Validation And Functional Analysis Of Early Diagnosis Marker MDK And Metastasis Associated Molecular GOLPH2for Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most common and aggressive malignancies worldwide, ranking the sixth most important cancer in terms of numbers of cases and the third in terms of cancer mortality. It has become the second cancer killer in China since1990s. Although tremendous efforts have been applied to improved HCC prognosis, unfortunately, the overall survival of patients with HCC is still dismal.There are two main reasons that contribute to the poor prognosis of HCC patients. One is attributed to the inability to diagnose HCC patients at early stage. Owing to diagnostic and therapeutic progress during the past decades, the HCC outcome is improved in a proportion of patients who were diagnosed at an early stage and received curative treatments. However, only about10-20%of patients are currently eligible for potentially curative therapies at the time of diagnosis and the prognosis of advanced HCCs remains very dismal. Alpha-fetoprotein (AFP) has been widely used as a serological tumor marker for HCC. However, there is debate regarding the roles of AFP in the early diagnosis and, particularly, surveillance of HCC.Meanwhile, though some of the patients have long term survival through early diagnosis and comprehensive therapy, recurrence and metastasis of tumor has been another major obstacle to improve HCC patients survival which is also another reason contributing to the poor prognosis of HCC patients. Therefore it is urgently needed to investgate the molecular basis underlying HCC metastasis and explore potential effective therapeutic targets. Only in this way, better prognosis and longer survival might be further achieved.In our previous studies, by comparing the gene expression profiles between40HCCs with and without intrahepatic metastases using cDNA micorarray, we proposed for the first time a new hypothesis that genes favoring HCC metastasis and progression were likely initiated from the primary tumors (Ye QH, et al. Nat Med2003).we also found that the established153genes’signature including osteopontin (OPN) could successfully predict the metastasis of HCC patients after curative resection as well as a potential therapeutic target.Based on the previous study, recently, the laser capture microdissection (LCM), gene expression array were introduced to explore the genomic aberration of paired HCCs with and without extra-hepatic metastasis. The new hypothesis that genes favoring HCC metastasis are initiated in primary tumors got further validated and some important candidate genes associated with HCC extrahepatic metastasis were disclosed. Of them, type II Golgimembrane protein (GOLPH2) was found to be one of the leading gene favoring HCC extra-hepatic metastasis.Moreover, genome-wide based bioinformatic analysis also indicate that MDK was one of the five important novel potential biomarkers (GPC3、PEG10、 MDK、SERPINI1和QP-C) for early detection of HCCs in our previous study.(Ja HL, et al. Clin Cancer Res2007).However, it is also important for us to validate the identified early diagnostic and metastasis associated gene signatures and further transmit to clinical use as well as explore the key molecular mechanisms for HCC carcinogenesis and metastasis.Bear this in mind, in the following study, we first evaluate the serum midkine (MDK) levels for the early detection and diagnosis of hepatocellular carcinoma (HCC), particularly for those with negative alpha-fetoprotein (AFP) and early-stage disease and finally elucidate the relationship and molecular basis of of GOLPH2with the invasion and metastasis phenotype of HCC.PART ONEMidkine Is a Novel Serum Marker Superior to Alpha-fetoprotein for the Early Detection and Diagnosis of Hepatitis B Virus-related Hepatocellular CarcinomaPurpose:The aim of this study was to evaluate the serum midkine (MDK) levels for the early detection and diagnosis of hepatocellular carcinoma (HCC), particularly for those with negative alpha-fetoprotein (AFP) and early-stage disease.Patients and Methods:MDK expression was assessed by immunohistochemistry in tissue microarrays containing paired tumor and cirrhotic liver tissues and cancer-free cirrhotic liver tissues from105patients with HCC or liver cirrhosis. Serum MDK levels were detected by enzyme-linked immunosorbent assay in501samples from445patients with HCC and different controls including HBV-related liver cirrhosis, benign liver tumor and healthy individuals from two medical centers in China. The sensitivity and specificity of serum MDK in diagnosing HCC according to AFP level, tumor size, and Barcelona Clinic Liver Cancer (BCLC) stage were analyzed.Results:MDK levels were significantly increased in HCC tissues compared with those in peri-tumor and cancer-free liver tissues (P<.0001). Serum MDK levels (1.346ng/ml; range,0.864-1.802) in HCC patients of the discovery cohort (n=84) were much higher than those of the different controls (n=193)(P=.0001). The areas under the receiver operating characteristic curves for MDK and AFP were0.87and0.69, respectively (P<.000). The sensitivity of MDK at the cutoff value of0.752ng/ml for HCC diagnosis in another large, independent cohort (n=163) was much higher than that of AFP (20ng/ml)(83.3%vs.51.2%, respectively), while their specificities were similar (81.1%vs.76.7%); even in those with negative AFP (<20ng/ml), the positive rate was still as high as82.9%. Moreover, in detecting early-stage HCCs (BCLC0/A), the sensitivity of MDK (82.3%) was also much higher than that of AFP (45.2%); even in detecting patients with very early stage (BCLC0), MDK showed a obviously higher sensitivity of76.2%comparing with38.1%of AFP. The combination of MDK and AFP could further improve the detection rate to90%for HCC at a very early-stage (BCLC0). After curative resection of HCCs, serum MDK levels (1.362±0.362ng/ml) were significantly decreased (0.482±0.281ng/ml; P<.001), while an increased MDK level was found in patients with tumor recurrence after HCC resection.Conclusions:Serum MDK level is significantly elevated in most HCC patients including those at early/very early stages and may serve as a novel diagnostic marker in early detection and postoperative monitoring of HCCs.PART TWO Spatial Regulation of Receptor Tyrosine Kinase(RTK) EGFR Endocytosis and Recycling Through GOLPH2Promoting Hepatocellular Carcinoma Growth and Metastasis Purpose:laser capture micro-dissection and gene expression array based study identifies a Golgi protein, GOLPH2, as a candidate metastasis favorite gene in Hepatocellular Carcinoma. The aim of this study was to elucidate the relationship and molecular basis of of GOLPH2with the invasion and metastasis phenotype of HCC.Patients and Methods:The expression levels of GOLPH2mRNA in HCC with paried adjacent non-tumorous liver tissue, normal liver tissues and different HCC cell lines with different metastatic potential was assessed by qRT-PCR. TMA(tissue micro-array)based immunohistochemical analysis was performed to detect the expression and the prognostic significance of GOLPH2in another independent HCC cohort (n=102). Lentivirus-mediated RNAi or over-expression was used to obtain a stable HCC cell line with low or high expression of GOLPH2. Both in vitro cell proliferation assay, transwell assay and wood healing assay combined with in vivo tumor growth and lung metastasis were investigated to validate the functional role of GOLPH2on HCC cell lines. To gain mechanistic insights into the biological functions of GOLPH2, we screened for GOLPH2-interacting proteins using IP-Pull Down plus mass spectroscopy (MS). Confocal microscopy and CO-IP assay were performed to validated the MS results. Finally, biotin-labeled membrane purification assay, western-blot and live cell spinning disk confocal microscopy was used to study the regulated role of GOLPH2on EGFR endocytosis and recycling.Results:RT-PCR showed that the expression of GOLPH2in HCC tissues was up-regulated compared with adjacent tissues and normal liver tissue ((P<.0001). Meanwhile, the expression of GOLPH2in HCC patients with metastasis was significantly higher than the group without metastasis(P<0.05), especially for those patients with extra-hepatic metastasis(P<0.01).Both the mRNA and protein expression levels of GOLPH2in two high-metastatic HCC cell lines were found to be significantly increased in comparison to three low-metastatic HCC cell lines. Immunohistochemistry results indicated that GOLPH2which mainly expressing in tumor tissues were significantly correlated with patients prognosis. The time to recurrence rate(TTR) and overall survival rate(OS) of patients with low GOLPH2expression was significantly higher than patients with high expression (TTR:P=0.019;OS:P=0.007). Multivariate analysis indicates that GOLPH2is a optimal independent predictor of outcome (P=0.023). Gain-and loss-of-function studies both in vitro and in vivo validated GOLPH2as a potent oncogene for HCC proliferation and metastasis. GOLPH2-interacting proteins screening assay links GOLPH2to EGFR endocytosis and recycling network. Physically, GOLPH2localizes to the trans-Golgi network(TGN) and interacts with EGFR during endocytosis and recycling. Mechanistically, GOLPH2promots cell proliferation, invasion and metastasis through regulating growth factor-induced EGFR endocytosis/recycling and its down stream signaling in vitro and vivo.Conclusions:GOLPH2expression levels positively relate to the hepatocellular carcinoma metastatic potential and prognosis; inhibition of GOLPH2expression can significantly reduce the the capacity of HCC cell proliferation and invasion. Mechanically, GOLPH2promots cell proliferation, invasion and metastasis through regulating growth factor-induced EGFR endocytosis/recycling and its down stream signaling which may become a new therapeutic targets.