Hepatocellular Carcinoma (HCC) is one of the most common malignancies in the world, and is well-known for its bad prognosis. Potassium calcium-activated channel subfamily N member 4 (KCNN4) is a type of intermediate conductance calcium-activated potassium channel, and increasing evidence suggests that KCNN4 contributes to the regulation of invasion and metastasis in a number of cancers. However, its clinical significance and biological function remain unclear in the HCC disease process. In this study, the expression levels of KCNN4 in 86 HCC samples were compared with corresponding paracancerous tissues. sh-RNA was used to reduce the expression of KCNN4 in Hep3B HCC cells in vitro; this was confirmed by Real time-PCR and western blotting. Wound healing, transwell assays and high content analysis were performed to investigate the tumor-promoting characteristics of KCNN4 in Hep3B HCC cells. As results, KCNN4 expression was significantly associated with preoperative serum alpha-fetoprotein level (p=0.038) and TNM stage (p=0.039). Additionally, patients with high KCNN4 amplification in HCC tissue exhibited shorter disease-free survival, whereas there was no statistical significance between KCNN4 amplification and overall survival. Wound healing and transwell assays showed that knockdown of KCNN4 expression could reduce migration and invasion abilities of HCC cells. High content analysis result showed that down-regulated KCNN4 could inhibit the ability of HCC cell proliferation. The mitogen-activated protein kinase (MAPK) pathway is active in cell proliferation, differentiation, migration, senescence, and apoptosis. Matrix metallopeptidase 9 and extracellular signal regulated kinase 1/2 (ERK1/2) were important biomarkers of MAPK/ERK pathway, knockdown of KCNN4 reduced the expression of MMP9 and ERK1/2. These findings showed that KCNN4 promotes HCC invasion and metastasis through the MAPK/ERK pathway.
- carcinoma, hepatocellular
- biological markers
- medical research
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Significance of this study
What is already known about this subject?
Potassium calcium-activated channel subfamily N member 4 (KCNN4) contributes to the regulation of invasion and metastasis in a number of cancers such as breast cancer and pancreatic cancer.
Several studies have demonstrated that increased KCNN4 expression is correlated with a poor clinical outcome in patients with various malignant tumors.
The clinical significance and biological function of KCNN4 remain unclear in the HCC disease process.
What are the new findings?
This is the first study to illustrate a role for KCNN4 in the invasion process of hepatocellular carcinoma (HCC) cells, through the mitogen-activated protein kinase/extracellular signal regulated kinase pathway.
KCNN4 expression was significantly associated with preoperative serum alpha-fetoprotein level and TNM stage.
The predictive value of KCNN4 in HCC patients was also investigated.
How might these results change the focus of research or clinical practice?
The higher expression of KCNN4 will result in worse disease free survival in HCC patients.
KCNN4 level may be used to supervise the relapse and metastasis in HCC patients.
Dysregulated KCNN4 channels could serve as therapeutic molecular targets with clinical potential value in HCC.
Liver cancer is one of the most common malignancies worldwide, especially in Asian countries; more than 7,80,000 people die from liver cancer each year, and approximately 8,40,000 patients are newly diagnosed with liver cancer.1 2 Hepatocellular carcinoma (HCC) is the most common pathological type of primary liver cancer and exhibits clinical features of occult onset, high degree of invasiveness, and high rates of recurrence and metastasis. Because of these features, most patients diagnosed with advanced stages of HCC are unable to undergo curative anticancer treatment.3 Surgery remains the primary therapy for HCC in early stages; however, approximately 70% of patients with HCC relapse within 5 years of surgery.4 HCC is largely insensitive to chemotherapeutic drugs.5 Sorafenib, a tyrosine kinase inhibitor, is the first systemic tumor-targeting therapy for HCC. Several international multicenter clinical trials of sorafenib have demonstrated that it provides a survival benefit in patients with HCC.6 7 Thus far, various treatments have been applied for HCC, such as surgery, chemotherapy, and radiotherapy; however, these have shown poor therapeutic effects. Therefore, it is necessary to identify biomarkers predictive of relapse among patients with early-stage HCC who have undergone surgery; moreover, new types of therapeutic drugs are urgently needed for treatment of HCC.
Potassium calcium-activated channel subfamily N member 4, also known as both KCNN4 and intermediate conductance calcium-activated potassium channel (KCa3.1), is a novel potential molecular target; this protein contributes to the regulation of intracellular calcium homeostasis and cell volume.8 KCNN4 is expressed in different cell types such as liver, pancreas, vascular smooth muscle, endothelial cells, and hematological cells.9 KCNN4 is a channel for intracellular Ca2+ influx and has great clinical significance in cell cycle progression, cell migration, epithelial transport, and other physiological activities.10 KCNN4 is reportedly overexpressed in various cancers, including lung cancer, endometrial cancer, breast cancer, glioblastoma, prostate cancer, and pancreatic cancer.11–16 In addition, several studies have demonstrated that increased KCNN4 expression is correlated with a poor clinical outcome in patients with various malignant tumors, such as renal carcinoma, pancreatic ductal adenocarcinoma, and lung cancer.11 16 17 Thus, KCNN4 channel blockers have been closely studied for their therapeutic potential in a variety of diseases, including autoimmune diseases and tumors, due to the involvement of KCNN4 in many important cellular functions.11 18 19 However, the role of changes in KCNN4 channel expression in HCC has remained largely unknown.
To our knowledge, this is the first study to illustrate a role for KCNN4 in the invasion process of HCC cells, through the mitogen-activated protein kinase (MAPK)/ extracellular signal regulated kinase (ERK) pathway. The predictive value of KCNN4 in patients with HCC was also investigated.
Materials and methods
In this study, HCC tissue microarrays were used to analyze 86 HCC specimens and corresponding paracancerous tissues from patients who underwent curative hepatectomy between February 2006 and May 2007. Eligibility criteria were as follows: (1) all patients who underwent radical hepatectomy due to an initial diagnosis of HCC without distant metastasis, and corresponding paracancerous tissues must have originated from normal tissues more than 5 cm from the edge of the HCC tumor; (2) patients who had no history of anticancer therapy, such as radiation therapy, chemotherapy, or molecular targeting therapy before surgery; (3) patients for whom detailed data were available regarding clinicopathological features and follow-up visits; and (4) patients from whom written informed consent had been obtained (ie, from patients or their legal representatives) before surgery. Importantly, clinical research ethics committee and legal regulations were observed during specimen collection. This study conformed to the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards.
Tissue microarray and immunohistochemistry
Human HCC tissue microarrays (LivH180Su07) used in this study were made and bought from BioChip (Shanghai, China). Immunohistochemistry was performed as described previously.20 Then we regarded the tissues scored 0 and 1 as low expression while others were regarded as high expression.
HCC cell lines and cell culture
The human HCC cell line Hep3B was originally obtained from the Chinese Center for Type Cultures Collections (CCTCC, Wuhan, China). Hep3B cells were cultured in RPMI 1640 media (Life Technologies, Beijing, China), supplemented with 10% fetal bovine serum (FBS) (Life Technologies, Beijing, China). All cells were maintained at 37°C in a humidified atmosphere with 5% CO2.
A total of 2×105 Hep3B cells per well were seeded into six-well plates, then incubated for 24 hours. Hep3B cells were then transfected with sh-KCNN4 or negative control shRNA and mixed with Lipofectamine 2000 transfection reagent (Invitrogen, California, USA). The sh-KCNN4 and negative control shRNA were constructed by GenePharma (Shanghai, China).
Wound healing assay
Scratch wound healing assay was performed as described previously.21 Hep3B cells used in this part were transfected with sh-KCNN4 or negative control shRNA.
Transwell migration assay
Transwell cell culture inserts (Costar Corporation, Corston, UK) were used to test invasion abilities of Hep3B cells: Hep3B cells transfected with sh-KCNN4 or negative control shRNA were plated into each transwell cell culture insert in serum-free Dulbecco’s modified Eagle medium (DMEM); the lower chambers were filled with 700 µl of DMEM with 10% FBS. After incubation for 48 hours, the cells in the upper surface of the upper chambers were removed with cotton swabs; the inner migrated cells were fixed with 500 µl of 4% paraformaldehyde for 15 minutes. The transwell membranes were washed with PBS and stained with crystal violet solution for 20 minutes. Migrated cells were counted and photographed by microscopy (Olympus, Tokyo, Japan).
High content analysis
Cells transfected with sh-KCNN4 or negative control shRNA were seeded in a 96-well plate at a density of 2000 cells/100 µl/well. After incubated overnight at 37°C and 5% CO2, the number of cells were measured over 5 days using a Celigo image cytometer (Nexcelom Bioscience, Lawrence, Massachusetts, USA). The cell proliferation rate was calculated based on the number of cells and the time point, and a growth curve based on the cell proliferation factor was drawn.
Western Blot was performed as described previously.21 The blots were treated with the respective antibodies overnight: anti-MMP-9 antibody (1:500 dilution, Proteintech Group, Wuhan, China), anti-ERK1/2 antibody (1:2000 dilution, Bimake, Houston, Texas, USA), anti-KCNN4 antibody (1:500 dilution, Biorbyt, Cambridge, UK).
Total RNA was extracted from Hep3B cells, which had been transfected with sh-KCNN4 and negative control shRNA, using TRIzol (Invitrogen, California, USA); cDNA was then synthesized using PrimeScript RT reagent kit (Takara, Beijing, China). Real-time PCR was performed with SYBR Premix Ex Taq (Takara, Beijing, China) on the LightCycler 480 system (Roche Diagnostics, Shanghai, China), in accordance with the manufacturer’s protocol. The qRT-PCR reaction included an initial denaturation step at 95°C for 30 seconds, followed by 40 cycles of 92°C for 5 seconds , 55°C for 30 seconds, and 72°C for 30 seconds. The relative gene expression levels were calculated using the relative quantitation value. The sequences for forward (F) and reversed (R) primers were as table 1.
Overall survival (OS) was defined as the interval between the date of curative hepatectomy and death. Disease-free survival (DFS) was defined as the interval between the date of surgical operation and tumor relapse. Data were censored at the last follow-up (February 2012) for patients without recurrence, metastases, or death. Two patients were lost to follow-up from among the 86 cases; both were eliminated at the stage of survival analysis.
Statistical analyses were performed by using SPSS statistics software V.24.0 (IBM) or GraphPad Prism V.6.0 (GraphPad Software, San Diego, California, USA). Cumulative survival time was calculated by the Kaplan-Meier method and analyzed by the log-rank test. Differences in the expression of KCNN4 between groups and the associations between KCNN4 and clinicopathological variables were evaluated by log-rank test. Univariate analyses were conducted by log-rank test. Multivariate Cox regression analyses were performed to identify factors that significantly influenced survival by Cox proportional hazards models. P values less than 0.05 were considered statistically significant.
Immunohistochemical analysis of KCNN4 expression and its relationship with clinicopathological parameters
More than 90% of pathological types of primary liver malignancies in this study were HCC; corresponding paracancerous tissues were normal liver tissues. Immunohistochemical staining of the HCC tissues and corresponding paracancerous tissues showed that KCNN4 protein was primarily located in the cell membrane and cytoplasm. Representative staining of KCNN4 expression in HCC tissues and corresponding paracancerous tissues is shown in figure 1. Among the 86 specimens of HCC, 55 (64.0%) showed high KCNN4 expression, while 31 (36.0%) exhibited low KCNN4 expression. In contrast to HCC tissues, analysis of corresponding paracancerous tissues showed that only 42 (48.8%) exhibited high KCNN4 expression. KCNN4 expression was upregulated in HCC tissues, compared with corresponding paracancerous tissues (p=0.046). We further analyzed correlations between KCNN4 expression and some clinicopathological parameters of patients with HCC. KCNN4 expression was significantly associated with preoperative serum alpha-fetoprotein level (p=0.038) and TNM stage (p=0.039). However, KCNN4 expression was not significantly correlated with the clinicopathological features of age, gender, tumor size, number of tumors, cirrhosis, tumor capsular invasion, Edmonson grade, Hepatitis B virus infection, preoperative serum alanine aminotransferase and serum gamma-glutamyl transferase in patients with HCC (p>0.05, see table 2).
Prognostic value of KCNN4 in human hepatocellular carcinoma
Among the 86 patients with HCC, two were lost to follow-up. Kaplan-Meier analysis demonstrated that patients with HCC with high KCNN4 expression had worse DFS than those with low KCNN4 expression (n=86, p=0.032). The mean survival time of patients with HCC with low KCNN4 expression was 53.0 months, whereas those with high KCNN4 expression demonstrated a shortened mean survival time of 41.0 months; however, there was no significant correlation between KCNN4 amplification level and OS (see figure 2).
Knockdown of KCNN4 expression reduces the expression of MAPK (ERK1/2) and MMP-9
Real-time PCR and western blotting were used to detect the expression of KCNN4. In this study, we found that Hep3B cells transfected with sh-KCNN4 had lower KCNN4, MMP9 and ERK1/2 mRNA expression levels than cells transfected with control sh-RNA(figure 3A). Moreover, transfection of Hep3B cells with sh-KCNN4 reduced the protein expressions of ERK1/2 and MMP9 (see figure 3B). The difference in expression between the two groups was statistically significant.
KCNN4 channel blockade reduces HCC cell line invasion and migration
Transwell and wound healing assays were used to test the invasion and migration abilities of the Hep3B cell line. As shown in figure 4 (see figure 4A,C), there were fewer Hep3B cells in the wound area after sh-KCNN4 transfection into Hep3B cells, compared with transfection with control sh-RNA at 0 , 8 , and 24 hours. Moreover, the transwell assay showed that the density of Hep3B cells in the group transfected with sh-KCNN4 was significantly lower than that in the group transfected with control sh-RNA (see figure 4B,D). Both of these experiments supported a role for KCNN4 in promoting the migration and invasion of these HCC cells.
KCNN4 channel blockade reduces HCC cell line proliferation
High content analysis was used to detect the proliferation ability of Hep3B cells. Compared with the cell numbers of the first day, the group transfected with sh-KCNN4 was 5.32 times larger while the number of the control group was 8.63 times. (see figure 5) The proliferation rate of sh-KCNN4 group was significantly lower than the control group after 5 days (p<0.001). It suggested that to knockdown the expression of KCNN4 would weak the proliferation ability of Hep3B cells.
Ion channels are transmembrane proteins that facilitate the transport of ions across biological membranes. Open ion channels cause redistribution of intracellular and extracellular ions, leading to a series of functional changes in cells; this is a key point in maintaining cell proliferation, migration, and apoptosis in normal tissues. Therefore, dysregulated expression and/or function of ion channels can impair physiological processes in healthy cells, thus leading to the transformation of normal cells into malignant tumor cells that exhibit uncontrolled proliferation, invasion, and migration. This may also be a mechanism of malignant tumor development.22 Increasing evidence supports the hypothesis that changes in ion channel function may lead to malignant tumor proliferation. The activation or inhibition of ion channels may affect many functions of malignant tumors.23–26 Dysregulation of ion channels greatly affects the phenotypes of malignant tumors, such as tumor cell proliferation, invasion, and distant metastasis. Cell migration is essential for metastasis of malignant tumors, and involves various types of proteins, including the KCNN4 channel; most types of cells with migration capabilities are able to express the KCNN4 channel,27–29 which belongs to the Ca2+-activated K+ channel superfamily. KCNN4 is reportedly overexpressed in various tumor tissues, including lung cancer, breast cancer, glioblastoma, prostate cancer, clear cell renal carcinoma, pancreatic cancer, and serous ovarian cancer.13–17 30 HCC is a highly malignant cancer arising from the liver, which demonstrates a poor prognosis.31 In the present study, immunohistochemistry analyses significant upregulation of KCNN4 expression in HCC tissue and the expression of KCNN4 has connection with serum alpha-feto protein level. The result was accordant with previous study.32 Importantly, increased expression of KCNN4 was associated with an increased risk of relapse in patients with early-stage HCC. This observation was consistent with the overexpression of KCNN4 mRNA or protein in HCC, as reported previously.33 Additionally, with respect to the prognostic value of KCNN4 expression, several studies have shown that KCNN4 is correlated with an unfavorable clinical outcome in patients with various types of cancer, such as renal carcinoma, pancreatic ductal adenocarcinoma, or lung cancer.11 16 17 However, there has not yet been a report regarding the role of KCNN4 in the prognosis of HCC. We characterized the expression pattern of KCNN4 in HCC; to our knowledge, this study is the first to reveal an association between increased expression of KCNN4 and worse DFS in patients with HCC, which suggests that KCNN4 plays an unfavorable role in HCC. Therefore, although it may not influence OS, patients with increased expression of KCNN4 should be monitored more frequently for earlier identification of tumor recurrence.
Generic MAPK comprise four parts that include signal-related kinases (ERK1/2), Jun amino-terminal kinases (JNK1/2/3), p38-MAPK, and ERK5. The MAPK pathway is active in cell proliferation, differentiation, migration, senescence, and apoptosis.34 There is extensive evidence that the MAPK/ERK pathway plays important roles in various types of cancer, such as prostate cancer, gastric cancer, and renal cell carcinoma.35–37 Some scholars speculate that the MAPK/ERK pathway is associated with resistance to chemotherapeutic agents in human cancers, such as head and neck squamous cell carcinoma and lung cancer, as well as HCC.38 In the present study, we found that silencing the expression of KCNN4 led to reductions in the levels of ERK1/2 and MMP-9. ERK1/2 and MMP-9 are both known as important biomarkers in the MAPK/ERK pathway. MMP-9 can induce degradation of the extracellular matrix, which reduces the stability of cancer cells, such that they become more likely to undergo metastasis. ERK1/2 is a key factor in the phosphorylation of Elk-1, which leads to the transactivation of downstream target genes involved in cell proliferation, migration and metastasis.39 The expression of ERK1/2 and MMP-9 was influenced by expression of KCNN4 in the present study; this suggests that a KCNN4 inhibitor can be used to prevent migration and invasion in HCC.
We further attempted to determine whether inhibition of KCNN4 channels could inhibit migration and proliferation of Hep3B cells. The results of transwell and wound healing assays showed that transfection with sh-KCNN4 could indeed inhibit the migration of human HCC cells, similar to the result of previous reports.33 The result of high content analysis showed that to knockdown the expression of KCNN4 would weaken the ability of HCC proliferation. Therefore, dysregulated KCNN4 channels could serve as therapeutic molecular targets with clinical potential value in HCC.
In conclusion, our data provide evidence for the differential expression of KCNN4 channels during malignant transformation in HCC. Furthermore, increased expression of KCNN4 channels was independently correlated with worse OS in patients with HCC and was associated with aggressive behaviors of HCC, such as invasion and migration. In addition, KCNN4 promoted the migration of Hep3B cells through the MAPK/ERK pathway, which facilitated tumor migration and invasion.
Q-TL and Y-MF contributed equally.
Contributors Conceived and designed the experiments: QT Li, ZF Xiong and LL Shi. Performed the experiments: ZH Ke, QT Li, MJ Qiu and XX He. Analyzed the data: YM Feng, MM Wang, YN Li and JX. Contributed reagents/materials/analysis tools: ZF Xiong and LL Shi. Wrote the paper: QT Li, YM Feng.
Funding This study was supported by the National Natural Science Foundation of China (No.81702885) and the Fundamental Research Funds for the Central Universities: the Independent Innovation Fund of Huazhong University of Science and Technology (No:2018KFYYXJJ106).
Competing interests None declared.
Ethics approval This study was approved by the Ethics Committee of the Union Hospital Affiliated with Tongji Medical College of Huazhong University of Science and Technology.
Provenance and peer review Not commissioned; externally peer reviewed.
Patient consent for publication Not required.