 6C). These results suggested that overexpression of PBK induced OXA resistance in HCC cells. Figure 6. Open in new tabDownload slide Overexpression of PBK inhibited SNU-182/OXA-s and HepG2/OXA-s cell apoptosis SNU-182/OXA-s and HepG2/OXA-s cells were stimulated with OXA (10 μg/ml) after transfection. (A,B) The cell apoptosis rate was evaluated by flow cytometry in PBK-overexpressing SNU-182/OXA-s and HepG2/OXA-s cells. (C) Expressions of Bax, Caspase-3, and Bcl-2 were determined by western blot analysis in SNU-182/OXA-s and HepG2/OXA-s cells after PBK overexpression. ***P <0.05 vs vector. OXA-s, OXA-sensitive. Figure 6. Open in new tabDownload slide Overexpression of PBK inhibited SNU-182/OXA-s and HepG2/OXA-s cell apoptosis SNU-182/OXA-s and HepG2/OXA-s cells were stimulated with OXA (10 μg/ml) after transfection. (A,B) The cell apoptosis rate was evaluated by flow cytometry in PBK-overexpressing SNU-182/OXA-s and HepG2/OXA-s cells. (C) Expressions of Bax, Caspase-3, and Bcl-2 were determined by western blot analysis in SNU-182/OXA-s and HepG2/OXA-s cells after PBK overexpression. ***P <0.05 vs vector. OXA-s, OXA-sensitive. Discussion Chemotherapy remains the major treatment for most patients with advanced HCC. However, chemoresistance is a prominent obstacle for effective treatment of HCC. Although OXA is widely used in the treatment of advanced HCC, the long-term therapeutic outcome is far from satisfactory, because most patients ultimately developed drug resistance. The sensitivity of HCC patients to OXA is a crucial factor for a curative effect. However, the mechanisms underlying chemoresistance for OXA in HCC have not been completely elucidated. In the present study, we found that PBK is upregulated in OXA-resistant HCC cell lines and that PBK is related to HCC cell proliferation, migration, and invasion. PBK is related to tumor growth in some cancers, including gliomas [32], T-cell leukemia [33], lung cancer [34], and colorectal cancer [35]. Similarly, we found that overexpressing PBK promotes proliferation in SNU-182 and HepG2 cells. In addition, our data from the Transwell assay also proved that silencing PBK inhibits HCC cells metastasis. More importantly, we found that overexpression of PBK inhibits expression of PTEN and activates the AKT signaling pathway. HepG2 cells do not have tumorigenic ability in nude mice (exposed by ATCC), which is also consistent with the lowest expression of PBK in HepG2 cells among the six HCC cell lines mentioned above. A previous study reported that PBK promotes cell migration via modulation of the PI3K/PTEN/AKT pathway and is associated with poor prognosis in lung cancer [36]. Whittaker et al. [37] also found that PI3K/AKT pathways contributed to HCC development and tumorigenesis. There are, of course, other pathways that may be related to the PBK function in HCC. Elucidation of these signaling mechanisms is interesting from a therapeutic perspective, since targeting them may aid in reversing, delaying, or preventing the occurrence of HCC [38]. This study revealed that the expression of PBK was significantly increased in SNU-182/OXA-r and HepG2/OXA-r cells when compared with that in SNU-182/OXA-s and HepG2/OXA-s cells, respectively, which suggested that PBK plays an important role in OXA resistance of HCC. It was reported that PBK confers cisplatin resistance in high-grade serous ovarian carcinoma [23], and overexpression of PBK attenuates the sensitivity of ovarian cancer cells to cisplatin treatment through inducing autophagy. Increased activity of PBK is also related to gefitinib resistance in non-small-cell lung cancer [39]. Moreover, forced expression of PBK induced the resistance to OXA treatment in colorectal cancer [40]. Hence, expression of PBK may be a marker to indicate sensitivity in many cancers. PBK suppressed the antitumor effects of doxorubicin by interacting with p53 and p21 inhibition [41]. PBK represents a new therapeutic target in multiple myeloma [42]. Our results also indicated the strong efficacy of PBK in chemoresistance. In conclusion, our study showed that PBK is highly expressed in HCC based on the TCGA database and HCC cell lines. High PKB expression is associated with a poor prognosis and OXA resistance in HCC. Overexpression of PBK attenuates OXA-resistant cell sensitivity and promotes cell viability, migration, and invasion through the PTEN/AKT axis. Study on PBK expression may be useful for the diagnosis, prognosis, and target therapy of HCC. PBK might be a predictive and prognostic marker for HCC patients with OXA resistance. Supplementary Data Supplementary Data is available at Acta Biochimica et Biophysica Sinica online. Acknowledgements Authors would like to thank Dr. Edward C. Mignot (Shandong University) for his linguistic advice. Funding This work was supported by the grants from the Social Science and Technology Development Major Project of Dongguan (No. 2018507150241638), the Medical Scientific Research Foundation of Guangdong Province of China (No. B2018243), the Medical Scientific Research Foundation of Guangdong Province of China (No. B2018016), and the Social Science and Technology Development General Project of Dongguan (Nos. 201950715024926 and 2018507150241501). Conflict of Interest The authors declare that they have no conflict of interest. References 1. Siegel RL , Miller KD. Cancer statistics, 2020 . CA Cancer J Clin 2020 , 70 : 7 – 30 . doi: 10.3322/caac.21590 Google Scholar Crossref Search ADS PubMed WorldCat 2. Rawla P , Sunkara T, Muralidharan P, Raj JP. Update in global trends and aetiology of hepatocellular carcinoma . Contemp Oncol (Pozn) 2018 , 22 : 141 – 150 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 3. Takahashi H , Kawaguchi T, Yan L, Peng X, Qi Q, Morris LGT, Chan TA, et al. Immune cytolytic activity for comprehensive understanding of immune landscape in hepatocellular carcinoma . Cancers (Basel) 2020 , 12 : E1221. doi: 10.3390/cancers12051221 Google Scholar OpenURL Placeholder Text WorldCat Crossref 4. Malla RR , Kumari S, Kgk D, Momin S, Nagaraju GP. Nanotheranostics: their role in hepatocellular carcinoma . Crit Rev Oncol Hematol 2020 , 151 : 102968. Google Scholar OpenURL Placeholder Text WorldCat 5. Huang F , Wang BR, Wang YG. Role of autophagy in tumorigenesis, metastasis, targeted therapy and drug resistance of hepatocellular carcinoma . World J Gastroenterol 2018 , 24 : 4643 – 4651 . doi: 10.3748/wjg.v24.i41.4643 Google Scholar Crossref Search ADS PubMed WorldCat 6. Lu JC , Liu LG, Lin L, Zheng SQ, Xue Y. Incident hepatocellular carcinoma developing during tenofovir alafenamide treatment as a rescue therapy for multi-drug resistant hepatitis B virus infection: a case report and review of the literature . World J Clin Cases 2018 , 6 : 671 – 674 . doi: 10.12998/wjcc.v6.i13.671 Google Scholar Crossref Search ADS PubMed WorldCat 7. Saeki I , Yamasaki T, Maeda M, Hisanaga T, Iwamoto T, Fujisawa K, Matsumoto T, et al. Treatment strategies for advanced hepatocellular carcinoma: sorafenib vs hepatic arterial infusion chemotherapy . World J Hepatol 2018 , 10 : 571 – 584 . Google Scholar Crossref Search ADS PubMed WorldCat 8. Ren WW , Li DD, Chen X, Li XL, He YP, Guo LH, Liu LN, et al. MicroRNA-125b reverses oxaliplatin resistance in hepatocellular carcinoma by negatively regulating EVA1A mediated autophagy . Cell Death Dis 2018 , 9 : 547. doi: 10.1038/s41419-018-0592-z Google Scholar OpenURL Placeholder Text WorldCat Crossref 9. Raymond E , Lawrence R, Izbicka E, Faivre S, Von Hoff DD. Activity of oxaliplatin against human tumor colony-forming units . Clin Cancer Res 1998 , 4 : 1021 – 1029 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 10. Qin S , Bai Y, Lim HY, Thongprasert S, Chao Y, Fan J, Yang TS, et al. Randomized, multicenter, open-label study of oxaliplatin plus fluorouracil/leucovorin versus doxorubicin as palliative chemotherapy in patients with advanced hepatocellular carcinoma from Asia . J Clin Oncol 2013 , 31 : 3501 – 3508 . Google Scholar Crossref Search ADS PubMed WorldCat 11. Kelland L . The resurgence of platinum-based cancer chemotherapy . Nat Rev Cancer 2007 , 7 : 573 – 584 . doi: 10.1038/nrc2167 Google Scholar Crossref Search ADS PubMed WorldCat 12. Endo T , Yoshikawa M, Ebara M, Kato K, Sunaga M, Fukuda H, Hayasaka A, et al. Immunohistochemical metallothionein expression in hepatocellular carcinoma: relation to tumor progression and chemoresistance to platinum agents . J Gastroenterol 2004 , 39 : 1196 – 1201 . Google Scholar Crossref Search ADS PubMed WorldCat 13. Ding K , Fan L, Chen S, Wang Y, Yu H, Sun Y, Yu J, et al. Overexpression of osteopontin promotes resistance to cisplatin treatment in HCC . Oncol Rep 2015 , 34 : 3297 – 3303 . doi: 10.3892/or.2015.4306 Google Scholar Crossref Search ADS PubMed WorldCat 14. Abe Y , Matsumoto S, Kito K, Ueda N. Cloning and expression of a novel MAPKK-like protein kinase, lymphokine-activated killer T-cell-originated protein kinase, specifically expressed in the testis and activated lymphoid cells . J Biol Chem 2000 , 275 : 21525 – 21531 . doi: 10.1074/jbc.M909629199 Google Scholar Crossref Search ADS PubMed WorldCat 15. Abe Y , Takeuchi T, Kagawa-Miki L, Ueda N, Shigemoto K, Yasukawa M, Kito K. A mitotic kinase TOPK enhances Cdk1/cyclin B1-dependent phosphorylation of PRC1 and promotes cytokinesis . J Mol Biol 2007 , 370 : 231 – 245 . doi: 10.1016/j.jmb.2007.04.067 Google Scholar Crossref Search ADS PubMed WorldCat 16. Herbert KJ , Ashton TM, Prevo R, Pirovano G, Higgins GST. LAK cell-originated protein kinase (TOPK): an emerging target for cancer-specific therapeutics . Cell Death Dis 2018 , 9 : 1089. Google Scholar OpenURL Placeholder Text WorldCat 17. Brown-Clay JD , Shenoy DN, Timofeeva O, Kallakury BV, Nandi AK, Banerjee PP. PBK/TOPK enhances aggressive phenotype in prostate cancer via β-catenin-TCF/LEF-mediated matrix metalloproteinases production and invasion . Oncotarget 2015 , 6 : 15594 – 15609 . Google Scholar Crossref Search ADS PubMed WorldCat 18. Chang CF , Chen SL, Sung WW, Hsieh MJ, Hsu HT, Chen LH, Chen MK, et al. PBK/TOPK expression predicts prognosis in oral cancer . Int J Mol Sci 2016 , 17 : 1007. doi: 10.3390/ijms17071007 Google Scholar OpenURL Placeholder Text WorldCat Crossref 19. Dong C , Fan W, Fang S. PBK as a potential biomarker associated with prognosis of glioblastoma . J Mol Neurosci 2020 , 70 : 56 – 64 . doi: 10.1007/s12031-019-01400-1 Google Scholar Crossref Search ADS PubMed WorldCat 20. Dou X , Wei J, Sun A, Shao G, Childress C, Yang W, Lin Q. PBK/TOPK mediates geranylgeranylation signaling for breast cancer cell proliferation . Cancer Cell Int 2015 , 15 : 27. doi: 10.1186/s12935-015-0178-0 Google Scholar OpenURL Placeholder Text WorldCat Crossref 21. Kwon CH , Park HJ, Choi YR, Kim A, Kim HW, Choi JH, Hwang CS, et al. PSMB8 and PBK as potential gastric cancer subtype-specific biomarkers associated with prognosis . Oncotarget 2016 , 7 : 21454 – 21468 . Google Scholar Crossref Search ADS PubMed WorldCat 22. Luo Q , Lei B, Liu S, Chen Y, Sheng W, Lin P, Li W, et al. Expression of PBK/TOPK in cervical cancer and cervical intraepithelial neoplasia . Int J Clin Exp Pathol 2014 , 7 : 8059 – 8064 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 23. Ma H , Li Y, Wang X, Wu H, Qi G, Li R, Yang N, et al. PBK, targeted by EVI1, promotes metastasis and confers cisplatin resistance through inducing autophagy in high-grade serous ovarian carcinoma . Cell Death Dis 2019 , 10 : 166. Google Scholar OpenURL Placeholder Text WorldCat 24. Yang QX , Zhong S, He L, Jia XJ, Tang H, Cheng ST, Ren JH, et al. PBK overexpression promotes metastasis of hepatocellular carcinoma via activating ETV4-uPAR signaling pathway . Cancer Lett 2019 , 452 : 90 – 102 . Google Scholar Crossref Search ADS PubMed WorldCat 25. Sun G , Ye N, Dai D, Chen Y, Li C, Sun Y. The protective role of the TOPK/PBK pathway in myocardial ischemia/reperfusion and H2O2-induced injury in H9C2 cardiomyocytes . Int J Mol Sci 2016 , 17 : 267. doi: 10.3390/ijms17030267 Google Scholar OpenURL Placeholder Text WorldCat Crossref 26. Goyal RK , Tulpan D, Chomistek N, González-Peña Fundora D, West C, Ellis BE, Frick M, et al. Analysis of MAPK and MAPKK gene families in wheat and related Triticeae species . BMC Genomics 2018 , 19 : 178. Google Scholar OpenURL Placeholder Text WorldCat 27. Guo YJ , Pan WW, Liu SB, Shen ZF, Xu Y, Hu LL. ERK/MAPK signalling pathway and tumorigenesis . Exp Ther Med 2020 , 19 : 1997 – 2007 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 28. Chen CY , Chen J, He L, Stiles BL. PTEN: tumor suppressor and metabolic regulator . Front Endocrinol (Lausanne) 2018 , 9 : 338. Google Scholar OpenURL Placeholder Text WorldCat 29. Zhang J , Xiang Z, Malaviarachchi PA, Yan Y, Baltz NJ, Emanuel PD, Liu YL. PTEN is indispensable for cells to respond to MAPK inhibitors in myeloid leukemia . Cell Signal 2018 , 50 : 72 – 79 . Google Scholar Crossref Search ADS PubMed WorldCat 30. Carnero A , Blanco-Aparicio C, Renner O, Link W, Leal JF. The PTEN/PI3K/AKT signalling pathway in cancer, therapeutic implications . Curr Cancer Drug Targets 2008 , 8 : 187 – 198 . doi: 10.2174/156800908784293659 Google Scholar Crossref Search ADS PubMed WorldCat 31. Dougherty JD , Garcia AD, Nakano I, Livingstone M, Norris B, Polakiewicz R, Wexler EM, et al. PBK/TOPK, a proliferating neural progenitor-specific mitogen-activated protein kinase kinase . J Neurosci 2005 , 25 : 10773 – 10785 . doi: 10.1523/JNEUROSCI.3207-05.2005 Google Scholar Crossref Search ADS PubMed WorldCat 32. Joel M , Mughal AA, Grieg Z, Murrell W, Palmero S, Mikkelsen B, Fjerdingstad HB, et al. Targeting PBK/TOPK decreases growth and survival of glioma initiating cells in vitro and attenuates tumor growth in vivo . Mol Cancer 2015 , 14 : 1476 – 1598 . doi: 10.1186/s12943-015-0398-x Google Scholar Crossref Search ADS WorldCat 33. Ishikawa C , Senba M, Mori N. Mitotic kinase PBK/TOPK as a therapeutic target for adult Tcell leukemia/lymphoma . Int J Oncol 2018 , 53 : 1791 – 2423 . Google Scholar OpenURL Placeholder Text WorldCat 34. Xu X , Mei J, Qi W, Shen H, Li Y, Wan L, Yan Z, et al. PBK/TOPK expression in non-small-cell lung cancer: its correlation and prognostic significance with Ki67 and p53 expression . Histopathology 2013 , 63 : 696 – 703 . doi: 10.3390/ijms17071007 Google Scholar PubMed OpenURL Placeholder Text WorldCat 35. Su TC , Chen CY, Tsai WC, Hsu HT, Yen HH, Sung WA-O, Chen CJ. Cytoplasmic, nuclear, and total PBK/TOPK expression is associated with prognosis in colorectal cancer patients: a retrospective analysis based on immunohistochemistry stain of tissue microarrays . PLoS One 2018 , 13 : 1932 – 6203 . doi: 10.1371/journal.pone.0204866 Google Scholar OpenURL Placeholder Text WorldCat Crossref 36. Shih MC , Chen JY, Wu YC, Jan YH, Yang BM, Lu PJ, Cheng HC, et al. TOPK/PBK promotes cell migration via modulation of the PI3K/PTEN/AKT pathway and is associated with poor prognosis in lung cancer . Oncogene 2012 , 31 : 2389 – 2400 . doi: 10.1038/onc.2011.419 Google Scholar Crossref Search ADS PubMed WorldCat 37. Whittaker S , Marais R, Zhu AX. The role of signaling pathways in the development and treatment of hepatocellular carcinoma . Oncogene 2010 , 29 : 4989 – 5005 . doi: 10.1038/onc.2010.236 Google Scholar Crossref Search ADS PubMed WorldCat 38. Liu YC , Yeh CT, Lin KH. Cancer stem cell functions in hepatocellular carcinoma and comprehensive therapeutic strategies . Cells 2020 , 9 : E1331. doi: 10.3390/cells9061331 Google Scholar OpenURL Placeholder Text WorldCat Crossref 39. Xiao J , Wang F, Lu H, Xu S, Zou L, Tian Q, Fu Y, et al. Targeting the COX2/MET/TOPK signaling axis induces apoptosis in gefitinib-resistant NSCLC cells . Cell Death Dis 2019 , 10 : 777. doi: 10.1038/s41419-019-2020-4 Google Scholar OpenURL Placeholder Text WorldCat Crossref 40. Zou J , Kuang W, Hu J, Rao H. miR-216b promotes cell growth and enhances chemosensitivity of colorectal cancer by suppressing PDZ-binding kinase . Biochem Biophys Res Commun 2017 , 488 : 247 – 252 . doi: 10.1016/j.bbrc.2017.03.162 Google Scholar Crossref Search ADS PubMed WorldCat 41. Hu F , Gartenhaus RB, Eichberg D, Liu Z, Fang HB, Rapoport AP. PBK/TOPK interacts with the DBD domain of tumor suppressor p53 and modulates expression of transcriptional targets including p21 . Oncogene 2010 , 29 : 5464 – 5674 . doi: 10.1038/onc.2010.275 Google Scholar Crossref Search ADS PubMed WorldCat 42. de Boussac H , Bruyer A, Jourdan M, Maes A, Robert N, Gourzones C, Vincent L, et al. Kinome expression profiling to target new therapeutic avenues in multiple myeloma . Haematologica 2020 , 105 : 784 – 795 . doi: 10.3324/haematol.2018.208306 Google Scholar Crossref Search ADS PubMed WorldCat Author notes † Hongmin Cao and Mei Yang contributed equally to this work. © The Author(s) 2021. Published by Oxford University Press on behalf of the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - PBK/TOPK promotes chemoresistance to oxaliplatin in hepatocellular carcinoma cells by regulating PTEN
JO - Acta Biochimica et Biophysica Sinica
DO - 10.1093/abbs/gmab028
DA - 2021-03-27
UR - https://www.deepdyve.com/lp/oxford-university-press/pbk-topk-promotes-chemoresistance-to-oxaliplatin-in-hepatocellular-VqL1EOZ5Va
SP - 1
EP - 1
VL - Advance Article
IS - 
DP - DeepDyve
ER -