Recurrence Of Liver Cancer Metastasis Related Factors Mina And Transcription Factor Gata Family Of Basic And Clinical Research

Primary liver cancer is the fifth most prevalent malignancy and the third most common cause of cancer-related deaths worldwide. China accounts for about53%of new cases reported globally each year,80%of which are hepatocellular carcinoma (HCC) as the second most common cause of cancer-related deaths. Despite the great advancement during the last decades, overall survival of the whole population is still as low as about5%, mainly due to high recurrence rate after curative resection. Thus, it is of great importance to seek molecular mechanisms responsible for HCC recurrence/metastasis for the improvement of clinical outcome.Mina (Myc Induced Nuclear Antigen, Mina53) encodes a protein with a molecular mass of53kDa, which is localized in the nucleus and partially concentrated in the nucleolus. The first study in human promyelocytic leukemia HL60cells revealed that Mina functioned as direct downstream target of c-myc gene and was involved in cellular proliferation. Following research correlated over-expression of Mina in tumor tissues with poor prognosis of several human cancers including lung, esophagus and colon cancer, probably associated with enhanced activity of cellular proliferation as determined by Ki-67or PCNA immunostaining. However, as myc induced downtream target, Mina expression in tumor tissues was not always in line with that of c-myc. In some early-stage lung cancers, over-expression of Mina in tumor tissues was associated with favorable prognosis. These evidences couldn’t be explained by its identity as c-myc co-expressing gene and widely-reported proliferation promoting function. As predicted by chemical structure, the128-271amino acids of Mina gene represent a JmjC domain, making it as potential candidate of histone methylation modulating enzyme. Indeed, Mina was reported to remove tri-methylation of histone H3lysine9(H3K9me3) and modulate ribosomal RNA synthesis in non-small cell lung cancer. Besides, Mina was also expressed in stroma cells including alveolar macrophages from coal miners and tumor-infiltrating lymphocytes in HCC tissues. Recently, Mina was reported to suppress expression of IL-4gene and tip the immune imbalance from Th2to Thl response, which has been correlated with favorable outcome of several human cancers. Evidences mentioned above indicated the diversity of biological function of Mina and the possibility that Mina could be involved in other processes rather than cellular proliferation through c-myc independent mechanisms. It has been reported by Japanese colleagues analyzing53surgical resected HCC specimens through immunohistochemistry that Mina was commonly over-expressed in HCC tissues, which significantly correlated with poor cellular differentiation and enlarged tumor size (>2cm). Nevertheless, this report provided no information about prognostic importance of Mina expression for hepatocellular carcinoma, neither was the impact of Mina over-expression on HCC invasiveness demonstrated by in vitro and in vivo experiments. Therefore, correlation about Mina expression with HCC progression is still largely unknown.The GATA family of transcription factors, consisting of conserved proteins that contain one or two C2-C2-type zinc-finger motifs that recognize the consensus DNA sequence A/T-GATA-A/G, play crucial roles in organ development and lineage specification. There are six members of mammalian GATA families, which have distinct and restricted tissue expression patterns. GATA1and GATA2are mainly thought as hematopoietic factors and intensely studied in hematopoietic malignancies. GATA3has been widely accepted as a classical modulator of T helper type2(Th2) immune response, which is reported to promote progression of breast and pancreatic cancers. Meanwhile, GATA3has been demonstrated as a tumor suppressor gene of breast tumor. GATA4, GATA5and GATA6are mainly thought as endodermal factors and proven to be differentially expressed in normal and malignant tissues of endodermal origin. Loss of function of GATA4-6by promoter hypermethylation or nucleocytoplasmic mislocalization is a common event in carcinoma of lung, digestive tract, pancreas and ovarian, which causes loss of expression of epithelial-specific markers (disabled-2, collagen Ⅳ and laminin) leading to cellular dedifferentiation and down-regulates potential targets of tumor suppressor genes (the trefoil factors, inhibin and disabled-2). Targeting promoter methylation or nuclear trafficking of GATA transcription factors therefore exhibits potential antitumor effect. Liver belongs to the organ systems of endoderm origin. In vivo foot-printing study of mouse embryonic endoderm cells has demonstrated occupancy of DNA-binding site for GATA factors on a liver-specific transcriptional enhancer of the serum albumin gene. This phenomenon persists during hepatic development and is necessary for the activity of albumin gene enhancer. GATA transcription factors have been reported to be expressed by human hepatoma cell lines HepRG/HepG2/Hep3B and associated with EGF-mediated induction of nucleotide excision repair activity and ERCC1expression, which is an important mechanism related to platinum resistance in many cancer types. Nevertheless, limited information has been known about the expression pattern and prognostic influence of GATA transcription factors in human hepatocellular carcinoma.In the present study, we analyzed the expression of Mina and GATA transcription factors in HCC cell lines. We also evaluated their expression pattern in paired tumor and peritumor tissues by immunohistochemistry of tissue microarrays and correlated them with important clinicopathologic factors as well as clinical outcome. By employing lentivirus transfection method, subcutaneous tumor implantation model and in situ tumor implantation model, we also evaluated the impact of Mina expression on the activity of cellular proliferation, migration and invasion in vitro as well as the capability of growth and metastasis in vivo of HepG2and SMMC-7721cell lines. We hope to find potential biomarkers predicting recurrence and death of HCC patients receiving curative resection.PART I. Decreased intratumoral Mina expression correlated with poor prognosis of HCCPURPOSE:This part aimed to evaluate the expression pattern of Mina protein in HCC cell lines with different metastatic potential as well as in paired tumor and peritumor tissues, correlate it with clinicopathologic features and clinical outcome of HCC.METHODS:We first analyzed the expression level and subcellular localization of Mina protein in HCC cell lines with different metastatic potential by Western blot and immunocytochemistry. We also evaluated the expression pattern of Mina protein in paired tumor and peritumor tissues and correlated it with clinicopathologic features and clinical outcome of HCC.RESULTS:Western blot demonstrated decreased expression of Mina protein and mislocalization from nuclear to cytoplasm as metastatic potential of HCC cell lines elevated, which was confirmed by immunocytochemistry. Immunohistochemistry of tissue microarrays indicated decreased positivity rate of Mina staining in tumor compared with peritumor tissues (training cohort:171/230,74.3%vs.229/230,99.6%; validation cohort:61/86,70.9%vs.85/86,98.8%). In tumor tissues, Mina was diffusely expressed in cytoplasm of cancer cells; while in peritumor and normal liver tissues, Mina was concomitantly expressed in both cytoplasm and nuclear of hepatocytes. In the training cohort, decreased intratumoral Mina expression (Staining Index≦1) significantly correlated with elevated serum AFP level (p=0.004), tumor size larger than5cm (p=0.002), absence of tumor capsule (p=0.001), poor cellular differentiation (p=0.001), presence of tumor thrombi (p=0.001), advanced TNM (p=0.001) or BCLC stage (p=0.001). In the validation cohort, decreased intratumoral Mina expression significantly correlated with age of less than52years (p=0.005), poor cellular differentiation (p=0.007), presence of tumor thrombi (p=0.016) and advanced TNM stage (p=0.040). In contrast, expression of Mina protein in peritumor tissues had no significant correlation with any clinicopathologic factors either in training cohort or in validation cohort. Kaplan-Meier analyses of training cohort showed that decreased intratumoral Mina expression was significantly associated with shorter recurrence-free survival time (median:16.0vs.60.8months, p=2.0E-05) and overall survival time (median:17.0vs.90.9months, p=7.1E-08), which was demonstrated by multivariate analyses as independent predictor of recurrence (HR=0.57,95%CI=0.39～0.84, p=0.004) and death (HR=0.60,95%CI=0.41～0.87, p=0.008) for HCC patients receiving resection. In contrast, peritumoral Mina expression had no significant association with either tumor recurrence or patient death in training cohort. Similarly, Kaplan-Meier analyses of validation cohort indicated that decreased intratumoral Mina expression significantly correlated with shorter recurrence-free survival time (p=0.002) and overall survival time (p=7.7E-05), which was further demonstrated by multivariate analyses as independent prognostic factor of recurrence (HR=0.46,95%CI=0.24～0.88, p=0.019) and death (HR=0.35,95%CI=0.19～0.64, p-0.001).CONCLUSIONS:Results of this part demonstrated decreased expression of Mina protein and mislocalization from nuclear to cytoplasm as metastatic potential of HCC cell lines elevated. Similarly, Mina exhibited aberrant mislocalization and decreased expression in HCC tumor tissues compared with peritumor, which significantly correlated with increased metastatic potential (poor cellular differentiation, without tumor capsule, presence of tumor thrombi, et al) and independently predicted elevated risks of recurrence or death for HCC patients.PART II. Mina suppressed proliferation, migration and invasion of HepG2and SMMC-7721cell lines in vitroPURPOSE:This part aimed to evaluate the impact of Mina expression on the activity of proliferation, migration and invasion of HepG2and SMMC-7721cells in vitro.METHODS:We employed lentivirus transfection system to specifically up-regulate or down-regulate Mina expression. We used CCK-8kit, scratch assay, transwell migration assay and transwell matrigel invasion assay to analyze the impact of Mina expression on the activity of proliferation, migration and invasion of HepG2and SMMC-7721cells in vitro.RESULTS:Immunocytochemistry and Western blot results demonstrated stable up-regulation or down-regulation of Mina protein in SMMC-7721transfected with over-expression vehicle or HepG2transfected with vshRNA vehicle. CCK-8kit assay demonstrated enhanced proliferation activity of HepG2cell with down-regulation of Mina protein or declined proliferation activity of SMMC-7721with up-regulation of Mina protein at different time points. Scratch assay showed that when expression of Mina protein was down-regulated in HepG2cell, the migrated distance significantly increased at both24hours (81.2±22.7vs.139.6±22.6μm, p=0.011) and48hours time points (292.0±5.6vs.351.3±38.4μm, p=0.022). By contrast, up-regulation of Mina protein in SMMC-7721cell significantly shortened the migrated distance at both24hours (280.4±27.9vs.210.1±22.0μm, p=0.008) and48hours time points (359.0±50.4vs.239.3±43.4μm, p=0.011). Transwell migration assay confirmed this tendency:down-regulation of Mina expression in HepG2cell caused significantly more migrated cell counts (48hours,262±48vs.459±16, p=0.002), while up-regulation of Mina expression in SMMC-7721cell led to significantly less migrated cell counts (48hours,485±33vs.215±12, p<0.0001). Transwell matrigel invasion assay demonstrated increased invaded cell counts (48hours,431±30vs.590±36, p=0.007) in HepG2cell with Mina down-regulation and decreased invaded cell counts (48hours,355±27vs.181±32, p=0.002) in SMMC-7721cell with Mina up-regulation.CONCLUSIONS:Results of this part demonstrated potent suppressive effect of Mina expression for the activity of proliferation, migration and invasion of HepG2and SMMC-7721cells in vitro.PART Ⅲ. Mina suppressed intrahepatic metastasis of SMMC-7721tumor of in situ implantation modelPURPOSE:This part aimed to analyze the effect of Mina expression for the growth of HepG2cell derived subcutaneous tumor or the growth and metastasis of SMMC-7721derived in situ tumor in vivo.METHODS:Using lentivirus transfection system, subcutaneous tumor model and in situ tumor implantation model of nude mice, we evaluated the impact of Mina expression on the growth and/or metastasis of HepG2derived subcutaneous tumor and SMMC-7721derived in situ tumor in vivo.RESULTS:In HepG2derived subcutaneous tumor model:compared with control group (n=5), RNAi group with Mina down-regulation (n=5) had increased tumor weight (1.9±0.65vs.2.4±0.51g, p=0.267) or volume (2.8±1.28vs.3.4±1.78cm3, p=0.559), which didn’t reach statistical difference. Similarly, in SMMC-7721derived in situ implantation model, there was no significant difference for tumor weight between control group and over-expression group (0.8±0.44vs.0.6±0.46g, p=0.414). However, up-regulation of Mina protein in SMMC-7721cell significantly suppressed diffuse intrahepatic metastasis of in situ implantation tumor (over-expression group vs. control group:2/9vs.9/9, p=0.002).CONCLUSIONS:Results of this part demonstrated potent suppressive effect of Mina expression for the intrahepatic metastasis of SMMC-7721derived in situ implantation tumor. However, Mina expression had no significant effect for the growth of either HepG2derived subcutaneous tumor or SMMC-7721derived in situ tumor.PART Ⅳ. Expression Pattern and Prognostic Importance of GATA Binding Proteins for Hepatocellular CarcinomaPURPOSE:GATA family of transcription factors are critical for organ development and associated with progression of various cancer types. In this part, we aimed to study the expression pattern and prognostic value of GATA transcription factors for hepatocellular carcinoma (HCC).METHODS:We evaluated the expression of GATA1, GATA2, GATA4, GATA5and GATA6in hepatic/HCC cell lines and tissues by RT-qPCR and western blot, and correlated them with clinicopathologic factors and prognosis of HCC by immunohistochemistry of tissue microarrays containing240patients undergoing resection.RESULTS:Expression of GATA1was completely lost in hepatic/HCC cell lines and tissues. RT-qPCR and western blot demonstrated decreased expression of GATA2in HCC cell lines compared with normal hepatocytes as well as in HCC tissues with recurrence compared with those without recurrence. Immunohistochemistry showed decreased expression of GATA2(p=1.27E-25) and increased expression of GATA4(p=6.12E-08), GATA5(p=2.39E-44) and GATA6(p=6.99E-19) in tumor tissues compared with peritumor. Decreased expression of intratumoral GATA2protein was significantly associated with female patients (p=0.008), older age (p=0.028), elevated pre-operative serum AFP level (p=2.7E-05),larger tumor size (>5cm, p=0.049), absence of tumor capsule (p=0.002), poor cellular differentiation (p=0.005), presence of tumor thrombi (p=0.005) and advanced TNM stage (p=0.001). Decreased expression of intratumoral GATA4protein was correlated with normal serum AFP level (p=0.014) and well tumor differentiation (p=0.047). Besides, peritumoral GATA2was more abundantly expressed in male patients compared with females (p=0.003). Kaplan-Meier analysis showed that decreased expression of intratumoral GATA2protein was significantly associated with higher cumulative recurrence rate and lower overall survival rate (low vs. high,5-y:69%vs.47%, p=1.6E-04for TTR;36%vs.57%, p=1.7E-04for OS), which was further confirmed as independent predictor of tumor recurrence (H.R.=0.63,95%CI=0.43-0.90, p=0.012) and patient death (H.R.=0.67,95%CI=0.47-0.95, p=0.026) in multivariate analysis. By contrast, expression of peritumoral GATA2(p=0.180and0.104) and intratumoral/peritumoral GATA4(tumor:p=0.089and0.183; peritumor:p=0.114and0.381), GATA5(tumor:p=0.221and0.281; peritumor:p=0.088and0.174) and GATA6(tumor:p=0.273and0.228; peritumor:p=0.173and0.953) showed no significant influence for TTR and OS.CONCLUSIONS:Results of this part demonstrated that ectopic expression of hematopoietic factor GATA2was significantly associated with prognosis of HCC following resection.CONCLUSIONS1. As the metastatic potential of HCC cell lines increased, Mina showed decreased expression and aberrant mislocalization from nuclear to cytoplasm. Mina exhibited aberrant mislocalization and decreased expression in HCC tissues compared with peritumor or normal liver tissues, which significantly correlated with increased metastatic potential (poor cellular differentiation, without tumor capsule, presence of tumor thrombi, et al) and independently predicted elevated risks of recurrence or death for HCC patients.2. Mina potently inhibited the activity of proliferation, migration and invasion of HepG2and SMMC-7721cells in vitro. Mina also suppressed intrahepatic metastasis of SMMC-7721derived in situ implantation tumor. However, it had no significant effect for the growth of either HepG2derived subcutaneous tumor or SMMC-7721derived in situ tumor.3. In HCC cell lines or tissues, hematopoietic factor GATA1was completely lost of expression while GATA2exhibited abundant expression. Expression of endoderm factors GATA4and GATA5were commonly lost while GATA6was partially maintained. Decreased expression of intratumoral GATA2significantly correlated with several clinicopathologic factors indicative of enhanced invasiveness and independently predicted worse outcome.NOVELTY1. For the first time, we analyzed the expression pattern and prognostic value of Mina and GATA transcription factors in paired tumor and peritumor tissues of HCC by high-throughout tissue microarray technique.2. For the first time, we systematically evaluated the impact of Mina expression on the activity of HCC proliferation, migration, invasion in vitro and metastasis in vivo.POTENTIAL APPLICATION1. Mina and GATA2could be potential candidates of new biomarkers predicting recurrence or metastasis of HCC following resection.2. Research about molecular mechanisms associated with suppressive effect of Mina expression for HCC invasion and metastasis would help unveil its true identity.