Abstract
Background/Aim: Gallbladder cancer (GBC) is a refractory cancer with poor prognosis. Recently, therapy targeting the tumor microenvironment (TME) has gained attention. Cancer hypoxia is a significant factor in the tumor microenvironment (TME). Our research has shown that hypoxia activates several molecules and signaling pathways that contribute to the development of various types of cancer. Our analysis indicated that C4orf47 expression was up-regulated in a hypoxic environment and had a role in the dormancy of pancreatic cancer. There are no other reports on the biological significance of C4orf47 in cancer and its mechanism is still unknown. This study analyzed how C4orf47 affects refractory GBC to develop a new effective therapy for GBC. Materials and Methods: Two human gallbladder carcinomas were used to examine how C4orf47 affects proliferation, migration, and invasion. C4orf47 was silenced using C4orf47 siRNA. Results: C4orf47 was over-expressed in gallbladder carcinomas under hypoxic conditions. C4orf47 inhibition increased the anchor-dependent proliferation and decreased the anchor-independent colony formation of GBC cells. C4orf47 inhibition reduced epithelial-mesenchymal transition and suppressed migration and invasiveness of GBC cells. C4orf47 inhibition decreased CD44, Fbxw-7, and p27 expression and increased C-myc expression. Conclusion: C4orf47 enhanced invasiveness and CD44 expression, and reduced anchor-independent colony formation, suggesting that C4orf47 is involved in plasticity and the acquisition of the stem-like phenotype of GBC. This information is useful for the development of new therapeutic strategies for GBC.
The most prevalent biliary tract cancer is gallbladder cancer (GBC). GBC accounts for 1.25 and 3.49 percent of cancer-related deaths in men and women in Japan, respectively (1). Radiation therapy and chemotherapy are not very promising treatments; open surgery is still the best option (2). GBC can manifest with a variety of symptoms, most of which do not manifest until late in the clinical course of the disease (3). Due to this, the diagnosis is typically only possible when the cancer is at an advanced stage (4). The development of new treatments for GBC has consequently become a crucial topic in Japan and worldwide.
In recent years, hypoxia has been shown to affect both normal and tumor cells, driving the activation of key transcription factors, which is an important cause of carcinogenesis (5). At the same time, hypoxia is also a very important factor for the conversion of GBC to a malignant tumor (6, 7). Cellular signaling pathways and a large number of molecules may be up-regulated or activated under hypoxic conditions, which may induce malignancy and metastasis of cancer cells.
We have previously shown that under hypoxic conditions, the expression of the centrosome-associated protein chromosome 4 open reading frame 47 (C4orf47) is up-regulated in pancreatic cancer, which simultaneously inhibits pancreatic cancer (PC) proliferation and promotes PC invasiveness and chemotherapy resistance (8, 9). C4orf47 has been reported to be a centrosome-associated protein, but its biological significance in cancer is completely unknown. In the present study, we analyzed the biological role of C4orf47 in GBC to aid in the development of a new effective therapeutic strategy against GBC.
Materials and Methods
Cell lines. We used two human GBC cell lines, NOZ and TGBC2TKB. NOZ cells were purchased from the Japanese Collection of Research Bioresources (JCRB) bank (Osaka, Japan). TGBC2TKB cells were purchased from Riken Cell Bank (Tsukuba, Japan). All cell lines were maintained in RPMI-1640 medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal calf serum (FCS; Life Technologies, Grand Island, NY, USA) and antibiotics (100 units/ml of penicillin and 100 μg/ml of streptomycin) as described previously (10, 11). The normoxic environment consisted of 5% CO2 and 95% air, whereas the hypoxic one comprised 1% O2, 5% CO2, and 94% N2; both were applied using a multi-gas incubator (Sanyo, Tokyo, Japan) as described in detail previously (12).
Cell transfection C4orf47 (ON-TARGETplus TM SMART pool, No. L-033933) and control non-targeting siRNA (ON-TARGETplus™ Control non-targeting siRNA, No. D-001810) were purchased from Dharmacon (Lafayette, CO, USA). NOZ, TGBC2TKB cells (2.0×105 cells/well) were seeded in 6-well plates and transfected for 48 h at 37°C under normoxic condition using Lipofectamine RNAiMAX (Invitrogen; Thermo Fisher Scientific) reagent according to the manufacturer’s protocol as previously described (13). Two days after the transfection, cells were used in the experiments.
Cell proliferation assay. Cell proliferation assay was performed as previously described (14). Briefly, the GBC cell line was transfected with C4orf47 siRNA or negative control siRNA and inoculated into 96-well plates (2.0×103 cells/well) for 2 days. These cells were then re-seeded and incubated at 37°C for 0, 24, 48, or 72 h under normoxic or hypoxic conditions. Cell counting reagent SF (Nacalai Tesque) was added to the cells and incubated for 1 h at 37°C. Cell proliferation was assessed by measuring the absorbance at 492 nm using a plate reader (Biotrak visible plate reader; Amersham Biosciences, Cytiva, MA, USA).
Western blotting analysis. Western blotting was performed as previously described (15). The protein transferred membranes were incubated overnight at 4°C with primary antibodies; C4orf47 (1:500, No. APR69924_P050; Aviva System Biology, San Diego, USA), CD44 (1:1000, No. ab243894, Abcam, Cambridge, UK), e-cadherin (1:200, No. sc-8426; Santa Cruz Biotechnology, Santa Cruz, CA, USA), vimentin (1:1,000, No. sc-6260; Santa Cruz Biotechnology), Twist (1:200, No. sc-81414; Santa Cruz Biotechnology), Snail-1 (1:200, No. sc-271977; Santa Cruz Biotechnology), Fbxw-7 (1:1000, No. ab109617; Abcam), P-27 (1:100, sc-528; Santa Cruz Biotechnology), and C-myc (1:200, No. sc-40; Santa Cruz Biotechnology). The membranes were incubated for at least 3 h at room temperature with secondary antibodies; horseradish peroxidase-linked anti-mouse antibody (1:10,000, No. NA931; Amersham Biosciences; Cytiva), horseradish peroxidase-linked anti-rabbit antibody (1:10,000, No. NA934; Amersham Biosciences; Cytiva), and horseradish peroxidase-linked anti-goat antibody (1:10,000, No. sc-2020; Santa Cruz Biotechnology). Immunocomplexes were detected with Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, Tokyo, Japan) and visualized with EZ Capture ST (ATTO, Tokyo, Japan). α-Tubulin (1:1,000, Sigma-Aldrich, St. Louis, MO, USA) was used as a protein loading control.
Colony formation assay. Colony formation assay was performed as described previously (15). Briefly, GBC cells (100 cells/well) transfected with C4orf47 or control non-targeting siRNA were cultured in 96-well plates at 37°C under normoxic or hypoxic conditions for 1 week, and colonies (>50 cells) were fixed with chilled 70% methanol for 10 min. After methanol fixation, the colonies were stained with 1% crystalline violet for 5 min. The number of colonies was counted using a fluorescence microscope at 100× magnification (BZ-X800, Keyence, Tokyo, Japan).
Cell migration and invasion assay. The migration ability of the GBC cell line was investigated with a Matrigel-free invasion assay as described previously, and the invasion ability of the GBC cell line was additionally assessed with a Matrigel invasion assay (16). siRNA-transfected cells (2.0×105) were placed in the upper chamber of the Transwell chamber and incubated for 18 h at 37°C under normoxic or hypoxic conditions. Cells on the lower side of the invasion filter were fixed with chilled 70% methanol for 10 min. Diff - Quik reagents (Sysmex Corporation, Kobe, Japan, Diff - Quik fixative, Diff - Quik solution I, Diff - Quik solution II) were used for cell staining. Stained cells were counted under a light microscope at 200X magnification (Nikon Eclipse TE 300, Nikon Corporation, Tokyo, Japan).
Statistical analysis. All data are expressed as the mean±standard deviation (SD). The unpaired Student’s t-test was utilized for the comparison of mean values between two groups. Calculations were performed using JMP 14.0 software (SAS Institute) or Microsoft Excel software (Microsoft). p-Values of <0.05 were considered statistically significant.
Results
C4orf47 is over-expressed in hypoxic conditions and reduces the anchor-dependent proliferation of GBC cell lines. First, we examined C4orf47 expression in two GBC cell lines (NOZ, TGBC2TKB) under normoxic and hypoxic conditions using western blotting. The expression of C4orf47 protein was significantly increased in the two types of GBC cells under hypoxia in a time-dependent manner (Figure 1A). Next, we used siRNA against C4orf47 to reduce the expression of C4orf47 in the two GBC cell lines under normoxia and hypoxia (Figure 1B) and investigated whether C4orf47 affects the anchor-dependent proliferation of GBC cells. When C4orf47 expression was inhibited, proliferation of GBC cell lines increased significantly under normoxia (Figure 1C) and hypoxia (Figure 1D).
Expression of centrosome-associated protein chromosome 4 open reading frame 47 (C4orf47) in gallbladder cancer cells in hypoxic condition and its effect on the proliferation of gallbladder cancer cells. A) The expression of C4orf47 in two gallbladder cancer (GBC) cell lines in normoxic, hypoxic culture for one day, and hypoxic culture for two days using western blot analysis. B) Inhibition of C4orf47 in two GBC cell lines using siRNA under normoxia and hypoxia was analyzed using western blot. C) The proliferative effects of C4orf47 on two GBC cell lines incubated for 24, 48, and 72 h under normoxic conditions. D: The proliferative effects of C4orf47 on two GBC cell lines incubated for 24, 48, and 72 h under hypoxic condition. Data are presented as means±standard deviations. *Significantly different at p<0.05. N.S: Not significant.
Inhibition of C4orf47 decreases the expression of CD44 and reduces the colony formation of GBC. Anchor-independent colony assay was used to explore the functions of C4orf47 in GBC. When the expression of C4orf47 in GBC was reduced using siRNA, the number of GBC cell colonies decreased significantly (Figure 2A and B). CD44 is a non-kinase transmembrane glycoprotein that is commonly expressed on a variety of cell types (17). The function of CD44 largely depends on cell adhesion, and CD44 up-regulation is associated with tumor progression and metastatic phenotypes in many cancers (18, 19). We examined CD44 expression in the two GBC cell lines where C4orf47 levels were reduced using siRNA under normoxia and hypoxia by western blotting. As expected, when C4orf47 expression was inhibited, CD44 expression in the GBC cell lines was also decreased under normoxia and hypoxia (Figure 2C), which was consistent with our colony assay data.
Effect of centrosome-associated protein chromosome 4 open reading frame 47 (C4orf47) on colony formation by gallbladder cancer cell lines. A) The effect of C4orf47 on gallbladder cancer (GBC) cells under normoxia and hypoxia was observed using the colony formation method. B) Quantification of the data on colony formation assays. C: Western blot assays for CD44 protein expression in two GBC cell lines under normoxia and hypoxia. Colony assay group, N=5. Original magnification: 200×. **Significantly different at p<0.01. Data are mean±SD.
C4orf47 enhances GBC cell migration and invasion through epithelial-mesenchymal transition (EMT). Next, we investigated whether C4orf47 is involved in the migratory and invasive properties of GBC cells. When C4orf47 was inhibited using siRNA, migration of GBC cells was significantly reduced under normoxia and hypoxia (Figure 3A). In addition, when C4orf47 was inhibited using siRNA, the invasiveness of GBC cells through matrigel was also significantly reduced under normoxia and hypoxia (Figure 3A). Furthermore, we found that when C4orf47 expression was inhibited, the expression of the EMT-related protein E-cadherin increased and protein expression of vimentin and SNAl-1 was suppressed, whereas twist expression was not affected (Figure 3B).
Centrosome-associated protein chromosome 4 open reading frame 47 (C4orf47) is involved in GBC cell migration and invasion through enhanced epithelial mesenchymal transition (EMT). A) The migratory and invasive ability of gallbladder cancer (GBC) cells transfected with siRNA control or siRNA C4orf47 was assessed using a non-matrigel migration assay or matrigel invasion assay under normoxia and hypoxia. B) Expression of EMT related molecules (E-cadherin, vimentin, Twist, SNAI-1) in GBC cell lines treated with C4orf47 or control siRNA under normoxia and hypoxia was examined using western blot assay. In the migration and invasion assay, all cells that had migrated from the upper to the lower side of the filter were counted. Data are presented as means±standard deviations. *Significantly different at p<0.05. **Significantly different at p<0.01.
The regulation of the expression levels of Fbxw-7, p-27, and C-myc contributes to the anchor-dependent proliferation of GBC. Our previous results showed that C4orf47 inhibits GBC cell proliferation and enhances migration/invasion. To analyze how C4orf47 decreases anchor-dependent proliferation, we selected Fbxw-7, P-27, and C-myc as our target molecules (8). Western blotting assay shows that when C4orf47 was inhibited, the protein expression of Fbxw-7 and P-27 was decreased and the protein expression of C-myc was enhanced (Figure 4A).
Centrosome-associated protein chromosome 4 open reading frame 47 (C4orf47) affects the expression of cancer-associated proteins in gallbladder cancer. A) Gallbladder cancer (GBC) cells were transfected with control or C4orf47 siRNA and then cultured under normoxia for 48 h. The cells were divided into normoxia and hypoxia groups and cultured for another 24 h. Expression of F-box/WD repeat-containing protein 7 (Fbxw-7), cyclin dependent kinase inhibitor p27, and cellular-myelocytomatosis (C-myc) was analyzed using western blotting. B) Conclusion schema of the results of this study. C4orf47 can enhance the malignant potential of GBC cells by affecting related molecules (Fbxw-7, p27, C-myc), CD44, and epithelial mesenchymal transition in hypoxia. Up line: Up-regulation; Down line: down-regulation.
Discussion
This study on GBC research confirmed that C4orf47 expression is up-regulated in hypoxic environments. C4orf47 is a centrosome-associated protein, and centrosomes are primarily associated with cell division, migration, and polarization (20). In recent years, no significant correlation between C4orf47 expression and disease pathology has been reported. Here, we found that C4orf47 promotes proliferation and decreases invasiveness of GBC. Similar to our previous study, which showed that C4orf47 contributes to the dormancy (21, 22) of pancreatic cancer, C4orf47 may also be involved in the dormancy of GBC. In dormancy, tumor cells remain inactive in the host for many years. However, they can become activated at both metastatic and secondary sites of the primary tumor, relapse and metastasize (23).
In this study, we found that C4orf47 inhibits proliferation of GBC cells, promotes colony generation, and mediates EMT, which is induced in hypoxic environment. The Fbxw-7 (F-box with 40 WD7 tandem) protein is one of the key components of the ubiquitin-ligase complex called Skp1-Cullin1-F-box (SCF) complex, which helps degrade many cancer proteins through the ubiquitin-proteasome system (UPS), thereby regulating cell growth (24). Recent studies have shown that P-27 is a cell cycle suppressor that may help predict the biological behavior of various human tumors (25). Our observations show that the expression of the tumor suppressor Fbxw-7 and the cell cycle-arresting factor P-27 are up-regulated, indicating that GBC cells enter a state of dormancy, resulting in a reduction in GBC cell proliferation. This may be due to the arresting effect induced by the inhibition of C-myc expression. CD44 is a receptor for Hermes antigen and lymphocyte homing receptor 11 that is involved in physiological and pathological processes which include inflammation, angiogenesis, cell adhesion and tumor development (26). In our study, we found that C4orf47 promotes CD44 expression, which leads to the efficient formation of colonies by GBC cells. In addition, C4orf47 is involved in EMT-related effects, leading to up-regulation of the epithelial marker E-cadherin and down-regulation of the mesenchymal markers vimentin and SNAI-1, that play a key role in migration and invasion of GBC.
In summary, the results of this study support the following conclusion (Figure 4B): first, C4orf47 expression was up-regulated under hypoxic condition in GBC; second, C4orf47 induced inhibition of GBC cell proliferation through up-regulation of cell cycle inhibitory factor Fbxw-, and P-27 and down-regulation of cell cycle promoter C-myc; third, C4orf47 promotes GBC cell colony formation of by promoting the up-regulation of CD44, which leads to enhanced migration of GBC cells; fourth, C4orf47 can up-regulate the expression of epithelial marker E-cadherin, down-regulate the expression of mesenchymal marker vimentin and EMT-associated factor SNAI-1, thus promoting EMT and enhancing the migration and invasion of GBC. Our results suggest that C4orf47 is involved with the plasticity and the acquisition of stem-like phenotype of GBC. These results help to elucidate the pathophysiology of refractory GBC and develop new therapeutic approaches.
Acknowledgements
The Authors thank Ms. Emi Onishi for her skillful technical assistance. This study was supported by JSPS KAKENHI Grant Numbers JP22H03163, JP21K08712, JP21K08906 and JP21K08673.
Footnotes
Authors’ Contributions
Lin Na was involved in the analysis of all experiments. Shogo Masuda, Shinjiro Nagao, Shinji Morisaki were involved in the acquisition and analysis of data. Naoya Iwamoto, Keita Sakanashi were involved in the gene transfection and interpretation of data.
Conflicts of Interest
The Authors declare no financial or commercial conflicts of interest regarding this study.
- Received February 13, 2023.
- Revision received February 28, 2023.
- Accepted March 2, 2023.
- Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
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