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Golgi protein 73 activation of MMP-13 promotes hepatocellular carcinoma cell invasion.

Golgi Protein 73 (GP73) is a serum biomarker for hepatocellular carcinoma (HCC), however its role in HCC is not clear. We report that GP73 promotes cell invasion, the hallmark of malignancy, through the upregulation of matrix metalloproteinase-13 (MMP-13). GP73 enhances MMP-13 expression through cAMP responsive element binding protein (CREB)-mediated transcription activation. Levels of GP73 and MMP-13 are increased and positively correlated in human HCC tissues. Augmented MMP-13 potentiates HCC cell metastasis. Thus, the GP73-CREB-MMP-13 axis potentiates cancer cell invasion and may be a target for HCC treatment.


INTRODUCTION
Hepatocellular carcinoma (HCC) is the fifth-most common cancer worldwide and the third-leading cause of cancer death [1]. The high mortality rate of HCC is mainly attributable to a late diagnosis at an advanced stage. Even though alpha-fetoprotein (AFP) is a specific serum marker for HCC, it is of minimal use as a diagnostic tool because its sensitivity is only around 50% [2]. Among other reported markers, Golgi protein 73 (GP73), also named Golgi membrane protein 1 (Golm1) or Golgi phos phoprotein 2 (GOLPH2), is proving to be a better biomarker for HCC [3][4][5].
GP73 is a 73-kDa type-II Golgi transmembrane glycoprotein that was originally cloned from a library derived from the liver tissue of a patient with adult giantcell hepatitis [6]. Knowledge on the structure and function of GP73 is limited. Aberrant over-expression of GP73 has been reported to correlate with many diseases such as viral infections, Alzheimer's disease, Wilson's disease and cancer [6][7][8][9][10]. Despite its steady-state localization in the Golgi ap paratus, GP73 can be secreted into the extracelluar space by cleavage at a proprotein convertase (PC) site and is present in the culture media of sev eral cell lines [11,12]. Block and colleagues reported that serum GP73 (sGP73) levels were up-regulated in patients with hepatitis B virusrelated HCC [12]. We, and others, demonstrate that the sensitivity and specificity of sGP73 for the identification of HCC are superior to those of alpha-fetoprotein (AFP), especially in early HCC [4,13]. Thus, we propose sGP73 as a novel marker for HCC diagnosis.
The discovery of GP73 as a potential biomarker for hepatocellular cancer begs the question as to its relevance in human HCC development. GP73 is a contributor to fibrogenesis in patients with chronic HBV infections, and GP73 recombinant protein can prompt the proliferation of LX2 cells (hepatic stellate cell line) [14]. Clinical results from several groups showed that an elevated level of GP73 protein is strongly associated with tumor size, vein invasion, tumor differentiation, epithelial-mesenchymal transition and overall survival [15][16][17]. Recently, we found that GP73 promotes cell proliferation, migration, invasion and metastasis [18]. Our studies suggest that GP73 plays an important role in tumor growth and metastasis. Nevertheless, the underlying mechanism is largely unknown.
In this study, we found that GP73 potentiated MMP-13 expression and that both GP73 and MMP-13 were higher in human HCC tissues compared to adjacent liver tissues. We then identified cAMP responsive element binding protein (CREB) as the mechanistic link between GP73 and MMP-13. GP73 increased CREB expression and then the enhanced CREB transcriptionally activated MMP-13. This GP73-CREB-MMP-13 signaling pathway was able to promote HCC cell invasion. Lastly, augmented MMP-13 potentiated HCC cell metastasis in nude mice.

GP73 potentiates MMP-13 expression in HCC cells
We first used quantitative real-time PCR (qRT-PCR) and Western blots to examine GP73 expression in 4 human liver cancer cell lines that had different metastatic potentials ( Figure 1A, 1B). Higher GP73 expression was observed in the more invasive cell lines, such as HCCLM3 and MHCC97L, than in HepG2 and Bel-7402. We therefore selected a higher GP73 expression cell line, HCCLM3, and a lower one, HepG2, for further investigation. To determine how GP73 facilitates HCC cell invasion, we checked for the expression of several proteolytic enzymes involved in degrading basement and Western blot B.. qRT-PCR analysis of MMPs mRNA level in HepG2 cells with GP73 overexpression C. or HCCLM3 cells with GP73 knockdown D.. Cellular levels of GP73 and MMP-13 in HepG2 cells with GP73 overexpression (E., top) or HCCLM3 cells with GP73 knockdown (F., top). The levels of secreted MMP-13 (active form) in the culture supernatants were measured by ELISA (E., F., bottom). A t-test was used to evaluate the statistical significance of these experiments, as compared to the control. *P < 0.05; **P < 0.01. membranes. These included 7 matrix metalloproteinases (MMPs) that we identified by qRT-PCR analysis and Western blot in HepG2 cells with ectopic GP73 expression and in HCCLM3 cells depleted of GP73. We found that 4 MMPs, MMP-1, MMP-2, MMP-13 and MMP-14, were increased after ectopic GP73 expression ( Figure 1C) and decreased after depletion of GP73 expression. Three other MMPS, MMP-3, MMP-7, and MMP-9, were not affected by GP73 state ( Figure 1D). Since alteration of MMP-13 was most prominent among the first 4 MMPs, we confirmed the ability of GP73 to increase MMP-13 levels with Western blot of cell lysates and ELISA of conditioned media ( Figure 1E-1F). To determine the potential correlation of GP73 and MMP-13 in HCC tissues, we checked the expression of GP73 and MMP-13 in primary tumors from HCC patients with Western blot (Figure 2A) (Table 1). In comparison with adjacent normal liver tissues, both GP73 and MMP-13 were both overexpressed in most of the tumors we checked ( Figure  2B).

Overexpression of MMP-13 promotes HCC cell invasion and metastasis
Upregulation of MMP-13 has been linked to lymph node metastasis of HCC [20]. We next examined the effect    Figure 3A) and metastasis in nude mice ( Figure 3B). In contrast, knockdown of the endogenous MMP-13 using MMP-13 specific shRNAs (shMMP-13) decreased the invasion of HCCLM3 cells in vitro ( Figure 3C) and metastasis in nude mice ( Figure 3D). Notably, MMP-13 increased GP73 expression in HepG2 cells ( Figure 3A). On the other hand, knockdown of endogenous MMP-13 decreased GP73 levels in HCCLM3 ( Figure 3C). Therefore, MMP-13 also influences the expression of GP73.
Even though GP73 is an integral Golgi membrane protein, it is also a secreted protein. The N-terminal 1-55 amino acids of GP73 encompass the N-terminal cytoplasmic domain, transmembrane domain, and a PC recognition site, which are crucial for protein Golgi localization and secretion. To test the direct effect of steady-state localization of GP73 on the invasive properties of hepatocellular carcinoma cells, we prepared HepG2 cells that expressed a non-secreted GP73 by transfecting a GP73-Δ(1-55) cDNA, which is devoid of the nucleic acids that encode for the N-terminal 1-55 amino acids needed for secretion [12,19,20]. We found that non-secreted GP73 potentiated HepG2 cell invasion ( Figure 6).

GP73 increases HCC cell invasion via activation of CREB-MMP-13-signaling pathway
CREB (cAMP responsive element binding protein) is a nuclear transcription factor that regulates the genes involved in cell survival and cell death. It has been reported that CREB promotes the expression of MMP-13 in human articular chondrocytes and osteoarthritis  [21]. We hypothesized that GP73 activates MMP-13 expression via upregulation of CREB. Both total and phosphorylated CREB were increased in the cells with GP73 overexpression, while knockdown of GP73 led to a drastic reduction of CREB expression in HCCLM3 cells ( Figure 7A). In addition, knockdown of CREB led to a drastic reduction of MMP-13 expression in HepG2-GP73 cells (Figure 7B), supporting the idea that GP73 increases MMP-13 expression through CREB. We thereby tested whether CREB stimulated MMP-13 expression as a transcriptional activator. We used qRT-PCR analysis of ChIP DNA and found that binding of CREB to a DNA region immediately upstream of the exon 1 of the MMP-13 gene was higher in HepG2-GP73 cells compared to binding in HepG2-Vector cells ( Figure 7C). Silencing the expression of CREB partially abrogated the invasive effect of GP73 on HepG2 cells ( Figure 7D). These results demonstrate that GP73 promotes HCC invasion through activation of the CREB-MMP-13 signaling pathway.

DISCUSSION
GP73 is a promising serum biomarker for HCC [4,22].However, its role in HCC progression was unclear.
Here, we suggest that GP73 promotes invasion of hepatocellular carcinoma cells. This promoting effect of GP73 on cell invasion is achieved through transactivation of MMP-13 expression by CREB. The abundance of GP73 and MMP-13 is positively correlated and enhanced in human HCC tissues.
Cell invasion is the major feature of malignancy. The Golgi apparatus plays an important role in cell migration and acts as a hub for different signaling pathways [23,24]. Therefore, elucidation of the regulatory networks of the Golgi apparatus will provide important information for the development of therapeutic strategies against cancer migration, invasion and metastasis. GP73 is a Golgiassociated protein and is overexpressed in HCC. We found that overexpression of GP73 promoted the invasion of HCC cells. These data suggest that augmented GP73 functions as an oncogene.
MMPs play important roles in tumor invasion because their proteolytic activities assist in degradation of the extracellular matrix and basement membrane. MMP-13 is expressed in a very restricted manner in the human body, but is often upregulated under pathological conditions, such as cancer and arthritis [25]. It was  reported that MMP-13 is upregulated in human HCC [26], however its role in HCC is unclear. We present here that MMP-13 potentiates human HCC cell lung metastasis in nude mice. GP73 accelerates cell invasion through activation of MMP-13 expression. MMP-13 also potentiates GP73 expression. It appears that there is a feedback loop between GP73 and MMP-13. Aberrant MMP-13 expression strongly associates with GP73 abundance in human HCC tissues. The levels of MMP-13 and GP73 are significantly higher in tumors than in their adjacent tissues, therefore, GP73 enhancement of MMP-13 expression may be implicated in HCC development.
Many transcription factors, such as CREB, STAT1, AP-1, and FOXO3a, may regulate the expression of MMP-13 [21,27]. CREB is one of the best understood phosphorylation-dependent transcription factors that has been shown to regulate the expression of many genes, such as MMPs [25]. In this study, we show that GP73 stimulates the expression CREB and MMP-13. CREB binds to the promoter of MMP-13 and the enhanced CREB is critical for GP73 mediated MMP-13 overexpression. Thus CREB serves as a bridge between GP73 and the In summary, we demonstrate that GP73 promotes cell invasion by potentiating CREB-MMP-13 expression. Augmented MMP-13 potentiates HCC cell metastasis. We suggest that GP73 enhancement of MMP-13 expression may have clinical relevance in HCC development. Therefore, inhibition of this GP73-CREB-MMP-13 signaling axis should provide a potential therapeutic strategy for HCC.

(II) RNA interference with siRNA
The CREB and negative control small interfering RNAs (siRNAs) were purchased from Dharmacon (Thermo Fisher Scientific, Pittsburgh, PA, USA). Cells were plated at a density of 8×10 4 cells/well in 6-well plates and transfected with 50 or 100 nM CREB or negative control siRNA oligoduplexes after preincubation for 20 minutes with DharmaFect transfection reagent in serumfree Opti-MEM I medium (Invitrogen, Carlsbad, CA, USA). The transfection mixture was replaced with DMEM medium containing 10% FBS (without antibiotics) 5 hours later. Forty-eight hours after transfection, cells were collected for invasion assays or for preparation of whole cell lysates.

Enzyme linked immunosorbent assay
HCCLM3 and HepG2 cells were transfected with the indicated plasmids for 48 hours. The levels of secreted MMP-13 (active form) in the culture supernatants were measured using MMP-13 Human enzyme linked immunosorbent assay (ELISA) Kit (Abcam, Cambridge, MA, USA) following the manufacturer's instructions.

RNA extraction, cDNA synthesis, and qRT-PCR
Total RNA was extracted from cell pellets using E.Z.N.A.® Total RNA Kit I (Omega Bio-Tek, Norcross, GA, USA) and reversely transcribed into cDNA using PrimeScript™ RT-PCR Kit (Takara Biotechnology, Dalian, China) according to the manufacturer's instructions. The target gene expressions were determined by qRT-PCR using specific primers of target genes ( Table  2). GAPDH was served as an internal control for total cDNA content. For qRT-PCR analysis, aliquots of doublestranded cDNA were amplified using a SYBR Green PCR Kit (TransGen Biotech, Beijing, China). The cycling parameters were 45 cycles of 95°C for 15 seconds, 57°C for 15 seconds, and 72°C for 15 seconds. A melting curve analysis was then performed. The Ct was measured during the exponential amplification phase, and the amplification plots were analyzed using CFX Manager 2.1 software (Bio-Rad Laboratories, Hercules, CA, USA). The relative expression level (defined as fold change) of the target gene was determined by the following equation: 2 -ΔΔCt (ΔCt = ΔCt target -ΔCt GAPDH ; ΔΔCt = ΔCt expressing vector -ΔCt control vector ). The expression level was normalized to the fold change detected in the corresponding control cells, which was defined as 1.0.

Cell invasion assay
Cell invasion was investigated using Matrigelcoated 8.0-μm filter invasion chambers (BD Biosciences, San Jose, CA, USA). Cells were incubated for 22 hours at 37°C in a humidified atmosphere of 5% CO 2 . Cells on the upper surface of the membrane were removed using cotton tips after the indicated incubation times. The migrant cells attached to the lower surface were stained with crystal violet (500μL of 5 mg/mL crystal violet dissolved in 20% methanol) and incubated for 30 minutes. The membrane was washed with phosphate-buffered saline, and the cells on the filter were counted under an optical microscope (magnification, 200×).

Metastasis assays
The pulmonary metastasis of tumors was studied using an intravenous injection model. Nude mice were injected with 2×10 6 cells in 0.2-mL Hank's solution via the lateral tail vein. After 70 days, mice were euthanized and the lungs were immediately removed and taken photographs for counting of pulmonary metastatic nodules in each of the five lobes. The metastatic tissues were analyzed with H&E staining.

Chromatin immunoprecipitation assay
Chromatin immunoprecipitation (ChIP) analysis was performed using EZ CHIP kit (Millipore, Billerica, MA, USA). HepG2 cells were grown on 15-cm plates until they reached 85% confluence. Then the cells were fixed and collected for ChIP assay. DNA-protein complexes were precipitated using specific antibody of CREB (Millipore, Billerica, MA, USA). DNA fragments were decross-linked and purified from complexes. Immunoprecipitated and input DNA were used as templates for real-time ChIP-PCR using Sybrgreen Mastermix (TransGen Biotech, Beijing, China). The enrichment of specific DNA sequences was calculated by using the comparative CT method [28]. PCR