Research ArticleExperimental Studies

Hypoxia Increases the Proliferative and Metastatic Ability of Canine Mammary Tumor Cells via Up-regulation of TSG-6

TAE-HYEON KIM, JEONG-HWA LEE, JU-HYUN AN, BYUNG-GEE KO, KYEONG-BO KIM and HWA-YOUNG YOUN
Anticancer Research December 2022, 42 (12) 5803-5812; DOI: https://doi.org/10.21873/anticanres.16087

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Abstract

Background/Aim: HIF1α-induced hypoxia is a major characteristic of solid tumors that plays an important role in cancer growth, metastasis, and chronic inflammation. Tumor necrosis factor (TNF) stimulated gene (TSG)-6 is a strong regulator of anti-inflammatory pathways, but its role in cancer cells remains unclear. We hypothesized that hypoxia up-regulates TSG-6, thereby increasing the metastatic and growth potential of cancer cells. Materials and Methods: Primary and metastatic canine mammary gland tumor (MGT) cell lines (CIPp and CIPm), were transfected with TSG-6 specific siRNA and treated with cobalt chloride (CoCl2) for 48 h to chemically induce a hypoxia environment. The expression of hypoxia-inducible factor-1-alpha (HIF1α) was evaluated by RT-qPCR and western blot analysis. The metastatic ability of cancer cells and cell cycle distribution were assessed with extracellular matrix invasion assays and flow cytometry. Results: HIF1α up-regulation, induced by CoCl2, was significantly inhibited in the TSG-6-knockdown group in both canine MGT cell lines. The change in the expression levels of HIF1α corresponded to the change of invading cells in the TSG-6-knockdown group. TSG-6-knockdown in the hypoxia group showed decreased proliferation, associated with G2/M phase arrest. Conclusion: HIF1α expression in hypoxic condition is regulated by TSG-6 expression in canine MGT. TSG-6-knockdown causes down-regulation of HIF1α, thereby reducing the metastatic and proliferative abilities of cancer cells. TSG-6 in canine MGT has a potential as a therapeutic target in anti-cancer therapy.

Key Words:

Hypoxia, a low oxygen level, is a common feature of tumors associated with proliferation and metastasis (1). Hypoxic areas are created by an imbalance between the oxygen supply and consumption. As tumor tissues proliferate and expand, the oxygen demand exceeds its supply. Moreover, oxygen diffusion is impaired by the increasing distance between cells and vasculature, resulting in an even more hypoxic environment (2).

In response to hypoxia, changes in gene expression affect angiogenesis and metabolism, thereby promoting tumor growth and survival (3). Hypoxia stimulates angiogenesis to alleviate hypoxic conditions, leading to increased, rapid, and chaotic vessel formation. Consequently, the tumor tissue becomes highly hypoxic with excessive, but dysfunctional vasculature (4). In breast cancer, the hypoxic environment is a fundamental driver of progression, and the expression of HIF1 is associated with high mortality and poor prognosis through increased tumor growth, migration, metastasis, and drug resistance (5, 6). Canine MGT has the highest incidence in female dogs and is a major research target due to its poor prognosis and high metastatic rate (7-9). In addition, it has 99% similarity in genome sequencing to human breast cancer (10). Therefore, exploring the factors affecting hypoxia in canine MGT cells is important in translational aspects.

TSG-6, a protein product of tumor necrosis factor-alpha (TNFα)-stimulated gene-6 is constitutively expressed in certain organs and is induced by inflammatory cytokines (11). It performs various functions such as matrix organization, immune regulation, and cell regulation. Especially, TSG-6 is expressed in mesenchymal stem cells and has an important anti-inflammatory activity (12-14). Recently, certain studies have reported the relationship between cancer and TSG-6. High expression of TSG-6 in gastric and urothelial carcinoma have been reported to be associated with a poor prognosis (15, 16), suggesting an interaction between tumor cells and TSG-6.

Also, one of the hallmarks of cancer tissues is chronic inflammation (17). HIF1α is related with transcription factors such as NF-kB and TNFα, which are key coordinators of tumor-associated inflammatory signaling (18-19). However, little is known on the role of TSG-6 in cancer, and whether it could regulate HIF1α and affect the chronic inflammation pathways. Evaluating the relationship between cancer hypoxia and TSG-6 will provide a better understanding of the microenvironment of solid tumors. In this study, we investigated whether TSG-6 induces HIF1α and increases cancer cell growth and metastasis in canine MGT cells, and whether TSG-6 could be a target for anti-cancer therapy.

Materials and Methods

Cell lines and cultures. CIPp (primary) and CIPm (metastatic), canine malignant MGT cell lines were obtained from the Laboratory of Clinical Pathology, College of Veterinary Medicine, Seoul National University (Seoul, Republic of Korea). Cells were tested for mycoplasma and were found negative. CIPp and CIPm cells were cultured in Roswell Park Memorial Institute medium (RPMI) (WELGENE, Namcheon, Republic of Korea) with 10% fetal bovine serum, 1% penicillin-streptomycin and 1% Kanamycin. Cells were incubated at 37°C in a humidified atmosphere of 5% CO2. Cell culture medium was replaced every 2-3 days, and subculture was performed at 80-90% confluency.

Cobalt chloride treatment. Cobalt(II) chloride (CoCl2) (Sigma Aldrich, St. Louis, MO, USA) was used to induce hypoxic condition during cell culture. CoCl2 was diluted to a concentration of 100 mM in Dulbecco’s phosphate buffered saline (DPBS) (WELGENE) and stored at −20°C while blocking the light. When CoCl2 was applied to cancer cells, it was diluted into desired concentrations using the culture medium.

Transfection with small interfering RNA (siRNA). Before siRNA transfection, CIPp and CIPm cells were cultured in antibiotic-free medium in a 6 well plate (2.5×105 cells/well). When 40% confluency was achieved, cells were transfected with TSG-6 specific siRNA or scrambled siRNA (Santa Cruz Biotechnology, Dallas, TX, USA; sc-39819 and sc-37007, respectively) for 48 h using Lipofectamine 2000 (Invitrogen, Waltham, MA, USA), according to the manufacturer`s instructions.

Cell viability testing. Cell viability was assessed according to the manufacturer’s protocol using D-Plus™ CCK cell viability assay kit (CCK-3000) (Dong-in Biotech, Seoul, Republic of Korea). To determine the maximum concentration of CoCl2 that does not induce toxicity in cells, CIPp and CIPm cells were seeded into 96 well plates at 1,000 cells/well and incubated for 24 h. Then, cells were treated with CoCl2 at 0, 50, 75, 100, 125 and 150 μM for 24, 48 and 72 h. Following incubation, cells were treated with 10 μl Cell Counting Kit solution (Dong-in Biotech) and incubated at 37°C for 1 h. After incubation, the optical density (OD) at absorbance 450 nm for each well were assessed in a Microplate reader (BioTek Instruments, Winooski, VT, USA).

RNA extraction, cDNA synthesis, quantitative real time PCR (RT-qPCR). Total RNA of CIPp and CIPm cells was extracted using the Easy-BLUE Total RNA Extraction kit (Intron Biotechnology, Gyeonggi-do, Republic of Korea). For each sample, the concentration and purity were measured by a spectrometer (Implen, Westlake Village, CA, USA). cDNA was synthesized using CellScript All-in-One 5X 1st cDNA Strand Synthesis Master Mix (CellSafe, Seoul, Republic of Korea). For reverse transcription, the reaction mixtures were incubated at 42°C for 15 min and then reverse transcriptase was inactivated by heat treatment at 85°C for 5 s. The cDNA samples were assayed in duplicate for RT-qPCR using the AMPIGENE qPCR Green Mix HI-ROX with SYBR Green Dye (Enzo Life Science, Farmingdale, NY, USA), according to the manufacturer`s instructions. RT-qPCR analysis was performed using the Quantstudio 1 Real-time PCR system (Applied Biosystems, Waltham, MA, USA). The reactions were performed at 50°C for 2 min then at 95°C for 10 min, followed by 40 cycles of 95°C for 30 s and 60°C for 1 min. mRNA expression levels were normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), using the method (20). The primer sequences used are listed in Table I.

View this table:
Table I.

Primer sequences for quantitative real time PCR (RT-qPCR).

Protein extraction and western blot. Total protein was extracted from cultured cells using the PRO-PREP protein extraction solution (Intron Biotechnology, Seongnam, Republic of Korea). Protein concentration was quantified using the DC protein assay kit (Bio-Rad, Hercules, CA, USA). For the western blot assay, 15 μg of protein were separated by SDS-PAGE gel electrophoresis. After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA) and then blocked with 5% skim milk in Tris-buffered saline. Membranes were incubated with primary antibodies against HIF1α (1:500; LifeSpan BioSciences, Seattle, WA, USA), TSG-6 (1:500; Santa Cruz Biotechnology), cyclooxygenase (COX)-2 (1:500; Santa Cruz Biotechnology), nuclear factor kappa light chain enhancer of activated B-cells (NF-κB) (1:1000; Cell Signaling Technology, Danvers, MA, USA) and β-actin (1:1,000, Santa Cruz Biotechnology) at 4°C overnight. Then, a secondary antibody was applied for 2 h at room temperature, and the bands were detected by using chemiluminescence (Advansta, Menlo Park, CA, USA).

Invasion assay. CIPp and CIPm cells transfected with siRNA were cultured with normal media or 50 μM CoCl2 media for 48 h. CIPp cells were resuspended with serum-free RPMI at a concentration 2×106 cells/ml and CIPm was resuspended with serum-free RPMI at a concentration 5×105 cells/ml. Eight μm pore transwell membrane (SPL Life Science, Gyeonggi-do, Republic of Korea) was coated with 0.125 M Matrigel (Corning Life Science, Glendale, AZ, USA) and incubated at 37°C for 30 min. Following this step, 2 ml of complete medium were added to the bottom chamber and 1 ml of serum-free cell suspension medium was added to the upper chamber. Then cells were incubated at 37°C with 5% CO2 for 72 h. The upper chamber with cells that did not invade was removed with a cotton swab, and the invaded cells at the lower chamber were fixed with 3.7% formaldehyde, permeabilized with 100% methanol and stained with Giemsa. Cells were counted in a 100X field of view under a light microscope.

Cell cycle assay. CIPp cells transfected with siRNA were seeded in 6 well plates at a density of 2.5×105 cells/well and treated with CoCl2 (0 μM or 50 μM) for 48 h. After harvesting cells for flow cytometry, 1×106 cells were prepared per group, fixed with 80% acetone, and washed with DPBS. The cells were stained with 500 μl of propidium iodide/RNase buffer (BD Biosciences, Franklin Lakes, NJ, USA) for 20 min at room temperature in the dark. Cell fluorescence was analyzed by a flow cytometer (FACS Aria II) (BD Biosciences).

Statistical analysis. The GraphPad Prism (version 5) software (GraphPad Software, San Diego, CA, USA) was used for statistical analyses. One-way analysis of variance (ANOVA) and Two-way analysis of variance (ANOVA) analysis of variance followed by Bonferroni multiple comparison test. Results are shown as the mean±standard deviation. A p-Value <0.05 was determined to be statistically significant.

Results

Cell viability analysis of mammary gland tumors when using CoCl2. To establish whether TSG-6 regulates HIF1α, it was necessary to determine the appropriate concentration of CoCl2 required to create a chemically-induced hypoxic environment. The appropriate concentrations of CoCl2 for the CIPp and CIPm cell lines were determined using the CCK-8 assay (Figure 1). CIPp showed a significant decrease in OD value at 150 μM when applied for 24 h (p<0.05), and at 125 μM when applied for 48 and 72 h (p<0.005 and p<0.001, respectively) (Figure 1A-C). CIPm cells showed a significant decrease at concentrations higher than 75 μM after 48 h (p<0.05) (Figure 1D-F). Therefore, 100 μM for CIPp and 50 μM for CIPm, which are the maximum concentrations that did not affect the viability of cancer cells, were applied in subsequent experiments.

Figure 1.
Figure 1.

Cell viability assay for cobalt chloride (CoCl2) in CIPp and CIPm. Canine mammary gland tumor cell lines CIPp (A-C) and CIPm (D-F) were treated with various concentration of CoCl2 for 24 h, 48 h and 72 h. Optical density (OD) measurements were obtained at 450 nm absorbance after treatment with 10 μl Cell Counting Kit solution. OD values are presented as the mean±SD of sextuplicate samples, representative of three independent experiments. *p<0.05 versus control group, **p<0.01 versus control group, ***p<0.005 versus control group, ****p<0.001 versus control group.

Expression of TSG-6 in tumor cells under hypoxic conditions. TSG-6 expression in CIPp and CIPm cells was analyzed by RT-qPCR and western blotting to confirm whether TSG-6-knockdown occurred even after CoCl2 was applied for 48 h after siRNA transfection. RT-qPCR showed a significant decrease in the expression of TSG-6 in both CIPp and CIPm after transfection (siTSG-6 groups) when compared to control siRNA transfected groups (scRNA groups) (CIPp, p<0.005; CIPm, p<0.05) (Figure 2A and B). The protein expression levels were also reduced in the siTSG-6 group when compared to scRNA groups (CIPp, p<0.01; CIPm, p<0.05) (Figure 2C-F).

Figure 2.
Figure 2.

TSG-6 expression in CIPp and CIPm cells with or without cobalt chloride treatment (CoCl2). TSG-6 expression was evaluated by RT-qPCR and western blotting. In RT-qPCR results, RNA expression of TSG-6 in TSG-6 specific siRNA transfected cells (siTSG-6 group) of both CIPp and CIPm cells was compared to control cells (naïve group) and scrambled siRNA transfected cells (scRNA group) (A, B). Protein TSG-6 expression in the CIPp and CIPm cells was confirmed using western blotting (C, D). Band densities were measured using Image J (E, F). Relative mRNA expression was normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), using the method, and presented as the mean±SD of duplicate samples, representative of three independent experiments. *p<0.05 versus control group, **p<0.01 versus control group, ***p<0.005 versus control group, ****p<0.001 versus control group.

Relationship between TSG-6 and HIF1α in tumor cells. Western blotting was used to determine whether TSG-6 regulates HIF1α. HIF1α protein expression levels were determined by investigating a CoCl2-untreated group, and a CoCl2 (50 μM)-treated group (Figure 3). In the treated group, the expression of HIF1α increased in both CIPp and CIPm cell lines. On the other hand, in the siTSG-6 groups, HIF1α expression was significantly lower than that in scRNA groups (CIPp, p<0.05; CIPm, p<0.01).

Figure 3.
Figure 3.

Reduction of HIF1α expression in the siTSG-6 group. HIF1α and β-actin (control) protein expression in CIPp (A) and CIPm (B) cells was evaluated by western blotting. Relative band densities are shown for CIPp (C) and CIPm (D). HIF1α band density was estimated using Image J and normalized to that of β-actin. Relative band density was presented as the mean±SD of duplicate samples, representative of three independent experiments. *p<0.05 versus control group, **p<0.01 versus control group, ***p<0.005 versus control group, ****p<0.001 versus control group.

Hypoxia increases the metastatic capability of the CIPp and CIPm cell lines but not in the TSG-6-knockdown groups. To examine whether TSG-6 regulates the metastatic potential of cancer cells by regulating the expression of HIF1α, an invasion assay was performed. The number of invading cells markedly increased in the group treated with CoCl2 (Figure 4). When compared within the CoCl2-treated groups, both CIPp and CIPm cells significantly decreased the number of invading cells in the siTSG-6 groups, compared to scRNA groups (CIPp, p<0.01; CIPm, p<0.001).

Figure 4.
Figure 4.

Invasion of CIPp and CIPm cells through an extracellular matrix under cobalt chloride (CoCl2) treatment. The invasive ability of CIPp (A) and CIPm (B) cells was evaluated with extracellular matrix invasion assay with or without TSG-6-knockdown, treated in either normal media or CoCl2 50 μM media for 72 h. Invading CIPp (C) and CIPm (D) cells were counted in ×100 field of microscopic view and at least 9 fields were counted. Invading cell number was presented as the mean±SD of duplicate samples and was representative of three independent experiments. *p<0.05 versus control group, **p<0.01 versus control group, ***p<0.005 versus control group, ****p<0.001 versus control group.

Cell cycle features were affected in TSG-6-knockdown CIPp. The CIPp cell line was selected for the cell cycle assay. The cell cycle distribution of CIPp cells after hypoxia and TSG-6-knockdown was determined using flow cytometry (Figure 5). Under normoxic conditions, CIPp cells were mostly found in the G0/G1 phase (57.3±4.9%), followed by the G2/M phase (26.8±5.2%) and S phase (14±1.13%). Under normoxic conditions, the differences in cell populations between groups were not significant. However, under hypoxic conditions, the G0/G1 phase of the control group and control siRNA transfection group were 61.8±4.73% and 57.3±5.91%, respectively, and the G0/G1 phase of the siTSG-6 transfection group was 49.2±0.99% (p<0.001, p<0.005, respectively). The G2/M phase of the naïve group and scRNA groups were 23.4%±3.35% and 25.1±6.09%, respectively, and the G2/M phase of the siTSG-6 group was 34.8±0.64% (p<0.001, p<0.005, respectively). The S phase of the naïve group and scRNA group were 14.23±0.76% and 14.5±1%, respectively, and the S phase of the siTSG-6 group was 15.6±0.44% (p<0.05, ns, respectively). Therefore, cell-cycle arrest was induced in the G2/M phase of CIPp cells by TSG-6-knockdown under hypoxia.

Figure 5.
Figure 5.

Cell-cycle arrest was induced in the G2/M phase of CIPp cells by TSG-6-knockdown under hypoxic conditions. CIPp cells with or without TSG-6-knockdown were treated with either normal media or CoCl2 50 μM media for 48 h. PI-stained cancer cells were analyzed by flow cytometry for cell cycle distribution analysis (A). The percentages of cells in G0/G1, G2/M, and S phases were compared (B). Data are presented as the mean±SD of duplicate samples, representative of three independent experiments. *p<0.05 versus control group, **p<0.01 versus control group, ***p<0.005 versus control group, ****p<0.001 versus control group.

Discussion

In this study, we investigated the effect of TSG-6 and HIF1α-induced hypoxia in canine MGT cells, focusing on the proliferative and metastatic potential. We used specifically designed commercial siRNA to induce stable knockdown of TSG-6. After determining the optimal CoCl2 concentration to induce hypoxia, we confirmed HIF1α expression in both RNA and protein levels. Hypoxia successfully increased HIF1α expression in primary and metastatic canine MGT cells. In the siTSG-6 group, up-regulation of HIF1α was significantly inhibited. Cells showed decreased metastatic ability and proliferation, making TSG-6 an effective target for reducing cancer growth and metastasis.

A hypoxia incubator or chamber is often used to study cancer cell characteristics in a hypoxic environment, but chemically induced hypoxia is also relevant. The advantages of chemically induced hypoxia are that it is less expensive than using a hypoxia chamber or incubator and is stable enough to maintain the hypoxic state. CoCl2 is mostly used to mimic hypoxic conditions (21), created by inducing HIF1α accumulation by replacing Fe2+, a cofactor required for prolyl hydroxylase domain (PHD) activation, to prevent PHD activation and suppress HIF1α degradation (21, 22). However, a high concentration of CoCl2 in culture media could cause cell damage (23). Thus, we confirmed a proper CoCl2 concentration at which the action of TSG-6 could be analyzed without affecting cell growth, using cell viability assay.

HIF is a transcription factor involved in the regulation of genes expressed in cells to adapt to hypoxic environments (24-26). HIF1α has a well-known mechanism and pathway compared to other HIFα subtypes, and many studies have used HIF1α as a marker for hypoxia. HIF1α is expressed throughout the body and is involved in a cascade of gene expression events for the adaptation to hypoxic environments (26). When expressed in normoxia, HIF1α is hydroxylated by PHD, tagged by the ubiquitination of the Von Hippel-Lindau tumor suppressor protein, and rapidly degraded by the proteasome. However, under hypoxic conditions, the activity of PHD decreases, and HIF1α cannot be hydroxylated; therefore, HIF1α is not degraded and stabilized, allowing it to bind to HIF1β and transcribe the target gene (27). HIF1α plays a key role in many critical aspects of cancer biology, such as angiogenesis, metastasis, and resistance to therapy (2, 27, 28). In Figure 3, the siTSG-6 group showed decreased HIF1α expression at both the RNA and protein level. This revealed that TSG-6 is a factor inducing HIF1α expression in cancer hypoxia. The potential for this mechanism has already been revealed in several papers showing that TSG-6 induces NF-kB activation in inflammatory conditions (29). In addition, the fact that it is involved in STAT3 and TNF-α signaling in various types of immune cells was verified (30-32). Accordingly, the possibility that TSG-6 secreted by tumor cells also possesses the same mechanism is sufficiently raised.

If hypoxia persists, cancer cells become necrotic in the tumor microenvironment, and inflammatory responses occur (1). Inflamed lesions often become more hypoxic due to increased metabolic demands of the cells, which leads to prolonged inflammation (33). Therefore, targeting tumor hypoxia is considered an effective treatment for solid tumors (34). Among them, HIF1α is also considered as a potential target (35-37). One study showed that in vivo metastasis of gastric cancer was considerably reduced by HIF inhibition, whereas in vitro HIF-deficient cells were less invasive and adhesive (38). But due to complexity of HIF1 pathway regulation, developing effective anti-HIF1α therapy has been declined so far (39, 40). Our data suggest that TSG-6 could regulate HIF1α expression in hypoxic condition, which is a quite encouraging result.

An association between TSG-6 and cancer has recently been reported. In colorectal cancer, TSG-6 overexpression induces CD44 stabilization and reprogramming into cancer-associated fibroblasts through paracrine activation (41). Since gastric carcinoma and chronic inflammation correlate, and there is also a correlation between TSG-6 and inflammatory diseases, it is suggested that TSG-6 may be associated with a poor prognosis related to inflammation control in cancer tissues (42, 43). Although we did not directly confirm the mechanism by which TSG-6 regulates HIF1α, we would like to confirm the mechanism by which TSG-6 regulates tumor cells through follow-up studies. It is also necessary to analyze the effect of TSG-6 secreted by tumors on the growth and metastasis of breast cancer cells at the in vivo level.

In conclusion, our data suggest that tumor cell-derived TSG-6 regulates HIF1α expression in hypoxic condition. TSG-6-knockdown canine MGT cells reduced cancer proliferative and metastatic abilities, revealing a therapeutic potential of targeting TSG-6 in anti-cancer therapy.

Acknowledgements

This research was supported by the National Research Foundation of Korea (No. 2020M3H1A107330413). This study was partially supported by the Research Institute for Veterinary Science, Seoul National University.

Footnotes

  • Authors’ Contributions

    JHL designed the study. JHL and THK collected, analyzed, and interpreted data, and wrote the article. HYY granted final approval of the manuscript. BGK, KBK, JHA and HYY reviewed the article. All Authors have read and approved the final manuscript.

  • Conflicts of Interest

    The Authors declare that they have no competing interests.

  • Received September 7, 2022.
  • Revision received September 29, 2022.
  • Accepted October 4, 2022.

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Hypoxia Increases the Proliferative and Metastatic Ability of Canine Mammary Tumor Cells via Up-regulation of TSG-6
TAE-HYEON KIM, JEONG-HWA LEE, JU-HYUN AN, BYUNG-GEE KO, KYEONG-BO KIM, HWA-YOUNG YOUN
Anticancer Research Dec 2022, 42 (12) 5803-5812; DOI: 10.21873/anticanres.16087
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