| Background and SignificanceAttributing to heavier air pollution and smoking, the incidence of lung cancer increased. In 2012, of the 14.1 million new cancer cases, lung cancer has become the most common (1.8 million,13%) and first leading cause of cancer deaths (1.6 million, 19.4%) worldwide. The 2014 American Cancer Society recent investigation exhibited that estimated incidence of new cases was 14%,13% in male and female separately; the estimated mortality was 26%. Non-small cell lung cancer (NSCLC) accounts for approximately 75-80% of cases. Despite recent therapeutic advances, the traditional surgical resection, chemotherapy and radiotherapy have not improved long-time survival of patients with NSCLC satisfactorily. Approximately 40% of patients with NSCLC die of recurrence or metastasis within 5 years after potentially curative resection. Owing to the findings of tumor biomarkers, the advanced molecular targeted therapy brings hope. However, further studies are required to discover and identify novel and specific biomarkers of NSCLC for patients’benefit in survival and quality of life.HSF1 is known as a main transcriptional regulator of heat shock response (HSR) that is involved in protection of cells and organisms from heat, ischemia, hypoxia, inflammation, and some other noxious stressors. When HSR is activated by some stressors, HSF1 trimerizes, translocates to nucleus, and binds to heat shock element (HSE) sites of promoters of heat shock protein (HSP) genes in a hyperphosphorylated form that is competent to activate transcription. In addition to mediate this process, lots of studies have revealed that HSF1 and its associated HSPs play critical roles in the cell transformation, tumorigenesis, tumor development and drug resistance in a wide range of human tumors. It reported that HSF1 was up-regulated in patients with hepatocellular carcinoma, which promoted invasion and metastasis of hepatoma cell by enhancing cell motility through Hsp27 and correlated significantly with a poor prognosis. Another in vitro study also indicated that knockdown of HSF1 with shRNA induced the expression of tumor suppressor retinoblastoma protein, resulting in attenuated hepatoma cell growth and colony formation. Immortalized mouse embryo fibroblast (MEF) cells derived from hsfl’’" animals were deficient in both basal and epidermal growth factor (EGF)-induced migration, and concluded that HSF1 was necessary for MAPK signaling which in turn affected EGF-induced cell migration. HSF1 deficiency impeded NF1-associated carcinogenesis by attenuating oncogenic RAS/MARK signaling. These data suggested that HSF1 may act as a novel candidate for the development of new cancer prognostic biomarkers.Tumor angiogenesis is a crucial and proved pathological process that is an essential requirement for tumorigenesis, progression and metastasis of human malignant tumors. The therapeutic potential of targeting tumor endothelium and vascular supply is now widely recognized, where solid tumors cannot grow beyond the size of a few millimeters without inducing the proliferation of endothelium and formation of new blood vessels. HSF1 promotes cardiac angiogenesis through suppressing p53 and downstream upregulation of hypoxia-inducible factor-la (HIF-la) to maintain cardiac adaption. In another pathway, HSF1 contributed to ischemia-induced angiogenesis by regulating the mobilization of bone marrow stem/progenitor cells in cardiovascular diseases. Little studies have been carried out to elucidate the correlation of HSF1 with intratumoral angiogenesis. In mammary cancers, elevated HSF1 increased angiogenesis significantly by promoting expression of HuR and decreasing degradation of HIF-1a indirectly, implying that HSF1 may be used as a potential anti-angiogenesis molecule candidate. But it is still unclear whether and how HSF1 contributes to angiogenesis in NSCLC up to now.Chemokines are a kind of low-molecular proteins (8-10kDa), which are divided into four classes (C, CC, CXC, and CX3C) based on the number and arrangement of conserved cysteine residues. The chemokine receptors (CKRs) nomenclature is based on chemokine subclass. To date, there have been 43 chemokines and 23 chemokine receptors (CKRs) identified in human cells. Some chemokines are able to bind to multiple CKR, whereas others bind exclusively to a single CKR subtype. Chemokines and CKRs are mainly involved in these physiological and pathological processes including inflammation, lymphatic homing, stem cell migration, embryonic development, tissue repair, and angiogenesis by forming a chemotactic gradient. Now, with the development of research, more and more research results demonstrate that chemokines and their CKRs also play a crucial role in the process of tumorigenesis and progression. As one of the most important CKRs, CXCR4 has been found in abundance in many different kinds of human malignant tumors such as ovarian cancer, breast cancer, esophageal cancer, lung cancer and so on. Through binding to the only ligand stromal cell-derived factor-1(SDF-1), also known as CXCL12, SDF-1/CXCR4 axis make a significant impact on invasion and metastasis in tumors. On the other hand, the high expression of SDF-1 in the most common sites for lung cancer metastasis liking brain, liver, and lymph nodes verifies indirectly the important function of SDF-1/CXCR4 axis in organ-specific metastasis of lung cancer. Nevertheless, the current study is mainly concentrated in the downstream signal transduction pathway which SDF-1/CXCR4 involves in, the mechanism leading to abnormal high-expression of CXCR4 in tumor cells is still not clear.Part I The relationship of HSF 1 protein to tumor angiogenesis and prognosis in patients with non-small cell lung cancerPurpose:To detect the expression of Heat shock factor 1 (HSF1) and Micro vessel density (MVD) in patients with non-small cell lung cancer, and examine the relevance between HSF1 and angiogenesis, clinicopathological factors and prognosis.Methods:Immunohistochemical staining method was used to examine the expression levels of HSF1 and CD34. MVD was used to detect the amounts of micro vessels by counting CD34-positive endothelial cells. Their relationship with clinicopathological factors and prognosis in patients with NSCLC were determined by SPSS statistical software.Results:The expression level of HSF1 were increased significantly in non-small cell lung cancer. HSF1 overexpression was observed in 45 cases and associated with MVD (P=0.005) significantly. The overexpression of HSF1 was not associated with patients’ gender, age, tumor size, histology type, as well as differentiation, but it was significantly associated with node metastasis (P=0.005) and clinical stage (P=0.006). HSF1 overexpression and high MVD were significantly associated with poor 5-year disease-free survival (P=0.001,0.006). Patients with HSF1 overexpression and high MVD had a significantly poor overall survival (P=0.006,0.019) and disease-specific survival (P=0.005,0.016). Multivariate analysis exhibited that HSF1 overexpression was an independent prognosticator for poor overall, disease-specific and disease-free survivals (P=0.040,0.046,0.004).Conclusions:Overexpression of HSF1 is common and significantly correlated with tumor angiogenesis in non-small cell lung cancer. Patients with high HSF1 expression had poorer overall, disease-free and disease-specific survival. These current findings suggested that HSF1 may serve as a novel prognostic marker and potential therapeutic target molecule for antiangiogenic therapy in patients with non-small cell lung cancer.PartⅡ The relationship of HSF1 protein to tumor invasion and metastasis abilities and mechanism in non-small cell lung cancerPurpose:To detect the expression levels of HSF1 and CXCR4 protein in NSCLC tissue and cell, and investigate the relationship between them. To further study the role of SDF-1/CXCR4 axis in the process of regulation of HSF1 protein on invasion and metastasis potential of NSCLC in vitro.Methods:we examined the different HSF1 and CXCR4 protein expression levels in 105 NSCLC tissues by immunohistochemical staining. Square test and Mann-Whitney U test were used to analyze the statistic correlation between HSF1 and CXCR4 protein; RT-PCR and Western blot methods were used to detect the expression of HSF1, CXCR4 in multiple NSCLC cell lines at the level of mRNA and protein respectively. FACS was performed to assess the membrane protein expression of CXCR4; HSF1 siRNA was transfected into NSCLC cell lines by liposome 2000. MTS assay was used to detect the proliferation ability; Transwell invasion assay was performed to detect invasive ability, wound healing assay was performed to detect the cell migration ability; cell adhesion experiment was performed to detect cell adhesive ability; Gelatio zymography was used to detect MMP-9 in cell supernatant. VEGF, Akt and phosphor-Akt were also detected by western blot in the different treated cell lines.Results:Both HSF1 and CXCR4 proteins have an overexpressed level in NSCLC tissues commonly. Square test showed that overexpression of HSF1 protein was significantly associated with CXCR4 (P=0.001). And Mann-Whitney U test determined there was a higher expression level of CXCR4 in HSF1 overexpression group than in non-overexpression group. Both Western blot and RT-PCR method proved the protein and mRNA levels of HSF1 were up-regulated in lung cancer cell lines A549, SPCA and HI975 compared to the human bronchial epithelial cell line BEAS2B. Western blot, Rt-PCR and FACS assays proved that CXCR4 expression was decreased in HSF1 interference group than control group. SPCA which transfected with HSF1-siRNA demonstrated a decreasing proliferation, adhesive, angiogenesis and invasion abilities significantly. In addition, the stimulation of SDF-1α enhanced the abilities of proliferation, migration and invasion. Both CXCR4 inhibitor AMD3100 and PI3K inhibitor LY294002 could obviously inhibit the abilities. HSF1 could enhance the expression levels of VEGF and MMP-9 via SDF-la/CXCR4 axis to phosphorylate Akt signal pathway.Conclusions:HSF1 and CXCR4 protein high-expression is common and significantly correlated with each other in NSCLC. HSF1 overexpression promotes the expression of CXCR4 and enhances the abilities of proliferation, adhesion, angiogenesis and invasion. HSF1 promotes the expression of VEGF and MMP-9 in lung cancer cells. Further study reveals that SDF-1α/CXCR4 axis mediates the process in which HSF1 protein activates the PI3K/Akt signal pathway and enhances the abilities of invasion and metastasis in NSCLC. |
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