P62 / Sqstm1 In Hepatocellular Carcinoma Of Oxygen, Nutrition, Lack Of The Role And Mechanism Of Micro Environment

Hepatocellular carcinoma (HCC) is the sixth most common and aggressive human malignancies worldwide, the third most common cause of cancer-related mortality. In China, HCC is the second major cause of cancer death and incidence, the mortality is26.3/100,000and accounting for more than50%of the liver cancer death worldwide. Although recent progress in early diagnosis and curative transplantation or resection due to surveillance programs had significantly improved the survival of patients with HCC, only30%～40%of patients with HCC are eligible for curative treatments. In well-selected patients, resection and liver transplantation provide5-year survival rates of70%. Unfortunately, there are still60%～70%of patients who accepted curative treatment experienced recurrence and metastasis within5years, the5-year overall survival of patients with HCC is still about30%. The high frequency of intrahepatic recurrence and extrahepatic metastasis are still the major obstacles for long-term survival. Therefore, it is urge to explore the mechanism of metastasis and recurrence of HCC, and find effective suppressor pathway to improve the survival of patients with HCC.Macroautophagy (hereafter referred to as autophagy), which is a lysosomal catabolic process responsible for the turnover and elimination of damaged organelles and macromolecules. This process is often used by cells to maintain cellular homeostasis and facilitate adaptation to adverse environments (e.g., hypoxia, nutrient deprivation, or growth factors deprivation). Recent discoveries have pointed to deregulation of autophagy as a novel feature that is central to the pathogenesis of human malignancy. Autophagy serves as a barrier to limit tumor initiation. In contrast, at the advanced stages, it appears that adaptive changes occur that result in positive roles for autophagy in malignant progression and in subsequent tumor maintenance. Since autophagy closely related to cell metabolism, protein and organelle turnover, and cell survival, the role of autophagy in cancer is likely dynamic and with a high degree of complexity.Sequestosome1(SQSTM1/p62, herein designated as p62) is a multifunctional ubiquitin-binding scaffolding protein that is gaining attention as it is intimately involved in the regulation of autophagy, trafficking of proteins to the proteasome, and activation of NF-κB. Overexpression of p62as a result of autophagy inhibition was shown to be important in the promotion of tumorigenesis through a variety of mechanisms, including deregulation of NF-kB signaling, accumulation of ROS, and increased DNA damage.In our study, we analyzed the p62expression in different malignant phenotype of HCC cell lines, compare with the expression of Atg5and beclin1. To investigate p62dynamic changes under the hypoxia or starvation environments, and the impact of p62on cell growth, apoptosis. Initially discuss the role of p62in HCC microenvironment. We used TMA to explore the p62expression within large-scale HCC samples and its relationship with clinical outcome. In the future, treatment targeted to clearance of p62and block the related functions of p62could be attractive therapeutic strategies against cancer prevention and treatment. Part One SQSTM1/p62expression in HCC cell lines and clinical samples of HCC patientsThe aim of this part is to investigate the expression of p62, autophgic genes Atg5and beclin1expression in HCC cell lines and clinical samples of HCC patients, and to preliminarily explore the relationship between p62and Atg5and beclin1.Firstly, we chosed a normal hepatic cell line L-02and7HCC cell lines Hep3B, HepG2, SMMC-7721, MHCC97-L, MHCC97-H and HCCLM3using Real-time PCR and Western blot to validate the mRNA and protein expression of p62, Atg5and beclin1. Immunofluorescence analyzed the p62location in Huh7, SMMC-7721, MHCC97-H and HCCLM3. Immunohistochemistry was performed to analyses the p62expression in HCC tumor and adjacent tumor tissue. Real-time PCR was used to detect the p62, Atg5and beclin expression in HCC tumor and adjacent tumor tissue. SMMC-7721-Beclin1-shRNA and MHCC97-H-Beclin1-shRNA were established, Western blot to validate the p62expression in autophagy deficiency cell lines.As a result, mRNA and protein expression of p62were observed in almost all HCC cell lines compared with a normal hepatic cell line L-02(P<0.001). Western blot and immunofluorescence staining of p62protein expression in line with Real-time PCR results. The p62protein level is quite high in MHCC97-H and HCCLM3cell lines compared with a normal hepatic cell line L-02. The p62protein is mainly existed in cytoplasm. Atg5and beclin1mRNA is lower in MHCC97-H and HCCLM3cell lines compared with a normal hepatic cell line L-02, however, the p62protein level is quite high in MHCC97-H and HCCLM3cell lines. In HCC tissue samples, the p62protein mainly existed in the cytoplasm of tumor cells and almost no p62staining was observed in adjacent normal tissue. Atg5and beclin1mRNA is quite lower in tumor tissue compared with adjacent normal tissue (P<0.001). However, the p62mRNA is highly expressed in tumor tissue compared with adjacent normal tissue (P<0.05). Western blot validated the results in randomly selected four HCC tissue samples.Taken together, these results indicated that the higher levels of baseline autophagy the lower level of p62expression. Autophagy deficient could cause p62accumulation and overexpression. Accumulation of p62may be biomarkers for defective autophagy in HCC. Part Two The role of SQSTM1/p62under hypoxia, starvation microenvironmentThe aim of this part is to investigate the importance of p62expression in HCC cell lines under hypoxia, starvation microenvironment.Firstly, SMMC-7721cell line was cultured under hypoxia (1%O2), the p62and LC3B protein level was detected by Western blot. Electron microscopy was used to detected autophagosome formation. SMMC-7721-GFP-LC3was cultured under hypoxia (1%O2) for12h and chloroquine (20μM) was used as an autophagy inhibitor, then GFP-LC3dots were calcutated under confocal laser microscopy. Immunofluorescence analyzed the p62and LC3protein co-location in SMMC-7721and MHCC97-H under hypoxia (1%O2) for12h. SMMC-7721-Beclin1-shRNA cell line was established, western blot to validate the p62expression in autophagy deficiency SMMC-7721cell line under hypoxia (1%O2). Secondly, in Huh7cell line, p62participates in the regulation of hypoxia-induced ERK phosphorylation, p62and the level of ERK phosphorylation were determinated by Western blot. Huh7cell line interfered with p62-siRNA caused ERK phosphorylation. Thirdly, We used EBSS to mimic nutrient deprivation, p62and two major downstream targets of mTOR (phosphorylated p70S6kinase and4E-binding protein-1) were determinated by Western blot in Huh7cell line. Huh7cell line were interfered with p62-siRNA and the role of p62in cell proliferation and apoptosis was determinate by CCK8and Hoechst33342staining, respectively. As a result, autophagy can be induced in SMMC-7721cell line under hypoxia, and degrades p62through autophagy. When hypoxia-induced autophagy occurred in SMMC-7721-GFP-LC3cell line, chloroquine (20μM), an autophagy inhibitor, GFP-LC3dots were significantly accumulated than in control group. Knock-down autophagy gene, beclin1, p62protein accumulated under normoxia and the degradation rate of p62was significantly reduced under hypoxia. In Huh7cell line, interfered the expression of p62caused inhibitation of mTOR, as manifested by the reduction of both phosphorylated p70S6kinase and4E-binding protein-1, major downstream targets of mTOR. Under nutrient-rich condition, the cell proliferation rate was reduced at24h and48h in Huh7cell line interfered with p62-siRNA. When nutrient deprivated for24h, the apoptosis rate was high in Huh7cell line interfered with p62-siRNA than that in control group (P<0.05).Taken together, these results indicated that the autophagy of HCC cell lines can be induced by hypoxia, p62degradated through autophagy. Knock-down autophagy gene, beclin1, p62protein accumulated under normoxia and the degradation rate of p62was significantly reduced under hypoxia. The protein p62participates in the regulation of hypoxia-induced ERK phosphorylation and has an essential role in the protection of cancer cells against hypoxic stress. The protein p62is necessary to mediate amino acid signaling for the activation of S6K1and4EBP1. In HCC cell lines, knock-down p62expression could inhibit cell proliferation and induce apoptosis. Part Three SQSTM1/p62in HCC tissues and corresponding clinical significanceThe aim is to explore the expression of p62in HCC tissues, and to analyse the relationship between its expression with clinical pathology, invasiveness and prognosis of HCC.Randomly selected40cases of HCC tissue, of which20cases of early recurrence,20cases recurrence-free, Real-time PCR was used to detect p62mRNA expression in two groups. The p62protein expression in HCC tissue between early recurrence group (12cases) and recurrence-free group (12cases) was validated by Western blot analyses. To further validate our proposed hypothesis, we used high-throughput TMA technology and immunochemistry to dectect p62expression in a group of311HCC patients, and then made correlation analysis between p62with other clinical-pathological characters and survival rates. We also compared p62expression in predicting the reccurence of HCC after curative resections, and evaluated the significance of p62as a new predictive marker for the prognosis of HCC.As a result, a significant increase of p62mRNA expression was observed in the early recurrence group compared with the recurrence-free group (P=0.019). The p62protein expression in HCC tissue in early recurrence group (12cases) is significant increased than that in recurrence-free group (12cases), which was validated by Western blot analyses (P=0.036). To further validate our proposed hypothesis, we used high-throughput TMA technology to assess the relationship between p62and prognosis of HCC in a group of311HCC patients who underwent curative resections. The over expression of p62were significantly associated with age, gender, AFP, tumor size, tumor number, microvascular invasion, Edmondson grade, pathologic satellites, tumor capsule, CLIP staging, TNM staging and BCLC staging (P<0.05). Patients with high expression of p62had a significantly poorer prognosis than low expression of p62patients. The1-,3-and5-year OS rates for p62high and p62low patients were75.8%,42%and28.7%versus95.8%,83.7%and71.9%, respectively. The1-,3-and5-year TTR for p62high and p62low patients were49.7%,77.1%and84.0%versus17.4%,41.7%, and54.3%, respectively. Multivariate analysis by Cox regression showed that with tumor size>5cm, multiple tumor numbers, CLIP stage (2/3), BCLC stage and p62high were independent predictors for OS. Gender, tumor size>5cm, microvascular invasion, CLIP stage (2/3) and p62high were independent predictors for TTR.Taken together, these results indicated that p62up-regulation is associated with the poor prognosis of HCC patients. p62can be used as an independent predictor for recurrence and metastasis in patients who have had surgery for HCC. Conclusions1. MHCC97-H and HCCLM3exhibite lower baseline autophagy and high expression of p62. Autophagy deficient could cause p62accumulation and overexpression.2. HCC cell lines can be induced by hypoxia, p62degradated through autophagy. Knock-down autophagy gene, beclin1, p62protein accumulated under normoxia and the degradation rate of p62was significantly reduced under hypoxia. The protein p62participates in the regulation of hypoxia-induced ERK phosphorylation and has an essential role in the protection of cancer cells against hypoxic stress.3. The protein p62is an integral part of the mTORC1complex and is necessary to mediate amino acid signaling for the activation of S6K1and4EBP1. In HCC cell lines, knock-down p62expression could inhibit cell proliferation and induce apoptosis.4. p62up-regulation is associated with the poor prognosis of HCC patients. p62can be used as an independent predictor for recurrence and metastasis in patients who have had surgery for HCC.Novelty1. Accumulation of p62may be a biomarker for defective autophagy in HCC.2. To our knowledge, this is the first report to demonstrate the prognostic value of p62in large cohorts of patients with HCC. p62up-regulation might be used as an independent predictor for recurrence and metastasis in patients who have had surgery for HCC.Potential Application1. Our research will be beneficial for further investigating the distinct role of autophagy in the HCC tumorogenesis and progression.2. The expression of p62can be easily detected in clinical HCC tumor sample by IHC and acted as a biomarker for defective autophagy in individual HCC patient.3. p62up-regulation is the novel independent predictor for recurrence and metastasis in HCC patients, which is beneficial in predicting which patients are at highest risk of recurrence, thus facilitating to surveillance programs and select high risk patient for more aggressive treatment.