Homeodomain-containing gene 10 inhibits cell apoptosis and promotes cell invasion and migration in osteosarcoma cell lines

Abstract

Homeodomain-containing gene 10 (HOXC10) belongs to the homeobox family, which encodes a highly conserved family of transcription factors that plays an important role in morphogenesis in all multicellular organisms. Altered expressions of HOXC10 have been reported in several malignancies. This study was aimed to reveal the expression profile of HOXC10 in osteosarcoma and evaluated whether HOXC10 is a molecular target for cancer therapy. We found that HOXC10 was up-regulated in osteosarcoma tissues compared with bone cyst specimens from The Cancer Genome Atlas database. Osteosarcoma MG63 cells were infected with HOXC10 shRNA expressing vector, and 143B cells were infected with HOXC10 expressing vector. We found that reduced expression of HOXC10 markedly impaired the ability of proliferation, invasion, and migration, and promoted cell apoptosis in vitro and in vivo. Up-regulated expression of HOXC10 promoted the proliferation, invasion, and migration, and inhibited apoptosis of 143B cells. Additionally, HOXC10 regulated apoptosis and migration via modulating expression of Bax/Bcl-2, caspase-3, MMP-2/MMP-9, and E-cadherin in both MG63 and 143B cells and in vivo. These results indicated that HOXC10 might be a diagnostic marker for osteosarcoma and could be a potential molecular target for the therapy of osteosarcoma.

Introduction

Osteosarcoma is the most common primary, malignant, musculoskeletal tumor in children and young adults, which has been regarded as a kind of differentiation disease caused by genetic and epigenetic changes that interrupt osteoblast differentiation from mesenchymal stem cells. The estimated survival rate for patients with osteosarcoma is approximately 30%, and 40% of it causes metastases at a later stage.^1,2 Despite the recent advance in therapeutic strategies, such as wide tumor excision, adjuvant chemotherapy, and radiotherapy, the clinical outcome is extremely poor due to recurrent or metastatic osteosarcoma. About 80% of patients present with metastatic disease following surgical treatment and have an extremely poor prognosis, with a long-term survival of less than 10%.³ Therefore, the prevention of metastases is critical for the improvement of the prognosis of patients carrying osteosarcoma, and various studies have been carried out to investigate the genes that are involved in osteosarcoma development. For example, metastasis-associated in colon cancer-1 (MACC1) was highly expressed in osteosarcoma tissues and contributes to osteosarcoma carcinogenesis via activation of Akt signaling pathway.⁴ FK506-binding protein 14 (FKBP14), a member of the family of FK506-binding proteins (FKBPs), is associated with cell cycle, apoptosis, and metastasis pathways and represents a new prognostic factor in osteosarcoma.⁵ However, the highly complex molecular mechanism of metastasis is still poorly understood.

Homeobox (HOX) genes play critical roles in cell differentiation and embryonic development through encoding transcription factors that bind to promoters of various target genes through their homeodomain controlling their expression.⁶ There is increasing evidence which implicates that HOX genes in cancers regulate many important processes such as differentiation, apoptosis, motility, receptor signaling, wound healing, and angiogenesis.^7–9 HOXC10 encodes a transcription factor containing a conserved DNA binding homeodomain and was found highly expressed in several cancer cells, including cervical,¹⁰ lymph node-positive breast,¹¹ thyroid cancer,¹² and leukemia.¹³ HOXC10 drives cell proliferation by facilitating transition from G1 to S phase and protects cells from apoptosis by binding to CDK7 and enhancing DNA repair in breast cancer cells.¹⁴ HOXC10 is an important mediator of invasion in the progression of high-grade squamous intraepithelial lesions (HSIL) to invasive cervical carcinoma.¹⁰ Clinicopathological features of patients with thyroid cancer revealed that the HOXC10 expression was positively correlated with advanced age and pathological stage and poor prognosis.¹² Although many studies implicated the role of HOXC10 in cancers, very little is known about HOXC10 expression level in osteosarcoma and its role in osteosarcoma development.

In this study, we demonstrated that HOXC10 expression significantly increased in osteosarcoma tissues compared with adjacent normal tissues. Moreover, down-regulated expression of HOXC10 promoted cell apoptosis and inhibited cell proliferation, migration, and invasion in human osteosarcoma cell lines and impede the development of tumors in vivo. We further verified that down-regulation of HOXC10 could induce the increase in expression of Bax, caspase-3, and E-cadherin and decreased Bcl-2, MMP-2, and MMP-9 expression both in vitro and in vivo. Taken together, this study indicated that HOXC10 plays an important role in osteosarcoma development and could be a potential therapeutic target for patients with osteosarcoma.

Materials and methods

Bioinformatics analysis

The Cancer Genome Atlas (TCGA) corresponding clinical data were downloaded from TCGA, following the approval of this project by the consortium. A total of 58 human osteosarcoma cases from the TCGA dataset were included, including 30 women and 28 men, aged between 15 and 69 years (median = 42 years).

Reverse transcription polymerase chain reaction analysis

RNA was extracted from frozen osteosarcoma tissues. cDNA was synthesized from RNA using a Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV RT) reagent kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). According to the manufacturer’s instructions, the DyNAmo Flash SYBR Green qPCR kit (Finnzymes Oy, Espoo, Finland) was used and analyzed on the 7900H Fast Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). For reverse transcription polymerase chain reaction (RT-PCR), primers targeting the HOXC10 (left: 5′-TGACTTCAATTGCGGGGTGA-3′, right: 5′-ACTAGGTGGGTAGGAGCAGG-3′) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; left: 5′-AATCCCATCACCATCTTC-3′, right: 5′-AGGCTGTTGTCATACTTC-3′) were generated. Relative quantification of genes’ expression levels was determined using the 2^−ΔΔCT method.

Cell lines and cell culture

Osteosarcoma cell lines HOS, 143B, U-2OS, SaoS2, and MG63 were purchased from Cell Bank of Type Culture Collection of Chinese Academy of Sciences, Chinese Academy of Sciences. Osteosarcoma cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Biowest, Riverside, MO, USA) and 1% penicillin–streptomycin solution (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in an atmosphere of 5% CO₂, except that SaoS2 cells were grown in DMEM with 15% FBS and U-2OS cells were grown in RPMI 1640 (Biowest).

Establishment of HOXC10 knockdown and stable expression cell lines

pLVX-Puro lentiviral construct containing human HOXC10 cDNA and pLKO.1-EGFP with shRNA against human HOXC10 was prepared as described previously.¹⁰ According to the manufacture’s instruction, the constructs were then transducted into HEK 293T cells with lentiviral packaging vectors using lipofectamine 2000 (Invitrogen). After 48 h transduction, lentivirus was collected and infected MG63 or 143B cells. A scramble shRNA cloned into pLKO.1-EGFP vector and black pLVX-Puro lentiviral vector were used as negative controls (NCs), respectively.

MTT assay

Cells were plated at 2 × 10³ cells/well in 96-well plates in DMEM containing 10% FBS 24 h after infection. Then, 20 µL of 5-mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; KeyGen Biotech Co., Nanjing, China) solution was added to each well and incubated for 4 h at 37°C. The optical density (OD) was measured using a microplate reader (BioTek, Winooski, VT, USA) at 490-nm wavelength.

Cell apoptosis assay

According to the manufacturer’s instructions, cell apoptosis was evaluated by flow cytometry using an Annexin-V-FITC Apoptosis Detection Kit (KeyGen Biotech Co.; Roche, Nanjing, China). Briefly, the cells (1 × 10⁵) were harvested and washed twice in phosphate-buffered saline (PBS) and resuspended in 500 µL of binding buffer and then stained in 10 µL of Annexin V and 5-µL propidium iodide (PI) and analyzed using a flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

Cell invasion and migration assay

For cell invasion assay, cells (5 × 10⁴) in 200 µL of serum-free DMEM were added to the upper layer which was coated with 20 µL of Matrigel (1:4 dilution; Costar). A volume of 600 µL of DMEM containing 10% FBS was added to the lower layer, and incubation was carried out for 48 h in a cell incubator. The cells that migrated to the lower surface of the membrane were fixed with 1 mL of 4% paraformaldehyde (Gibco) for 10 min and stained with 1 mL of 0.5% crystal violet for 30 min. Cells were photographed and counted under microscopy in random 10 fields with magnification of ×200. Migration assay were similar to Matrigel invasion assay except that the Transwell insert was not coated with Matrigel.

Western blot

Total proteins were extracted using radioimmunoprecipitation assay (RIPA)–phenylmethylsulfonyl fluoride (PMSF) solution and were quantified by the bicinchoninic acid assay (BCA; Beyotime, China). Proteins (10 µg) were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto a polyvinylidene difluoride membrane. The membranes were then blocked with 5% skim milk at room temperature for 1 h and incubated overnight at 4°C with the primary antibodies directly against HOXC10 (1:1000; Abcam), Bcl-2 (1:200; Santa; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), Bax (1:200; Santa), caspase-3 (1:1000; Abcam, Cambridge, MA, USA), MMP-2 (1:1000; Abcam), MMP-9 (1:500; Abcam), E-cadherin (1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA), and GAPDH (1:1500; Cell Signaling Technology). The intensity of the protein bands was quantified using an ultraviolet crosslinker (Bio-Rad Laboratories, Hercules, CA, USA) and normalized to GAPDH.

Tumor implantation

MG63 cells (2 × 10⁶ cells/mL) were injected subcutaneously into the right flank of 6-week-old male athymic nude mice (n = 6; Shanghai Laboratory Animal Co., Ltd., Shanghai, China). The tumor volume was measured 12, 16, 20, 24, 28, 32, and 36 days after the initiation of injection. At 36 days after injection, the mice were euthanized and the tumors were weighted.

Statistical analysis

Data were described as mean ± standard deviation (SD). Comparisons between different groups were undertaken using Student’s two-tailed t-test and one-way analysis of variance (ANOVA). All data were analyzed with GraphPad Prism Version 5.0 (GraphPad Software Inc., La Jolla, CA, USA). A value of p < 0.05 was considered a significant difference.

Results

HOXC10 is highly expressed in osteosarcoma tissues and cell lines

The real-time PCR showed that HOXC10 was significantly over-expressed in human osteosarcoma tissues when compared with the bone cysts samples in TCGA database (Figure 1(a)). Next, the protein levels of HOXC10 were detected in five osteosarcoma cell lines by western blot. The HOXC10 protein showed higher expression in osteosarcoma cell lines, with the highest expression detected in MG63 cells and the lowest expression detected in 143B cells (Figure 1(b)). These two cell lines were therefore used for our subsequent experiments. To explore the biological significance of HOXC10 in osteosarcoma tumorigenesis, we established HOXC10 knockdown and stably expressed cell lines of MG63 and 143B by lentivirus infection. Altered expression of HOXC10 was confirmed by western blot. We found significantly decreased HOXC10 expression by 66.1% in MG63 cells and increased by 1.83-fold in 143B cells compared with corresponding control cells (Figure 1(c) and (d)).

Figure 1.

HOXC10 expression in osteosarcoma tissues and cell lines. (a) HOXC10 expression level in osteosarcoma tissues was analyzed by real-time PCR. (b) HOXC10 expression level in five osteosarcoma cell lines was analyzed by western blot. (c) Expression of HOXC10 in MG63 with pLKO.1-EGFP-HOXC10 shRNA (shHOXC10) lentiviral infection and (d) in 143B cells with pLVX-Puro-HOXC10 (HOXC10) lentiviral infection was analyzed by western blot, respectively. GAPDH was also detected as the internal control. Representative western blots (upper panel) and quantitative results are shown (lower panel). *p < 0.05; ***p < 0.001 compared with 143B cells or corresponding control cells.

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Knockdown of HOXC10 inhibits cell proliferation and promotes cell apoptosis of osteosarcoma cells

Furthermore, MTT assay showed that knockdown of HOXC10 significantly decreased the proliferation of MG63 cells compared with controls, but increased cell proliferation was detected in 143B cells with HOXC10 over-expression (Figure 2(a) and (b)). These data suggest that HOXC10 suppresses in vitro proliferation of osteosarcoma cells.

Figure 2.

Suppressing HOXC10 expression-induced proliferation inhibition and apoptosis in osteosarcoma cells. (a, b) Cell proliferation was detected in MG63 and 143B cells by MTT assay, respectively. (c, d) Cell apoptosis was analyzed by Annexin V/PI staining and flow cytometry. *p < 0.05; **p < 0.01; ***p < 0.001 compared with corresponding control cells.

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To explore the potential mechanism by which HOXC10 knockdown suppresses osteosarcoma cell growth, we evaluated the cell apoptosis in MG63 and 143B cells using flow cytometry. The results showed that knockdown of HOXC10 in MG63 cells elicited an increased number of apoptotic cells as compared with control cells, and a significant decrease in apoptotic cells was detected in 143B cells with HOXC10 over-expression (Figure 2(c) and (d)). Collectively, our data suggest that suppression of cell growth by HOXC10 knockdown is partially attributable to increased cell apoptosis in vitro.

Knockdown of HOXC10 inhibits cell invasion and migration of osteosarcoma cells

Matrigel assay and Transwell assay were carried out to evaluate the effects of HOXC10 on invasion and migration capacity in osteosarcoma cells. As shown in Figure 3(a), silencing HOXC10 can significantly reduce the number of cells invading by 58.6%, and over-expressing HOXC10 can increase the number of cells invading by 2.12-fold as compared with corresponding control cells. Similarly, silencing HOXC10 can also significantly decrease the number of migrating MG63 cells by 55.9% (Figure 3(b)). However, high expression of HOXC10 led to 85.7% increase in the number of 143B cells migrating through the membrane (Figure 3(b)). These results reveal that HOXC10 promotes invasion and migration of osteosarcoma cells.

Figure 3.

Silencing of HOXC10 inhibited cell invasion and migration in osteosarcoma cells. (a) Cell invasion and (b) migration of MG63 and 143B cells were measured by Matrigel assay and Transwell assay. ***p < 0.001 compared with corresponding control cells.

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Knockdown of HOXC10 regulated apoptosis- and motility-associated protein expressions

To explore the potential mechanism by which HOXC10 regulated apoptosis and motility of osteosarcoma cells, we examined the levels of several apoptosis- and motility-associated proteins by western blot. As shown in Figure 4(a), the protein levels of active caspase-3, Bax, and E-cadherin significantly increased, whereas the expression level of Bcl-2, MMP-2, and MMP-9 remarkably reduced in MG63 cells with HOXC10 knockdown compared with control cells. However, high expression of HOXC10 led to decreased protein levels of caspase-3, Bax, and E-cadherin and increased protein levels of Bcl-2, MMP-2, and MMP-9 in 143B cells as compared with control cells (Figure 4(b)).

Figure 4.

Mechanisms of HOXC10 exert their functions in osteosarcoma cells. The expression of apoptosis and motility-related protein in (a) MG63 and (b) 143B cells was measured by western blot analysis. ***p < 0.001 compared with corresponding control cells.

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Knockdown of HOXC10 inhibits growth capacity of osteosarcoma cells in vivo

Next, we determined the effect of HOXC10 knockdown on the tumor growth in vivo. MG63 cells infected with shNC or shHOXC10 were subcutaneously injected in athymic nude mice, respectively, and tumor volumes were measured for 36 days. As shown in Figure 5(a), HOXC10-down-regulated tumors grew slowly in mice compared with the shNC tumors in mice. After 36 days, tumor volume, as well as tumor weight, in HOXC10-down-regulated mice was also examined, which showed that the tumor volume and weight in HOXC10-down-regulated mice significantly decreased compared with those in shNC mice (Figure 5(b) and (c)). The significantly decreased expression of HOXC10 is shown in Figure 5(d). Moreover, the protein levels of apoptosis- and motility-associated proteins, including Bcl-2, Bax, caspase-3, MMP-2, MMP-9, and E-cadherin, were also detected by western blot in vivo. As shown in Figure 5(d), the protein levels of active caspase-3, Bax, and E-cadherin significantly increased, whereas the expression level of Bcl-2, MMP-2, and MMP-9 was remarkably reduced in mice with injection of HOXC10-knockdown-treated MG63 cells compared with shNC-treated MG63 cells. Taken together, these results support the notion that HOXC10 can suppress the tumorigenesis of osteosarcoma cells in vivo.

Figure 5.

Knockdown of HOXC10 in osteosarcoma cells reduced tumor growth in vivo. MG63 cells infected with shRNA control (shNC) or shHOXC10 were subcutaneously injected in athymic nude mice. Tumor growth was shown 36 days after injection. (a) HOXC10 knockdown inhibits tumor growth in nude mice xenograft model in vivo. (b, c) Tumor volume and weight were also measured after HOXC10 knockdown. (d) The expression of HOXC10, Bcl-2, Bax, caspase-3, MMP-2, MMP-9, and E-cadherin in xenograft from the nude mice was determined by western blot analysis. ***p < 0.001 compared with corresponding shNC cells.

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Discussion

In this study, we investigated the biological function of HOXC10 in osteosarcoma. The bioinformatics data and in vitro data showed that HOXC10 was up-regulated in osteosarcoma tissues compared with bone cysts and in cell lines. The in vitro experiments also showed that down-regulating of HOXC10 in osteosarcoma cancer cells inhibited cell proliferation, migration, and metastasis, and promoted cell apoptosis. HOXC10 knockdown also inhibited tumor growth and apoptosis- and motility-related protein expression in vivo. Thus, HOXC10 may serve as a potential target for treatment of osteosarcoma.

HOXC10 is unique among the HOX genes required for efficient ubiquitylation and protein destruction by anaphase promoting complex (APC).¹⁵ Here, we found that HOXC10 was up-regulated in osteosarcoma tissues compared with normal bone cysts, which was in line with the previous studies. These results indicated that HOXC10 was a potent oncogene in osteosarcoma. Zhai et al.¹⁰ found that HOXC10 transcripts localized tumor cells, with relatively weaker expression in HSILs and virtual absence of HOXC10 expression in squamous epithelium from normal cervix. Ansari et al.¹³ also found that HOXC10 over-expressed in breast cancer and is transcriptionally regulated by estrogen.

Then, we examined the effect of HOXC10 on proliferation and apoptosis in MG63 and 143B osteosarcoma cells. We found that knockdown of HOXC10 in higher-HOXC10-expression MG63 cells significantly reduced cell proliferation and promoted cell apoptosis. Over-expression of HOXC10 in lower-HOXC10-expression 143B cells dramatically promoted cell proliferation and suppressed cell apoptosis. Further in vivo tumor formation study in nude mice indicated that knockdown of HOXC10 in osteosarcoma cells reduced the progress of tumor formation. These data may explain the inhibited cell proliferation of HOXC10 knockdown cells. Consistent with our findings, increased expression of HOXC10 in HeLa cells also resulted in increased colony number and size in soft agar assays;¹⁰ HOXC10 indirectly activates the E2F1 pathway to promote proliferation of breast cancer cells;¹⁴ and down-regulation of HOXC10 expression by shRNA inhibited cell proliferation and induced G1 phase cell cycle arrest in ARO and TT cells.¹² However, MCF-7 and ZR75B cells with HOXC10 knockdown increased growth and reduced apoptosis compared to control cells with non-silencing shRNA control, and no effect in MDA-MB-361 cells,¹⁶ suggesting that the different roles for HOXC10 may depend on the cell type and specific genetic background. Moreover, we also detected the apoptosis-related protein expression in response to the knockdown of HOXC10 in osteosarcoma cell line. Caspase activation is regulated by various cellular factors, including inhibitor of apoptosis proteins, pro-apoptotic Bcl-2 family members, and/or the Fas/Fas ligand (FasL) system.^17,18 In this study, knockdown of HOXC10 increased the expression of caspase-3 and the ratio of Bax/Bcl-2, whereas HOXC10 over-expression decreased them. Matrine inhibited the proliferation and induced apoptosis of osteosarcoma cell lines in vitro and in vivo through activation of caspase-3 and Bax and down-regulation of Bcl-2, which then trigger major apoptotic cascades.¹⁹

Moreover, it has been shown that HOXC10 promotes cell migration and invasion in several cancers. Zhai et al.¹⁰ found that HOXC10 transcripts localized tumor cells, with strongest expression in invasive carcinomas, and higher than all 39 HOX genes in distant metastasis correlated with short relapse-free or overall survival and resistance to chemotherapy.¹⁴ Knockdown of HOXC10 expression reduces the invasiveness of cervical cancer cells, indicating its key role in cervical cancer progression.²⁰ HOXC10 knockdown by shRNA conferred inhibition of migration and invasion through inhibiting the expression of transforming growth factor (TGF)-β1/2 and CXCL1/5 levels.¹² In our study, reduction in the HOXC10 expression in MG63 cells by treatment with specific shRNA decreased their migration and invasion capability both in vitro and in vivo, whereas over-expression of HOXC10 in 143B cells significantly promoted their migration and invasion. Contrast with our findings, some other studies showed that HOXC10 was expressed in breast cancer;²¹ however, this expression is lost in matched metastatic tissue,²² suggesting a negative role of HOXC10 in metastatic breast cancer. MMP-2 and MMP-9 promote tumor cell migration and invasion by degrading structural components of the extracellular matrix.^23,24 E-cadherin inhibits tumor metastasis by forming cell–cell adhesion.²⁵ In many carcinomas, expression of E-cadherin is correlated with tumor grade.^26,27 In line with our results, Jin et al.²⁸ reported that down-regulation of MMP-2 and MMP-9 inhibits osteosarcoma cell migration and invasion. E-cadherin was significantly less in osteosarcoma with metastasis, and its expression might be related to the prediction of metastasis potency and poor prognosis for patients with osteosarcoma.²⁹ These results indicate that HOXC10 may play an important role in promoting metastasis of osteosarcoma.

In summary, our study provides for the first time that HOXC10 played a key role in the proliferation, apoptosis, and metastasis of osteosarcoma cells. Moreover, HOXC10 might regulate these biological progresses through Bax/Bcl-2 ratio, caspase-3, MMP-2, MMP-9, and E-cadherin, thus may provide useful information for targeted therapy of osteosarcoma.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.