Pro-Inflammatory Homeobox Gene, ISX, Regulates Tumor growth and Survival in Hepatocellular Carcinoma

Chronic inflammation drives initiation of hepatocellular carcinoma (HCC) but the underlying mechanisms linking inflammation and tumor formation remain obscure. In this study, we compared the expression of IL-6 (IL6) and CYCLIN D1 (CCND1) with the IL-6 induced homeobox gene ISX (intestine specific homeobox) in 119 paired specimens of HCC and adjacent normal tissues, and also in paired specimens from 11 non-HCC patients. In pathological analysis, ISX exhibited a tumor-specific expression pattern and a high correlation to patient survival time, tumor size, tumor number and progression stage. Enforced expression of ISX accelerated cell proliferation and tumorigenic activity in hepatoma cells through CCND1 induction. In contrast, shRNA-mediated attenuation of ISX in hepatoma cells decreased cell proliferation and malignant transformation in vitro and in vivo. A high positive correlation existed in human hepatoma tumors between ISX and CCND1 expression. Together, our results highlight ISX as an important regulator in hepatoma progression with significant potential as a prognostic and therapeutic target in HCC. tumor cell lines and tumor masses. The cellular function and tumor-specific expression pattern suggests that Isx is an important activator in proliferation and tumor formation in hepatocellular carcinoma. expression and a redundant regulatory effect in the intestine aborted the cellular proliferation effects. The genomic alterations at different stages are still unclear. In this study, ectopic Isx expression was detected in HCC patients, and some non-HCC patients infected with HBV and/or HCV (with no hepatitis) also showed the decreasing trend in the level of Isx mRNA expression, although the difference did not reach not statistical significance. Isx expression in different liver disease stages was still unclear, because the limitation in sample collection and the detailed regulatory mechanism of Isx in HCC tumorigenesis needs further investigation. through activating JAK /STAT3 signaling, NF- κ B and downstream genes, such as Isx, are consequently activated to mediate cell survival and G1 to S cell cycle transition. In this study, one potential I(cid:5)B binding site (RNNYYCC) on the Isx promoter region (-294 to -318 bp) was found to respond to NF-(cid:5)B regulatory activation in Isx expression. The results of this element deletion on the Isx promoter showed that this potential I(cid:5)B element was essential to NF-(cid:5)B activation. Also, from the mouse model, increased IL-6 production has also HCC Further, in liver regeneration, IL-6 regulates cellular proliferation the activation of cyclin D1 D1


Introduction
Hepatocellular carcinoma (HCC), the fifth most commonly occurring cancer and the third leading cause of cancer-related deaths every year worldwide, has a multiple step progression with a high evidence of association with chronic inflammation exposure induced by environmental toxin intake and/or viral infection, such as HBV or HCV (1). The chronic inflammation often interacts with innate and adaptive immune responses, and the detailed regulatory mechanism leading to HCC tumor formation is unclear to date. D-type cyclins are the major regulators governing G1 progression to S phase in response to mitogenic and oncogenic signals and serve as markers for human malignancies (2,3). Through binding with and activation of their associated cyclin-dependent kinases, CDK4 and CDK6, cyclin D-CDK complexes phosphorylate the retinoblastoma tumor suppressor gene products, pRB, and pRB-related proteins, p130 and p107 (4,5). This phosphorylation aborts growth-inhibitory functions of pRB, which leads to release of the E2F1 transcription factors and allows induction of E2F1-target genes that are required for progressing into S phase (6). The growth-promoting functions and deregulation expression of D cyclins is a driving force toward increased tumor proliferation and transforming activity in several human cancers (2,7). D cyclin overexpression in human cancers is driven by several mechanisms including transcriptional activation, genome alteration, post-transcriptional regulation, and post-translational protein stabilization (2,3 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on December 5, 2012; DOI: 10.1158/0008-5472. CAN-12-2795 oncogenes to transform cells (8,9). The oncogenic characterization of cyclin D1 explored in cancer models and human cancers suggests that cyclin D1 and its associated CDKs' activity may be a potential therapeutic target (3).
Homeobox genes, a superfamily of transcription factors with homeo domains, play an essential role in controlling cell growth, differentiation, and morphogenesis during early embryonic development (10). Deregulation of homeobox genes appears to increase cell survival and proliferation, and inhibit cell differentiation (11,12). Recently, many homeobox genes were found to be aberrantly expressed in a wide variety of human tumor masses (13). Isx (Intestine-specific homeobox) is a newly identified pair-family homeobox transcription factor, which shows a intestine-specific expression pattern in both adult and fetal intestines (14). Phylogenetic analysis showed that the homeo domain of Isx belongs to the Paired sub-family and is homologous to Pax3, Pax7, and Prrx1. Targeted disruption of Isx in mice revealed that Isx was required for intestine-specific regulation of the high density lipoprotein (HDL) receptor and cholesterol transporter scavenger receptor class B, Type 1 (SRB1), for vitamin A metabolism (14).
In this study, we identified Isx as a pro-inflammatory cytokine-induced homeobox gene, which is ectopically expressed in hepatocellular carcinoma (HCC). By directly binding to the cyclin D1 promoter, Isx regulated cellular cyclin D1 expression which further increased cell proliferation and transforming activity. Furthermore, both Isx and cyclin D1 were upregulated in both hepatoma

Patient characteristics
Isx is an ectopically expressed homeobox gene found in hepatoma microarray analysis (16); however, the correlation between clinical outcome of HCC and cellular function of Isx is unknown to date. In order to characterize the regulatory effect and clinical outcome of Isx in HCC, 123 HCC patients from two medical centers were enrolled into the Isx cohort study from July 2004 to November 2009. Of these, 119 had adequate follow-up data for analysis. The baseline characteristics of HCC and non-HCC patients were compared between groups with non-HCC, low and high Isx expression and the results are shown in Table 1. The overall survival time of all patients was 48 months. There were significant associations in terms of albumin (p = 0.0040), bilirubin (p = 0.0282), ac sugar (p = 0.0281), tumor size (p < 0.0001), number of tumors (p < 0.0001), and tumor grade (p < 0.0001), but not in age, sex, GOT, GPT, alkaline phosphotase, α-fetoprotein, γ-GT, cholesterol, and triglycerides ( Table 1) Fig. 1E). These results suggest that Isx plays an important regulatory role in HCC progression and patients' survival. The highly correlated expression between Isx and cyclin D1 also suggests that Isx could be a predictive marker for cyclin D1 expression and hepatoma growth.

Pro-inflammatory cytokines induced Isx expression through NF-țB signaling pathway
To explore the association of Isx expression with inflammatory cytokines in HCC tumor formation, the pro-inflammatory cytokines elevated in serum or liver tissue of hepatoma patients in previous studies (17)(18)(19) were first used to treat six HCC cells (Hep G2, Hep 3B, SK-Hep1, Huh 7, PLC/PRF/5, and HA22T) to evaluate the induction activity of Isx. Pro-inflammatory cytokines, such as IL-6, IL-1 ѽ ҏ and TNF-Į, were shown to be able to increase the expression of Isx mRNA at both the lower and higher concentrations used (5 and 20 ng/ml, respectively) for 8 hours ( Fig. 2A).
Higher, but not lower, doses of IL-8, IL-1β and TNFβ also were shown to increase Isx mRNA expression. In order to further clarify the regulatory signaling of Isx by these cytokines, the Isx promoter region was sub-cloned into a luciferase assay system. IL-6, an elevated cytokine in serum sample of HCC patients (20) showed a dose-dependent regulatory effect on the promoter activity of Isx ( Fig. 2B and C). Also, IL-6 (10 ng/ml) was shown to increase Isx protein and cyclin D1 deleted to a length shorter than 220 bp (Fig. 3B). Different segments of oligonucleotides between -320 to -220 bp in the Isx promoter region were then synthesized to determine the NF-țB (p65) binding affinity by electrophoresis mobility shift assay (EMSA) in vitro. The nuclear proteins of IL-6-treated cells showed high binding affinity and were supershifted by the addition of an anti-p65 antibody in the -294 to -318 bp region on the Isx promoter (Fig. 3C). Deletion of this NF-țB binding element showed abortion of the luciferase activity induced by IL-6 ( Fig. 3 B). Further, the Isx promoter region (-320 to -220 bp) could be pulled down (3.91 folds) in p65 (Rel A) immunoprecipitates treated with IL-6 compared with vehicle-treated cells (Fig. 3D). This promoter binding activity of p65 induced by IL-6 was abated when the cells were treated with NF-țB-specific inhibitor, BAY 11-7085 (5 ȝM), MAPK kinase inhibitor (U0126, 5 ȝM) and JAK/STAT2 inhibitor (AG490, 20 ȝM). These results suggested that signals (MAPK and JAK/STAT2) activated by IL-6 could activate Isx mRNA expression through increasing NF-țB (p65) binding activity on Isx promoter. However, NF-țB signaling activated by IL-6 regulated Isx expression at both the transcriptional and translational level.
Moreover, dietary Vitamin A intake has been shown to regulate intestinal Isx expression (3), and its metabolites are suggested to play a role in tumorigenesis. We next examined the potential role of vitamin A and its related metabolites on IL-6-induced Isx expression. Higher levels of retinoid X receptor (RXR) binding to the Isx promoter were detected in hepatoma cells than those noted in normal hepatocytes which showed no detectable expression of Isx (Fig. 3E) IL-6 treatment increased Isx expression in hepatoma cells, the levels of RXRĮ's binding to the Isx promoter were significantly reduced (ranging from 52 to 58%) in IL-6-treated hepatocytes, SK-Hep1 cells and, to a lesser degree, in HepG2 cells (18% reduction) (Fig. 3F). These results suggested that the Isx expression induced by IL-6 was a retinoid-independent regulatory pathway

Isx expression enhanced cell proliferation in hepatocellular carcinoma cell lines
To characterize the cellular function of Isx, wild Isx and different truncated proteins tagged with GFP were transfected and characterized in Hep G2 cells. Overexpressed Isx protein (green) was mainly detected in nuclei (blue), but Isx protein with the deleted homeobox domain was detected in whole cells including cytoplasm and nuclei, with loss of its original nuclear localization (Fig. 4A).
Interestingly, the GFP protein fused with the Isx homeobox domain showed a nuclear translocation pattern similar to that of the wild Isx protein (Fig. 4A). The cell proliferation activity of Isx in Hep cycle population compared with mock transfected only (Fig. 4D). These results suggested that Isx increased cell proliferation to speed up cell cycle progression from G1 to S phase despite an increase in sub-G1 apoptotic cells.

Isx up-regulated cyclin D1 and E2F1 eexpression in HCC cells
To determine the direct targets regulated by Isx, established regulators of the G1/S transition were monitored by Western blot analysis. As shown in Fig. 5A promoter was through the binding sequence between -260 to -297 bp.

Isx increased cyclin D1 expression through directly binding to the cyclin D1 promoter in Vitro and in Vivo
To further delineate the regulatory elements of Isx on the cyclin D1 promoter region, the different regions of oligonucleotide between -260 and -297 bp in cyclin D1 promoter were synthesized for EMSA analysis in vitro. The nuclear proteins extracted from Hep G2 cells transfected with Isx could specifically bind to the region between -260 and -272 bp and the binding complex also showed a super shift (arrow) of the anti-Isx antibody in vitro (Fig. 5E). The cyclin D1 promoter fragment (-197 to -330bp) bonded by Isx was detected by ChIP assay in vivo and showed an increase up to 11.6-fold compared with mock transfected cells (Fig. 5F). The binding activity of Isx on cyclin D1 promoter abated after NF-țB inhibitor (BAY 11-7085, 5 ȝM) treatment (Fig 5F).
Interestingly, MAPK (ERK1/2) inhibitor (PD98059, 50 ȝM) also showed a decrease to baseline on the cyclin D1 promoter binding activity of Isx (Fig. 5F). The regulatory effect of Isx on cyclin D1 was also determined in HCC patients. Isx (green) showed highly expression localization with cyclin Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Fig. 2). These results showed that Isx directly bound to cyclin D1 promoter and activated cyclin D1 (E2F1) expression transcriptionally.

Isx expression was essential for cyclin D1 expression and proliferation activity in hepatocellular carcinoma cells
To further evaluate the essential role of Isx on cell proliferation and tumorigenic activity, two sequence-specific shRNAis of Isx were transfected into four types of hepatoma cells with ectopic Isx expression (Hep G2, Hep 3B, SK-Hep1, and HA22T) and the knock-down efficiency in these hepatoma cells was examined by Western blot (Supplemental Fig. 3 and Fig. 6A). Isx protein expression was decreased 85% in Hep G2 cells but the knock down was less efficient in Hep 3B cells. These hepatoma cells with Isx knock down were first examined to determine the cell proliferation activity by [H 3 ] thymidine incorporation assay. The [H 3 ] thymidine incorporation rate in Isx knocked down cells showed a significant decrease of 62%, 56%, 54%, and 68%, respectively, in thymidine incorporation activity compared with those that were mock-transfected (Fig. 6B).
Further, many G1/S transition regulators, such as cyclin D1 (84%), CDK4 (62%), and E2F1 (86%), were significantly downregulated in Isx shRNAi cells (Fig. 6C and Supplemental Fig. 4). Some apoptotic factors were also shown to elevated in Isx shRNAi cells (Supplemental Fig. 5).Other tumor suppressors, such as p19 and p21, did not show any regulatory effect. Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Discussion
In this study, we identified Isx as a proto-oncogenic homeobox gene specifically overexpressed in HCC is a chronic liver disease that occurs as a multistep process characterized by progressive accumulation of genetic alterations causing aberrant growth, malignant transformation, and metastasis (21). Homeobox genes, essential regulatory transcription factors for multiple body plan development (10), have been found to be deregulated in many malignancies and are thought to be potential oncogenes (11). Few homeobox genes have been found to be involved in HCC development, excluding Hox, Prox1, and CDX2; however, most of them are expressed both in normal and tumor cells, and are thought to act as tumor suppressors in the liver (22)(23)(24). In this study, we found Isx, in particular, ectopically expressed in tumor cells of HCC tumor mass, but not normal cells, and highly correlated to cyclin D1 expression in HCC tumor cells. This tumor-specific pattern and the gene silencing results suggest that Isx is an important proto-oncogenic protein in HCC development. Isx, a gut specific transcription factor, was a putative repressor for intestinal SRBI and BCMO1 expression that consequently repressed vitamin A production and downstream retinoic acid (RA) signaling (3,14). Retinoid-induced signaling is important in regulating cell growth, differentiation, and development (25), and found to abnormally express and highly associated with tumor development in many malignances, including HCC (26). Deficiency in retinol and RXRĮ expression were found in tumor cells, but not the adjacent normal hepatocytes in HCC patients and animal model, which was adversely to Isx expression in HCC patients (27).
Also, our study suggested that IL-6-induced Isx expression in hepatoma cells was independent of the retinoid signaling. These results appeared to be at variance with the previous studies showing that dietary vitamin A intake was able to increase intestinal Isx expression (3). IL-6 might regulate Vitamin A metabolism via Isx expression, but this is, at present,uncertain in the case of HCC. More studies would be needed to elucidate the regulatory effect. Further, the regulatory activity on Vitamin A by ectopic Isx expression is still unclear, although defects of vitamin A metabolism have been observed in Isx knockout mice.
In Isx -/mice, the cell growth regulatory and transforming activity was not observed in tissue development and the offspring were born in the expected numbers and appeared healthy for at least expression and a redundant regulatory effect in the intestine aborted the cellular proliferation effects.
The genomic alterations at different stages are still unclear. In this study, ectopic Isx expression was detected in HCC patients, and some non-HCC patients infected with HBV and/or HCV (with no hepatitis) also showed the decreasing trend in the level of Isx mRNA expression, although the difference did not reach not statistical significance. Isx expression in different liver disease stages was still unclear, because the limitation in sample collection and the detailed regulatory mechanism of Isx in HCC tumorigenesis needs further investigation.
Chronic inflammation induced by hepatitis B virus, hepatitis C virus, and alcohol are always correlated to HCC development and serves as the most common factor involved in HCC progression (28,29). Many pro-inflammatory cytokines, such as TNF , IL-1 ѽ ҏ and IL-6, are found at high serum concentrations in HCC patients; however, the pathological role of these cytokines in HCC development is unclear to date (18,20). In this study, we demonstrated that Isx was showed high expression correlation to IL-6 in HCC patients and was an activated proto-oncogene through inflammatory cytokines (IL-6 or TNFĮ) that then subsequently regulate downstream cell cycle regulators, such as cyclin D1 and E2F1, to progress to cell proliferation and transformation in HCC.
This result provides a positive linkage between inflammation and HCC development. IL-6, one of the pro-inflammatory cytokines, has been reported to have high expression levels in HCC patients (19,20) and plays an important regulatory role in HCC development (18). Many viral proteins from HBV or HCV, and also alcohol abuse, all increase the IL-6 expression level in liver cells (28,30). IL-6 activates many signaling pathways to transform cellular responses, including JAK/STATs, MAPK, and AKT/PI3K (17,31), and, through activating JAK /STAT3 signaling, NF-κB and downstream genes, such as Isx, are consequently activated to mediate cell survival and G1 to S cell cycle transition. In this study, one potential IțB binding site (RNNYYCC) on the Isx promoter region (-294 to -318 bp) was found to respond to NF-țB regulatory activation in Isx expression.
The results of this element deletion on the Isx promoter showed that this potential IțB element was essential to NF-țB activation. Also, from the mouse model, increased IL-6 production has also been implicated in the pathogenesis of HCC (32). Further, in liver regeneration, IL-6 regulates cellular proliferation through the activation of cyclin D1 expression in hepatocytes, which provides a model for cyclin D1 activation by pro-inflammatory cytokines in HCC development (33).
In summary, in this study we found that Isx was an important tumor-specific proto-oncogenic