Expression Of MACC1/HGF/c-Met In Hepatocellular Carcinoma And Clinical Study

[BACKGROUND]Hepatocellular carcinoma (HCC) ranks the fifth in malignancies, and 60 million patients died worldwide every year. Incidence of HCC in China accounts for 55% of that in the world. HCC is the second killer in tumor-related disease. It is serious threat to human health. HCC has early occult symptoms and a low rate of diagnosis at early stage. The incidence of metastasis and recurrence is extremely high after surgical resection (rate of recurrence is 60%-70% 5 years after resection). Rapid progression and poor prognosis seriously restrict the extension of survival. Effective and predictive factors of early recurrence and metastasis, as well as positive and effective interventions for extending survival and improving the prognosis are critical. Many researches are involved in the field in recent years. However, until now no specific and high-sensitive indicators were available in clinic for recurrence and metastasis of HCC can be used.Recurrence and metastasis is a multi-factor, multi-step complex process with many signal transduction pathways involved, HGF (hepatocyte growth factor) / c-Met (receptor tyrosine kinases) signaling pathway attracts more attention. c-Met is a key factor in this pathway. It has been demonstrated that c-Met affects invasion, metastasis and recurrence of HCC by upregulating expression of vascular endothelial growth factor (VEGF) and urokinase type plasminogen activator (uPA). Therefore, this pathway became a hotspot in the study of HCC.Stein identified the metastasis-associated in colon cancer 1 (MACC1) gene by a genome-wide search in human colon cancer tissues in 2009,and found that expression of MACC1 was closely related to colon cancer metastasis. Clinical study results suggested that MACC1 might be an important indicator to predicte metastasis and recurrence of colon cancer. In vitro, SW480 colon cancer cells was cultured, MACC1 could induce tumor cell migration, invasion and proliferation. In the allogeneic transplantation model, MACC1 could induce metastasis in lung and liver. Further studies revealed that the mechanisms of MACC1 inducing tumor cell migration, invasion and proliferation were closely related with HGF / c-Met pathway. First of all, MACC1 upregulated the expression of c-Met and enhance HGF / c-Met signal transduction. HGF changed the subcellular localization of MACC1. The fluorescence showed MACC1 mainly appeared in the cytoplasm of SW480 colon cancer cells, but after adding HGF, MACC1 mainly appeared in the nucleus. Clinical histological study showed that MACC1 only appeared in the cytoplasm of tumor cells in colon cancer without metastasis, once MACC1 appeared in the nucleus of tumor cell, distant metastasis would emerge in following-up study. The study suggested MACC1 and HGF / c-Met formed a positive feedback loop. HGF promoted MACC1 migrating from cytoplasm to nucleus and binded c-Met promoter to regulate transcription of c-Met , c-Met could enhance the role of HGF. MACC1 could promote cell movement, invasion and metastasis through the above mechanism.MACC1 expressed in human various tumors in addition to colon cancer, e.g, pancreatic cancer, ovarian cancer, breast cancer, and head-neck cancer. Expression of MACC1 was significantly higher in malignant tissues (colon primary and metastatic lesions) than in normal tissues (colon, liver, adenomas). Studys in HCC showed that MACC1 expressed in normal liver tissues and hepatocellular carcinoma tissues. Expression of MACC1 in HCC was higher than in normal liver tissue. However, there are no studies and reports about feature of MACC1 expression in HCC and mechanisms between MACC1 and HGF/c-Met signaling pathway, as well as recurrence, metastasis in HCC.In summary, to explore the relationship and interaction between MACC1 and HGF/ c-Met signaling pathway about recurrence and metastasis in HCC, we will observe expression of MACC1, HGF, c-Met in tumor tissues and non-tumor liver tissues. To analyze the relationship between MACC1 and clinical features including staging,metastasis,recurrence ect,and to evaluate predictive value of MACC1 in metastasis,recurrence and prognose of HCC.[OBJECTIVE]1. To observe MACC1, HGF, c-Met mRNA expression features in hepatocellular carcinoma.2. To analyze the relationship between expression of MACC1 and clinical characteristics including staging, recurrence, metastasis etc, and evaluate its predictive value of MACC1 in metastasis, recurrence and prognose of HCC.3. To analyze the relationship between MACC1 and HGF/c-Met in HCC.[METHODS]Patients: Fifty-five patiens who were hospitalized in 302 hospital from April 2006 to December 2009 were admitted into our study, including 50 cases with HCC patients and 5 cases with non-HCC patients. HCC patients: male 44 cases, female 6 cases, age> 50 years of old, 33 cases, age≤50 years of old, 17 cases, mean age 50.3±11.7 years. Twenty-one were with tumor thrombus, 29 cases without tumor thrombus, 27 cases with single tumor nodules, and 23 cases with multiple nodules. Twenty-one were with extrahepatic metastases, and 29 cases without metastasis. According to the stage standard of BCLC: 15 cases of early stage, 14 cases of mediate stage, and 21 cases of advanced stage. Patients were confirmed hepatocellular carcinoma by pathologic examination. Fifteen cases were of good differentiated, 22 cases of moderate differentiated, 13 cases of poorly differentiated. Twenty-eight cases had AFP≤400ng/ml, 22 cases had AFP> 400ng/ml. Forteen patients were with HBeAg-positive, 36 cases with HBeAg-negative. Tirty-one cases were with HBV DNA positive, 19 cases with HBV DNA negative. Thirty-six cases were of Child-Pugh A class, 14 cases of B class.Fresh liver tissues were obtained from patients. Some tissues were stored at -80℃refrigerator, and some were stored as formalin-fixed, paraffin-embedded sections. The total RNA was extracted from frozen tissues, reversed transcripted into cDNA, and detected for MACC1, HGF, and c-Met mRNA in different tissues of the liver via real-time PCR method. Tirthy-six HCC patient (comprised of 8 cases at early stage, 6 cases at mediate stage, and 13 cases at advanced stage), and 7 patients with liver cirrhosis. The expression of HGF, and c-Met were detected using immunohistochemistry (IHC).[RESULTS]1. Real-time PCR results of 11 cases showed that MACC1 mRNA expression was higher in adjacent tissues than in cancer tissues(adjacent tissues: -2.98±-3.07 vs cancer tissues: -3.28±-3.4,[Log2-ΔCt]P=0.041), HGF mRNA in adjacent tissues was higher than cancer tissues (adjacent tissues: -1.62±-2.03 vs cancer tissues: -1.93±-2.61,[Log2-ΔCt]P=0.01), while c-Met mRNA expression in cancer tissues is higher than in cancer tissues(adjacent tissues: -1.82±-1.88 vs cancer tissues: -1.63±-1.83,[Log2-ΔCt]P=0.155).2. The relationship between MACC1 mRNA expression and clinical featureswas as follows. (1) Patients aged≤50 years occupied 52% in the high expression group and 16% in the low expression group. (2) Sixty-four percent patients with tumor thrombus in high expression group, and 20% in the low expression group. (3) MACC1 mRNA expression in patients with metastasis was higher than those without metastasis (P = 0.035). (4) patients with metastasis expression group occupied 60% in the high and 24% in the low expression group. (5) MACC1 mRNA expression in patients at advanced stage is higher than at early and middle stage (P = 0.022), 60% at advanced stage in high expression group, 24% in the low expression group. (5) The high expression group had 12% with well differentiated tumor and 88% with poorly differentiated tumor. By contrast, the low expression group had 48% with well-differentiated tumor and 52% with poorly differentiated tumor. Above-mentioned clinical features in the high expression and low expression had statistical difference. MACC1 mRNA expression was independent of gender, tumor size, number of nodules, AFP, Child-pugh.3. Twenty nine patients at early and middle stage of HCC in the low expression group had longer tumor-free survival than those in the high expression group. Seventy percent(7/10) of patients in the high expression group recurred, and 60% (6/10) had metastasis. Their median tumor-free survival time was 12 months. By contrast, only one case in the low expression group recurred 18 months after section. Patiens in MACC1 mRNA and c-Met mRNA both low expression group had longer tumor-free survival time than those with high expression of both markers. Sixty percent(3/5) of patients with high expression of both markers recurred; and tumor-free median survival was 7 months for them.4. Immunohistochemistry showed that c-Met protein expression in hepatocyte membrane occupied 72% (26/36). There was no c-Met expression in hepatocyte membrance of adjacent cancer tissue; HGF expression in cancer tissues had a positive rate of 78% (28/36), HGF expression in adjacent tissue was 39% (14/36). The difference had statistical significant. HGF and c-Met expression together accounted for 28% (10/36), in which patients with poorly differentiated (7/10) tumor accounted for 70%, with multiple tumor nodules 80% (8/10).5. MACC1 mRNA expression and c-Met protein expression had 53% (19/36) consistency. MACC1 mRNA expression and c-Met mRNA expression had 46% (23/50) consistency. MACC1 mRNA expression and HGF mRNA expression had 66% (33/50) consistency. Their expression in HCC was all higher than that in normal liver tissues.[CONCLUSIONS]1. Increased expression of MACC1 mRNA in HCC may indicate rapid disease progression and poor outcome.2.Increased expression of MACC1 mRNA in HCC may be a predictive factor for recurrence and metastasis of HCC.3. MACC1 has potential correlation with HGF/c-Met signaling pathway in HCC.4. HCC patients with expression of both HGF and c-Met proteins by IHC has relative poorer prognosis.