HIF-1¦Á Liver Cancer Recurrence, Metastasis And Liver Cancer Inside And Outside Microenvironment

Hepatic cell carcinoma (HCC) is one of the most common cancer in China and often occurred in the male between 40 and 60 years old, charactered with fast relapse and high mortality. Liver resection gave HCC patients the chance to escaped from death and some patients might obtain a complete cure. However, the frequent recurrence especially the recurrence in residual liver after resection was the main problem and seriously reduced the HCC patients survival rate and made the outcome dismal. All things happened for its intrinsic factor and extrinsic factor. The event of intrahepatic recurrence after resection also determined by the feature of original caner and the residual liver tissue especially the peritumoral liver tissue.HCC as a human solid tumor, consist of the tumor cells and microenvironment where the tumor cells survive and proliferation. The previous study mainly focused on tumor cell itself, such as oncogene and antioncogene, differentiation and phenotype of tumor cells and so on. However, modern research revealed that the tumor microenvironment also crucial to the tumor progression. Tumor environment contained a lot of cells and molecular correlated with tumor cell, such as kinds of cytokines, growth factors, inflammation cells and factors, stromal cells, nutrition, pH and electrolytes and so on.Tumor progression or metastasis was described as "seed and soil". In HCC research area, traditional research mainly focused on the features of the primary tumor itself. However, the residual liver tissue, which used to be thought as normal tissue, was often neglected. In fact, the residual liver tissue especially the peritumor liver tissue not only serves as the most common field where a visible intrahepatic recurrence occurs after resection, but also provides the microenvironment of the chemoattracting direction and nourishment for original cancer cells dissemination and micrometastasis formation. The feature of peritumoral liver tissue microenvironment had existed even if the primary HCC was very small.In the field of tumor environment, hypoxia was confirmed to be a common character in human solid tumor including HCC. Hypoxia in solid tumor can be caused by two reasons:the increased oxygen consumption and the decreased oxygen supply. The character of tumor cells determined the high oxygen consumption rate in tumor tissue, such as the abnormal mitotic division, rapid proliferation, high metabolism rate, as well as extensive apoptosis, autophagy and necrosis which also consume a lot of oxygen. However, the most important reason for tumor hypoxia was the low supply of oxygen. Aberrant vasculogenesis, including caecum of vessels, thrombus, embolus from cancer cells and so on, make the ischemia aera in tumor tissue. The irregular arrangement of intratumor cells made the oxygen more differcultly diffuse to intracellar organelles of the target cells. Besides, the anemia or dysfunction of erythrocytes in tumor patients also reduced the oxygen supply. All these caused a hypoxia microenvironment in tumor.Tumor hypoxia was very important in the feature of tumor cell. A lot of research have found that tumor hypoxia is closely correlated to tumor cells proliferation, apoptosis, autophagy, metabolism and so on. It is the most obvious difference between tumor cells and normal cells that tumor cells survived in the hypoxia microenvironment all the time but the normal cells not. Tumor hypoxia was not only suitabled for the survival of tumor cells but also a necessary condition for keeping and developing the feature of the tumor cell itself. This may be correlated to the natural selection for tumor cells by hypoxia. Hypoxia can regulate the cell apoptosis. From the beginning of the tumor progression, only the specific malignant cell which can survive in the hypoxia condition was selected to continue to proliferation and formed the macroscopic tumor. While the others that can not be suitable for the hypoxia condition in tumor were disappeared in the process of tumor formation by apoptosis and other ways. Hypoxia not only affect the character of the tumor cell itself, but also have close correlation to the feature of intratumor pathologic reaction such as inflammation, immunity, angiogenesis and so on. "Existence in possible" tells us that hypoxia as a common "existence" in cancer may correlate to most of the feature and action of the malignant neoplasm.Although the research of tumor hypoxia had a long history, there was few good tools in this field until hypoxia induced factors (HIFs) were discovered. HIFs was a sort of transcription factors upregulated in hypoxia. They are endogenous factors regulating a lot of target genes which were different with those exogenous markers induced by hypoxia condition. In the family, HIF-1 was the one that had most importance and widely distribution with HIF-1αas the functional unit. HIF-1αexpression level is positively correlated to oxygen concentration. In the same kind of tissues, HIF-1αexpression level is higher in those cells with more severe hypoxia. And the intracellular HIF-1αprotein could reach a high level as soon as the cell was sensitive to the hypoxia.There were more than sixty HIF-1αdirect target genes were confirmed by previous research. And more possible target genes need to be confirmed. The proteins generated by HIF-1αtarget genes often play the crucial roles in cell actions and microenvironment variation that were responding to hypoxia. Intratumoral HIF-la had close correlations to the tumor progression. HIF-la could regulate the apoptosis and proliferation of tumor cells and provide the natural selection of those suitable for hypoxia to form the macroscopic tumor. HIF-1αcould regulate the key enzymes in anaerobic glycolysis which make the tumor cells survive in hypoxia microenvironment. More importantly, HIF-1αplay a important role in the metastasis or invasion of tumor. HIF-1αcould regulate the angiogenesis in the progression of tumor by upregulating kinds of related factors and receptors. HIF-1αalso increased the expression of matrix metalloproteinases (MMPs) which can degrade the matrix. Besides, HIF-1αcan regulate the receptors in the tumor cell surface related the cell adhesion and mobility or their ligands secretion, such as CXCR4, c-Met, SDF-1 and so on. HIF-1αas a transcription factor also correlated to the variation of oncogene in tumor cells such as MYC. In some research, hypoxia feature increased MYC expression which also affecting the HIF-1-depended regulating pathway in metabolism, angiogenesis and so on.In our research, we detected the expression of HIF-1αmRNA as well as protein. Combining with the complete clinical date, we analyzed the correlations between HIF-la and the clinicopahologic features, as well as the prognosis value in HCC after resection. We also detected the factors related to inflammation such as COX-2 MMP7和MMP9, those related to angiogenesis such as VEGF,PDGFRA and MYC oncogene. Then we analyzed the correlation between HIF-1αand these factors. The prognostic value of all factors in HCC after resection was assessed. On the other hand, we detected HIF-1αexpression in peritumor tissue as well as the chemokine SDF-1α. The correlation between peritumoral SDF-1αand HIF-1αwas analysed. Their prognostic value in recurrence and survival of HCC after resection was also discussed.Part One The clinical significance of intratumoral HIF-1αin survival and recurrence of HCC after resection Tumor hypoxia has been confirmed to be a crucial role in tumor invasion or metastasis in a lot of basic research. And in clinical study, HIF-la high expression was correlated to recurrence and survival in some kinds of tumor, such as gastric cancer, renal cancer, breast cancer and so on. However, few reports studied the clinical significance of both the HIF-1αmRNA and protein in HCC.In our study, we randomly chose 110 HCC patients under curative resection in Zhongshan Hospital of Fudan University between 2002 and 2005. The HIF-la mRNA in the tumor was detected by Real time RT-PCR. The cutoff value was determined by X-tile and the specimens were divided to high and low expression groups. Fisher s exact test was carried out to analyse the correlation with clinicopathologic features. Cox proportional hazards regression model was carried out to assese the prognostic value of HIF-la mRNA in the survival and recurrence of HCC after curative resection. We also detected the HIF-la protein expression in the tumor of these 110 patients by immnohistochemisty and tissue microarray. The degree of the expression was evaluated and classified by the criterion in previous researches. Then the specimens were separated into HIF-1αprotein staining positive and negative groups. The correlations between HIF-1αprotein and clinic pathologic features was analyzed in the same methods as HIF-1αmRNA, as well as the prognostic value of HIF-1αprotein for recurrence and survival in HCC patients after resection.The results showed that:(a) In multivariable analysis, intratumoral HIF-1αmRNA was an independent prognostic factor for survival (P=0.012) and recurrence (P=0.004) of HCC after resection. (b) HIF-1αmRNA was correlated to vascular invasion (P=0.033)and BCLC stage (P=0.041). Intratumoral HIF-1αmRNA high expression had more proportion of tumor cell vascular invasion and advanced BCLC stages. (c) Intratumoral HIF-1αprotein level was also an independent prognostic factor for OS (P=0.021) and DFS (P=0.007) in multivariable analysis. (d) HIF-1αprotein also correlated to vascular invasion and BCLC stages. HIF-1αpositive staining group was more likely to suffer vascular invasion and advanced BCLC stages.These results suggested that both HIF-1αmRNA and protein were independent prognostic factors for survival and recurrence of HCC after curative resection. Increased HIF-1αin tumor correlated to high recurrence rate and low survival. Intratumoral HIF-1αalso increased the probability of vascular invasion and high HIF-1αoften accompanied with advanced BCLC stages. A further investigation to HIF-1αmay bring us new and good diagnostic and therapeutic tools in HCC. Part TwoThe correlations between intratumoral HIF-la and inflammation, angiogenesis as well as MYCTumor hypoxia was an important feature and one of the crucial ingredient in the procession of malignant tumor. However, there were also other pathologic actions in the tumor tissue, such as inflammation, immunity, angiogenesis and so on. There were complicated relations between hypoxia and other pathologic actions in tumor and related factors also play an important role in tumor invasion and metastasis. The MYC oncogene was also showed complicated relation to HIF-la in some investigations. However, few research showed the correlations between hypoxia and inflammation, angiogenesis as well as MYC in the clinical view.In this part, we analyzed these problems. In part one, we randomly chose 110 HCC patients under curative resection in Zhongshan Hospital of Fudan University between 2002 and 2005. We carried out Real time RT-PCR to detected intratumral HIF-la mRNA. And in the same procedure, we detected the mRNA of other factors for this part analysis. This factors included inflammation related factors (COX-2, MMP7, MMP9), angiogenesis related factors (VEGF, PDGFRA) and oncogene MYC. Spearman's correlation test was carried out to assess the correlations between HIF-1αmRNA and these factors. The cutoff value of these factors also determined by X-tile. And all the specimens were divided into high and low expression group in each factor. Multivariable and univariable Cox proportional hazards regression model was carried out to assess the prognostic value in HCC survival and recurrence after curative resection.The results showed that:(a) Significant positive correlations were observed between HIF-1αmRNA and the putative markers of inflammation, angiogenesis and MYC:COX-2 (P＜0.001, r=0.708), MMP7 (P<0.001, r=0.593), MMP9 (P＜0.001, r=0.384), VEGF (P=0.183,r=0.128), PDGFRA (P＜0.001, r=0.493), MYC (P=0.016, r=0.230). However, there were no significant correlation between mRNA expression level of HIF-1αand VEGF (P< 0.183, r= 0.128). (b) In multivariable analysis, COX-2 (P=0.004),MMP7 (P=0.010) and PDGFRA (P=0.010) was an independent prognostic factor for OS. While COX-2 (P=0.010) and PDGFRA (P=0.038) was an independent prognostic factors for DFS. And of all the six parameters, COX-2, MMP7 and PDGFRA were the top three factors with the largest correlation coefficients to HIF-1α.These results suggested that intratumoral HIF-1αwas closed correlated to inflammation, angiogenesis and oncogene MYC. HIF-1αmay be a crucial role in the pathologic actions in tumor. The inflammation, angiogenesis related factors also advanced the tumor progress and increased the recurrence. In all the factors, HIF-1αwas an independent prognostic factor for both survival and recurrence in HCC with the smallest P value as well as the largest hazard ratio for recurrence. And other factors correlated to HIF-1αclosely also were the independent prognostic factor for OS or DFS, such as COX-2, PDGFRA and MMP7. We thought that HIF-1αwas the crucial factor affecting the prognosis of HCC, which may be in association with inflammation and angiogenesis. HIF-1αrelated factors including those functioned in inflammation, angiogenesis and so on may serve us new diagnostic markers and therapeutic targets in HCC.Part ThreeThe correlation between peritumoral HIF-1αand SDF-1αas well as their clinical significanceIntrahepatic recurrence and metastasis was the most common feature in HCC after resection. The residual liver tissue which used to be thought as normal tissue was often neglected. In fact, the peritumoral liver tissue not only serves as the field where a visible intrahepatic recurrence occurs after resection, but also affected intrahepatic recurrence and metastasis in more and more investigation. SDF-1α, as a chemokine ligand, played an important role in direct migration of HCC cells with CXCR4 positive. Some basic research also confirmed that SDF-1αwas a target gene of HIF-1αin hypoxia condition. Peritumoral live tissue also suffered hypoxia due to some reactions to tumor, such as direct presssion, inflammation, immunity and vascular invasion. In this study, we firstly confirmed the correlation between SDF-la and HIF-1αin the clinical view and firstly detected the clinical significance of peritumoral SDF-1αand HIF-1αin HCC after resection.In our study, we randomly chose 240 HCC patients under curative resection in Zhongshan Hospital of Fudan University between 2002 and 2006. The tissue microarray and immunohistochemical technology were carried out to detected the HIF-1αand SDF-1αprotein. The density of SDF-1αand HIF-1αpositive staining was assessed by mean optical density. Cut-point value of experiment results was determined by the median and the specimens were divided into HIF-1αor SDF-1αprotein staining positive and negative groups. The correlations between SDF-la and HIF-1αwas analyzed by Spearman's correlation test and Fisher s exact test. Cox Proportional Hazards Regression model was carried out in univariate and multivariate survival analyses.The results showed that:(a) Peritumoral SDF-1αwas an independent prognostic factor for survival and recurrence in HCC after resection (P=0.021 for OS, P=0.012 for DFS). In addition, peritumoral SDF-1αhad prognostic value for early recurrence of HCC (P=0.017) in multivariate analysis, but not for late recurrence (P=0.111 in univariate analysis). (b) Significant positive correlation was observed between HIF-1αand SDF-1αexpression in peritumoral tissue (P＜0.001, r=0.305). (c) However, peritumoral HIF-1αhad a propensity of reduced recurrence and prolonged survival with boardline significance in univariate analysis (P=0.062 for OS and P=0.064 for DFS in univariate analysis) which was different to peritumoral SDF-1α. Furthermore, the combination of negative HIF-1αand positive SDF-1αwas an independent prognostic factor for increased recurrence (P=0.022) and reduced survival (P=0.009) in multivariate analysis. While both positive had not significance just in unvariate analysis (P=0.418 for OS and P=0.394 for DFS).These results suggested that peritumoral SDF-1αcould increase the recurrence and reduce the survival, especially the early intrahepatic recurrence of HCC after resection which was mainly from the metastasis and proliferation of residual tumor cells derived from the original tumor. Peritumoral HIF-1αmight partly upregulate SDF-1α, but did not increase the recurrence. Attach importance to peritumoral SDF-1αmay develop new anti-metastatic strategies in HCC. The role of peritumoral HIF-1αshould not be neglected when we thought hypoxia was a therapeutic target for HCC.Conclusions1. Intratumroal HIF-1αmRNA and protein were correlated to the clinicopathologic features of HCC and were all independent prognostic factors in survival and recurrence of HCC after resection.2. Intratumoral HIF-1αmay influence HCC biological behaviors and affect the tumor inflammation, angiogenesis and act in concert with the oncogene MYC. 3. The inflammation and angiogenesis related factors such as COX-2 and PDGFRA, which were closely correlated to HIF-1α, also has prognostic value in survival and recurrence of HCC after resection.4. Peritumoral SDF-1αincreased the recurrence, especially the early recurrence of HCC after resection.5. Peritumoral HIF-1αmight upregulate SDF-1αexpression, but did not increase the recurrence of HCC after resection.Novelty1. For the first time, we detected clinical significance of the intratumoral HIF-1αin both mRNA and protein levels. It provided the basis for HIF-1αrelated diagnostic and therapeutic technology in the future.2. For the first time, we detected the correlations between intratumoral HIF-1αand the tumor pathologic actions such as inflammation and angiogenesis. It suggested that hypoxia was crucial in tumor microenvironment.3. For the first time, we reported the peritumoral SDF-1αcorrelated to the intrahepatic recurrence of HCC after resection. It confirmed again that peritumoral chemokines played an important role in the intrahepatic recurrence and metastasis of HCC.4. For the first time, we detected the clinical significance of peritumoral HIF-1αin survival and recurrence of HCC after resection. In provide a new viewpoint in tumor hypoxia related therapy.5. For the first time, we detected the correlations between peritumoral HIF-1αand SDF-1αin the clinical view by large quantity of patients, which provided the proof for the further related investigations.Potential application1. Intratumoral HIF-1αplay the crucial role in the pathologic actions in HCC such as inflammation and angiogenesis, which was valuable for the further related research.2. The clinical significance of intratumoral HIF-1αsuggested that related markers will be valuable in the diagnostic and therapeutic practice for HCC. 3. The clinical significance of peritumoral SDF-la suggested that it could be provided as the good diagnostic marker and therapeutic target for intrahepatic recurrence of HCC after resection.4. The complex affections of peritumoral HIF-la suggested that peritumor tissue should not be neglected when the tumor hypoxia was thought as a therapeutic target.