Homeobox B9 Promotes the Progression of Hepatocellular Carcinoma via TGF-β1/Smad and ERK1/2 Signaling Pathways

Objectives Homeobox B9 (HOXB9), a homeodomain-containing transcription factor, may play a role in hepatocellular carcinoma (HCC) progression. However, the exact mechanisms underlying its action remain unclear. Materials and methods. Immunohistochemistry was used to investigate the expression of HOBX9 and its prognostic values in HCC patients. HCC cells were transfected with pBabe-HOXB9 and shHOXB9 plasmids, and MTT assay, Transwell assays, and xenograft mouse models were employed to determine the effects of HOXB9 on HCC cell proliferation, migration, and invasion in vitro and in vivo. The biological mechanisms involved in the role of HOXB9 were determined with Western blot and RT-qPCR methods. Results HOXB9 expression was significantly increased in HCC tissues and cell lines. Patients with higher HOXB9 levels were associated with poor prognosis. Overexpression of HOXB9 in BEL-7405 cells promoted proliferation, migration, and invasion, whereas knockdown of HOXB9 in HepG2 cells significantly reduced cell proliferation, migration, and invasion abilities. Mechanically, a positive correlation was found between HOXB9 expression and transforming growth factor-β1 (TGF-β1) and extracellular signal-regulated kinase (ERK)1/2 pathway in HCC tissues. HOXB9 overexpression stimulated TGF-β1/Smads signaling pathway in BEL-7405 cells. In contrast, HOXB9 knockdown inhibited the TGF-β1/Smads signaling pathway in HepG2 cells. In addition, the treatment with TGF-β1 inhibitor, LY364947, significantly reserved HOXB9 overexpression-induced cell proliferation, migration, and invasion abilities. Conclusions These findings validated that HOXB9 promoted proliferation, migration, and invasion in HCC cells by stimulating the TGF-β1/Smads and ERK1/2 signaling pathway. HOXB9 could be a promising prognostic biomarker and a potential therapeutic target in HCC.


Introduction
Hepatocellular carcinoma (HCC) is the most prevalent primary liver cancer that has emerged as a critical global medical problem [1]. HCC can be caused by hepatitis B virus infection and cirrhosis [2]. Although substantial efforts have attempted to improve the prognosis of HCC, the 5-year survival rate is still less than 20% [3], and a better understanding of its molecular mechanisms is urgently needed. Tumorigenesis is mainly caused by the abnormal initiation of oncogenes, a mechanism that is equally important for the development of HCC progression [4]. The discovery of these genes associated with HCC progression makes them potentially applicable in the treatment of HCC in the future.
The homologous cassette gene HOX, a highly conserved gene family, acts to control cell proliferation and differentiation [5]. Recent studies have revealed that HOX not only plays a key role in growth and development, but is also closely related to the development of a variety of diseases, especially the process of tumor development [6]. HOXB9 is a member of the HOX gene family that regulates physiological processes such as cell proliferation, cell cycle and differentiation, embryonic segmentation, limb formation, and angiogenesis [7]. It has been found that HOXB9 could enhance the ability of lung cancer cells by regulating the proliferation and invasion through regulating epithelial-tomesenchymal transition [8]. In breast cancer, high expression of HOXB9 stimulates the expression of various vascular factors (such as transforming growth factor, TGF-β) that prepares tumor cells for proliferation and metastasis [9]. In HCC, Xiong et al. found that elevated HOXB9 were closely related to tumor invasion and metastasis [10]. On the contrary, HOXB9 was also reported to be served as a tumor suppressor gene in some cancer types, and elevated HOXB9 levels were found to predict good overall survival in colon cancer [11] and gastric cancer [12]. They found that HOXB9 acetylation may account for its inhibitory role in cancer cells [13,14]. Therefore, the role of HOXB9 in cancers may be more complicated than we assumed. However, the mechanism of HOXB9 in the generation and progression of HCC remains largely unknown. In our previous study, we found that the activation of transforming growth factor-β 1 (TGF-β1) contributes to HOXB9-induced proliferation activities in HCC [15]. Here, we further indicated that HOXB9 contributed to the HCC tumor growth by promoting cell proliferation, migration, and invasion through TGF-β1-mediated Smad2 signaling and extracellular signal-related kinases (ERK1/2) signaling pathway. The present study validated that HOXB9 may serve as an oncogene to promote HCC progression and be applied as an effective therapeutic target.

Materials and Methods
2.1. Patient Tissue Samples. A total of 89 HCC patients, who received the curative surgery treatment in our hospital between January 2017 and December 2018, were collected. HCC tumor tissue and paired adjacent tissue were obtained from HCC patients who underwent surgical resection at the First Affiliated Hospital of Dalian Medical University and Zhongshan Hospital of Dalian University. The tissues removed surgically were liquid nitrogen and transported to our laboratory and stored at −80°C. The clinicopathological data and survival information of included patients were collected. The study was approved by the Ethics Committee of the First Affiliated Hospital of Dalian Medical University and Zhongshan Hospital of Dalian University, and each participant signed informed consents before sample collection.
2.3. Cell Culture. The human hepatocellular carcinoma cell line (BEL-7402, BEL-7404, BEL-7405, HepG2, Hep3B, and SNU475) and the human normal liver cell line THLE-3 all were obtained from the Cell Bank of the Type Culture Preservation Committee of the Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco's modified Eagle's medium (DMEM) with glucose (4.5 g/l, Hyclone Laboratories, Logan, UT) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and were maintained at 37°C and 5% CO2. BEL-7405 and HepG2 cells were then chosen to investigate the underlying mechanisms of HOXB9 in HCC because of their high expression of HOXB9 and TGF-β1 expression.

Xenograft Model in Mice.
Female BALB/c nude mice (4-6 weeks of age, weighing 16-20 g) were purchased from SLAC Animal (Shanghai, China), housed under pathogenfree conditions, and randomly divided into the pBabe group, the HOXB9 group, the pSuper group, and shHOXB9 group (5 per group). The nude mice were subcutaneously injected with Bel-7405-pBabe, Bel-7405-HOXB9 or HepG-Super, HepG-shHOXB9 cells (2 × 10 7 in 0.5 mL PBS/mouse) on the left upper flank region of nude mice. The tumor volume was calculated using the following formula ðL × W 2 Þ/2 (L is the length, and W is the width of the tumor). After 4 weeks following implantation, mice were all sacrificed, and the tumors were excised, weighed, photographed, and harvested for subsequent RT-qPCR, Western blot, and IHC analysis.  10 BioMed Research International variance (ANOVA) and Student's t-test were used for pairwise comparisons. If P < 0:05, the difference was considered statistically significant.

HOXB9 Is Upregulated and Correlated with Unfavorable
Prognosis in HCC. We firstly determined the expression of HXOB9 in HCC tissues and nontumor tissues by using IHC assay. As shown in Figure 1(a), HXOB9 is located both in the nucleus and cytoplasm of HCC cells. By using IHC score, we separated the tumor tissues into three groups, including high, moderate, and low group. HXOB9 was highly expressed in the HCC tissues when compared with normal tissues (Figure 1(b)). Subsequent RT-qPCR and Western blot analysis showed that the expression of HOXB9  13 BioMed Research International in the low, moderate, and high group was upregulated gradually when comparable to those in the nontumor group (Figures 1(c) and 1(d)).
We further explore the association between the HXOB9 levels and clinical characteristics. Due to the relatively small samples, we assigned the patients with moderate and low HOXB9 as one group. As illustrated in Table 1, HOXB9 is positively correlated with tumor size (P = 0:028). Nevertheless, HXOB9 levels were not associated with other clinical characteristics, containing age, gender, histological differentiation, liver cirrhosis, metastasis, recurrence, HBsAg status, and serum AFP levels (P > 0:05 for all). In addition, Kaplan-Meier survival analysis then showed poorer percent survival of HCC patients with high HXOB9 levels (Figure 1(e)). The above data showed that increased HOBX9 in HCC indicated poor prognosis.

Correlation of HOXB9 and TGF-β1
Signaling Pathway in HCC Samples and Cell Lines. We then explored the potential mechanisms underlying the clinical significance of HOXB9 in HCC. Due to the involvement of TGF-β1 signaling in HCC progression [17], we determined the expression of phosphorylated-Smad2 in HCC tissues. As illustrated in Figures 2(a) and 2(b), the expression of p-Smad2 in the HCC tissues is higher than those in the nontumor tissues, and its expression in the high HOXB9 expressed tissues is higher than those in the low HOXB9 expressed HCC tissues. Meanwhile, the results of Western blot revealed that the expression of HOBX9, as well as the markers of TGF-β1 sig-naling, including Smad7, Smurf2, TGF-β1, and p-Smad2, was highly expressed in HCC tissues compared with those in the nontumor tissues. Smad7, Smurf2, TGF-β1, and p-Smad2 protein expression was higher in the high HOXB9 group than those in the low HOXB9 group (Figures 2(c) and 2(d)). Induction of metastatic phenotype by TGF-β1 signaling in tumor needs alteration in additional signaling cascades including MAPK [18]; thus, we detected the status of MAPK signaling pathway in HCC tissues. We found that the expression of p-ERK was in sync with HOBX9 and TGF-β1, expressed higher in the high HOXB9 group than those in the low HOXB9 group and in the nontumor group (Figures 2(e) and 2(f)). However, there was no significant change in the levels of p38 and JNK1/2 signaling pathway between the HCC tissues and nontumor tissues.
Subsequently, we analyzed the expression of HOBX9 in various HCC cell lines. RT-qPCR showed that HOBX9 was expressed at a low level in the normal human liver cells (THLE3 cells). The expression of HOBX9 mRNA was higher in all HCC cell lines when compared with THLE3 cells (Figure 3(a)). Equally, Western blot analysis showed that all HCC cell lines displayed higher HOBX9 protein levels compared to THLE3 cells. Moreover, except SNU475 cell, the expression of the markers of TGF-β1 signaling, including Smad7, Smurf2, TGF-β1, and p-Smad2, was highly expressed in HCC cell lines compared with those in THLE3 cells (Figures 3(b) and 3(c)). When referring to the MAPK signaling pathway, we found that the expression of p-ERK levels was significantly increased in HCC cell lines than

16
BioMed Research International those in the THLE3 cells (Figures 3(e) and 3(f)). However, there were no significant changes in the p38 and JNK1/2 signaling pathway between the HCC cell lines and THLE3 cells (Figures 3(d) and 3(e)). Taken together, the above data indicated a close relationship between HOBX9 and TGF-β1 and p-ERK signaling pathway in HCC.

HOXB9 Promotes Cell Proliferation, Migration and
Invasion of HCC Cells. To investigate the biological functions of HOXB9 in HCC, overexpression and knockdown of HOXB9 were performed in BEL-7405 and HepG2 cells, respectively, and the efficiency was verified by RT-qPCR and Western blot analysis. As shown in Figures 4(a) and 4(b), the expression of HOXB9 is marked increased in the HOXB9 group when compared with the pBabe group both at mRNA and protein levels. On the contrary, the expression of HOXB9 was decreased in the shHOXB9 group when compared with the pSuper group at mRNA and protein levels (Figures 4(c) and 4(d)). Subsequent CCK-8 assay showed that overexpression of HOXB9 enhanced the proliferative ability of BEL-7405 cells (Figure 4(e)), whereas inhibition of HOXB9 significantly inhibited cell proliferation of HepG2 cells (Figure 4(f)). Similarly, colony assay showed that overexpression of HOXB9 promoted cell growth in BEL-7405 cells (Figure 4(g)), and downregulation of HOXB9 suppressed cell growth in HepG2 cells (Figure 4(h)). Cell cycle analysis then suggested that overexpression of HOXB9 increased S phase arrest in BEL-7405 cells (Figures 4(i) and 4(j)), and downregulation of HOXB9 promoted the S phase transition in HepG2 cells (Figures 4(k) and 4(l)). Besides, Transwell migration assays reveled that upregulation of HOXB9 notably facilitated migration of BEL-7405 cells, while downregulation of HOXB9 suppressed migration of HepG2 cells (Figure 5(a)). Transwell invasion assays showed that upregulation of HOXB9 facilitated invasive ability of BEL-7405 cells, while downregulation of HOXB9 suppressed invasive ability of HepG2 cells ( Figure 5(b)).

HOXB9 Definitely Promotes HCC Cell Growth, In Vivo.
We then confirmed the function of HOXB9 in tumor growth, in vivo. IHC was used to confirm the changed HOXB9 expression in nude mice. As predicted, the expression of HOXB9 was increased in the BEL-7405-pBabe-HOXB9 group when compared with those in BEL-7405-pBabe group, while the expression of HOXB9 was significantly decreased in the HepG2-shHOXB9 group when compared with those in HepG2-Super group (Figures 6(a) and 6(b)). The size and weight of the tumors in the HOXB9 overexpressing group were markedly larger than its corresponding control group (Figure 6(c)), and the tumors in HOXB9 knockdown group were significantly lower than those in the control group ( Figure 6(e)). The tumor growth model showed that overexpression of HOXB9 facilitated tumor growth ( Figure 6(d)), whereas knockdown of HOXB9 notably inhibited tumor growth (Figure 6(f)).

3.5.
Correlation of HOXB9 and TGF-β1 Pathway, In Vivo. Moreover, we measured the levels of TGF-β1 and MAPK signaling in xenograft model, and the results of IHC showed that the p-Smad2-staining score was significantly higher in the HOXB9 overexpression group than in the BEL-7405-pBabe group, and significantly lower in the HOXB9 knockdown group than in the HepG2-Super group (Figures 7(a) and 7(b)). Tumors and extracted total proteins were isolated to evaluate the changes of TGF-β1 and MAPK signaling. Consequently, Smurf2, TGF-β1, and p-Smad2 expression was apparently increased, while Smad7 expression was decreased in the HOXB9 overexpressing group compared with the BEL-7405-pBabe group. Smurf2, TGF-β1 and p-Smad2 expression was decreased, and Smad7 expression was increased in the HOXB9 knockdown group compared with the HepG2-Super group (Figures 7(c) and 7(d)). Moreover, the results of immunofluorescence found that overexpression of HOXB9 in BEL-7450 cells increased the fluorescence intensity of p-Smad2 in the nucleus, whereas knockdown of HOXB9 in HepG2 cells decreased the fluorescence intensity of p-Smad2 in the nucleus.

HOXB9 Influences Cell Proliferation, Migration and
Invasion by Mediating the TGF-β Pathway. To further support that the TGF-β signaling pathway was involved in the mechanism of HOXB9-mediated tumorigenesis, HCC cells were treated with TGF-β type I receptor kinase inhibitor, LY364947. As shown in Figure 8(a), BEL-7405 cells treated with LY364947 effectively reverse HOXB9 overexpressionenhanced cell viability. In addition, FCM analysis found that the treatment with LY364947 could abrogate HOXB9 overexpression-induced S phase arrest (Figures 8(b) and 8(c)). Subsequent Transwell analysis confirmed that LY364947 successfully reversed HOXB9 overexpressionenhanced cell migration and invasion (Figures 8(d) and 8(e)). Furthermore, the result of immunofluorescence indicated that fluorescence intensity of p-Smad2 was obviously impaired in BEL-7405 cells with upregulation of HOXB9 (Figure 8(f)). Taken together, these results indicated HOXB9 promoted HCC progression by mediating TGF-β signaling pathway.

Discussion
Our previous study had validated that overexpression of HOXB9 stimulated the proliferation of HCC cells, whereas the knockdown of HOXB9 produced an opposite effect [15]. Further, in the present study, we validated the role of HOXB9 in HCC progression and revealed that its action was dependent on TGF-b1/Smad2 and ERK1/2 signaling pathways. The roles of HOXB9 in tumorigenesis and disease progression seem to vary between different tumor types. Elevated HOXB9 was correlated with the advanced clinicopathological staging and identified as a prognostic marker in lung cancer [19]. In breast cancer, increased HOXB9 was reported to promote the progression and metastasis of the cancer cells [20]. On the contrary, decreased HOXB9 was considered as a factor for poor prognosis in colon adenocarcinoma and pancreatic cancer, as well as gastric cancer [21][22][23]. The above data indicated the complex roles and mechanisms underlying tumorigenesis and disease progression of HOXB9. In the current study, we validated the elevated HOXB9 expression both in HCC tissues and cell lines. Consistent with our study, a previous study by Chiba et al. has identified that HOXB9 was upregulated in HCC tissues and increased HOXB9 levels in HCC predicts poor overall survival, but a beneficial response to sorafenib [24]. Similarly, the present survival of patients with high HOXB9 expression was lower than the patients with low HOBX9 expression in our study. Moreover, Xiong et al. further found that ubiquitin-like protein FAT10 could regulate HOXB9 expression by modulating the β-catenin/TCF4 pathway, therefore promoting the invasion and metastasis of HCC [25]. They also identified that glucose-regulated protein 78 activated the Wnt/HOXB9 pathway to promote maturation of low-density lipoprotein receptor-related protein 6 and to regulate the invasion and metastasis of HCC [10]. The above data strongly indicated the regulatory role of HOBX9 in HCC. However, to date, how HOXB9 participates the progression of HCC remains unclear, which promotes us to conduct our study. The association analysis between the degree of HOXB9 expression and clinicopathological characteristics revealed a significant correlation between HOXB9 expression and HCC tumor volume, suggesting that HOXB9 may facilitate the growth of HCC. Consistent with our previous study [15], HOXB9 promoted HCC cell proliferation and migration in vitro, as well as tumor growth in vivo. To elucidate the possible mechanisms by which HOXB9 regulates the malignant behavior of HCC cells, we also analyzed the relationship between HOXB9 and TGF-β1 and MAPKs signaling pathway, which are critical tumorigenic event contributing to hepatocarcinogenesis [26,27]. By applying RT-qPCR, Western blot, and IHC, we found that HOXB9 was positively correlated with TGF-β1 signaling pathway Smads-related proteins (TGF-β1, p-Smad2, Smurf2, and Smad7) and non-Smads pathway MAPK-related proteins (p-ERK1/2). The above data suggested that HOXB9 may affect the expression of TGF-β1, which in turn activates p-Smad2 and p-ERK1/2.
Currently, the role of TGF-β1 signaling in HCC remains controversial. TGF-β1 pathway may play a tumor suppressor role in the early phases of HCC development [28], whereas promoting epithelial-mesenchymal transition in favor of tumor growth in the later phases [29]. Abnormal activation of the TGF-β signaling pathway in Hep3B, BEL-7404, and BEL-7402 cells [30] indicates that the activation of the TGF-β1 pathway is a common feature of HCC cells. We found that TGF-β1 pathway was significantly activated in high HOXB9 expressed tissue, which may be because the HCC patients enrolled was not in an early stage. Previous studies have shown that in gliomas, HOXB9 can directly bind to the promoter of TGF-β1 to induce TGF-β1 expression [31]. Also, HOXB9 is able to activate TGF-β1 signaling pathway to promote the invasive migration of tumor cells in oral squamous cell carcinoma [32]. More importantly, our previous study has identified that HOXB9 overexpression could promote TGF-β1-induced epithelial-mesenchymal transition of HCC cells [33]. Here, our in vitro experiments further found that inhibition of TGF-β1 pathway by specific inhibitors could effectively abolish HOXB9 overexpression induced cell malignant ability. The phosphorylation level of Smad2 was reduced, and nuclear transport was inhibited after co-administration of TGF-β1 pathway inhibitors. The above data indicated that TGF-β1 pathway was essential for the role of HOXB9 in the hepatocarcinogenesis.
Taken together, our study suggested that aberrantly high HOXB9 expression in HCC could promote the proliferation, invasion, and migration ability of HCC tumor cells by activating TGF-β1-mediated Smads and ERK1/2 signaling pathways.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors confirm that there are no conflicts of interest.