Cross-Signaling Among Phosphinositide-3 Kinase, Mitogenactivated Protein Kinase And Sonic Hedgehog Pathways Exists In Esophageal Cancer

Esophageal cancer is a common primary gastrointestinal cancer, and it is the 8th most common cancer and the 6th most frequent cause of cancer death in the world. China is one of the country with the highest incidence of ESCC worldwide, and esophageal squamous cell carcinoma (ESCC) accounts for 90% of esophageal carcinomas in china. Since most esophageal cancers remain clinically silent until late in the course of disease, patients with ESCC are often associated with later diagnoses, poorer prognoses and higher mortality rates. ESCC is a disease which do serious harm to people's health. There are multiple signal pathways involved in the occurrence and development of ESCC, and the cross-talk among these signal pathways induce tumor cells to get unlimited proliferation advantage of growth, which promote tumor cells'development and progression.Recent reports have demonstrated that the tumorigenesis of esophagus, stomach, pancreas and other gastrointestinal tumors were closely associated with Sonic Hedgehog (Shh) signaling pathway. Shh pathway was highly activated in esophageal cancer, which can be used as a valuable biomarker for the diagnosis and in molecular classification of esophageal cancer. In addition, several studies have shown that Ras signaling pathway plays an important role in the pathogenesis of cancer. Phosphatidylinositol 3 - kinase (PI3K) / AKT signaling pathway and mitogen-activated protein kinase (MAPK) signaling pathway are two main downstream pathways of the Ras signal. The mitogenic and antiapoptotic signals induced by these two signaling pathways play an important role in the growth and prognosis of human cancer. Up to date, there are only a few independent researches at home and abroad concerning about PI3K/AKT, MAPK, and Shh signaling pathways which study in ESCC. There is no research concerning about the cross-talk among the Shh, PI3K/AKT and MAPK signaling pathway in ESCC which demonstrate the pathology, proliferation and other aspects behavior of biological.To explore the role of Shh, PI3K/AKT and MAPK signaling pathway in the carcinogenesis and the prognosis of ESCC, we examined the expression of Shh, Gli1, p-AKT and p-ERK in human ESCC , and studied the relationship between the expression of these proteins and the clinicopathological data as well as the prognosis in ESCC. We used ESCC cell line TE-1 and TE-10 in vitro. We first used the specific drugs which targeted inhibited the key factors of Shh, PI3K/AKT and MAPK pathways, then we detected the expression of the key factors of each pathway by western-bolt analysis. We hoped to clarify the mechanism of cross-talk among Shh, PI3K/AKT and MAPK signaling pathways exists in ESCC through this experimental design. We also probed the feasibility that Joint suppression of Shh and the PI3K/AKT signaling pathway would be a preferred target of gene therapy of ESCC, and discussed the possible mechanism by which Shh and PI3K/AKT signaling pathways influencing the biological characteristics of ESCC.PartⅠ: The expression of Shh, Gli1, p-AKT and p-ERK in human ESCC tissues and its significanceObjective: To examine the expression of Shh, Gli1, p-AKT and p-ERK in ESCC tissues, and explore the correlation among Shh, PI3K/AKT and MAPK pathway in ESCC.Methods: Tissue specimens were collected during thoracic surgerg from 30 patients and were divided into two groups: the ESCC tissues group and the adjacent normal esophageal tissue group. The protein expression of Gli1, p-AKT and p-ERK were detected by western-bloting in two groups. In addition, we collected 88 cases surgical specimens archived paraffin during January 2004 to December 2005 and divided them into two groups: the ESCC tissues group and the adjacent normal esophageal tissue group. The expression of Shh, Gli1, p-AKT and p-ERK were detected by immunohistochemical staining in two groups.Results: 1, Western-blot analysis showed that the expression of Gli1 and p-AKT were undetected in the adjacent normal esophageal mucosa, whereas the positive expression of Gli1 and p-AKT were detected in most ESCC. The expression of p-ERK protein was undetected or just weakly positive in the normal esophageal mucosa, whereas the positive expression of p-ERK was detected in most ESCC. 2, Immunohistochemical analysis showed that the expression of Shh, Gli1 and p-AKT were undetected in the adjacent normal esophageal mucosa, whereas the positive expression of Shh, Gli1 and p-AKT were detected in most ESCC. The expression of p-ERK protein was undetected or just weakly positive in the normal esophageal mucosa, whereas the positive expression of p-ERK was detected in most ESCC. Statistical analysis showed that the high expression of Shh target genes was associated with AKT activation in ESCC.Conclusion: The expression levels of Shh, Gli1, p-AKT and p-ERK were high in the majority of esophageal cancer, but negative in the adjacent normal esophageal tissues, indicating that Shh, PI3K/AKT and MAPK pathways were usually activitied in most esophageal cancers The expression of Gli1 in ESCC was positively correlated with p-AKT, indicating that the cross-talk may exist between Shh and PI3K/AKT signaling pathways in ESCC. PartⅡ: Cross talk among Shh, PI3K/AKT and MAPK signaling in vitroObjective: To explore wherether there exist cross talk among Shh, PI3K/AKT and MAPK pathways in ESCC and the mechanism of cross talk among them.Methods: ESCC cell linesTE-1/TE-10 were treated with EGF, Gβγand N-Shh to induce the activation of Shh, PI3K/AKT and MAPK pathways. We first used the specific drugs to target inhibit the activation of Shh, PI3K/AKT and MAPK pathways, and then detected the expression of the key factors of each pathway by western-bolt analysis. We also examined the activity of Gli1 by measuring its nuclear import ratio after treatment with inhibitors. In addition, we detected the influence of cell proliferation and apoptosis after treatment with inhibitors in TE-1/TE-10.Results: 1, The expression of p-AKT, p-ERK, and Gli1 in TE cells were all up-regulated after induced by EGF, Gβγand N-shh. 2, The expression effcets of p-AKT or p-ERK induced by EGF in TE cells could be inhibited by wortmannin or PD98059 respectively, but not by cyclopamine. 3, The activation of AKT or ERK by transfected with Gβγin TE cells could be blocked by GRK-ct, but not by cyclopamine. 4, The expression effect of Gli1 induced by EGF in TE cells was inhibited by wortmannin, but not by PD98059 or GRK-ct. 5, The expression effect of Gli1 in TE cells transfected with Gβγcould be abrogated by cyclopamine or wortmannin, but not by PD98059. 6, The expression effcet of p-AKT or p-ERK induced by N-shh in TE cells could be inhibited by wortmannin or PD98059 respectively, also it could be inhibited by cyclopamine. 7, In the presence of EGF, TE cells grew significantly faster than those without stimulation, and this effect was abrogated by pre-treatment with PD98059. The increased proliferation rate after EGF stimulation was only partially inhibited by pre-treatment with cyclopamine. Stimulation with N-Shh enhanced TE cell proliferation, and this effect was blocked by pre-treatment with cyclopamine. In addition, PD98059 almost completely suppressed cellular activity. 8, The expression of caspase 3 in TE cells was significantly increased after induced by serum starvation and the expression of p-AKT was significantly reduced at the same time, neither wortmannin nor cyclopamine treatment could block such effects. 9, The nuclear location effect of Gli1 in TE cells can be induced by EGF, and such effect can be blocked by wortmannin or cyclopamine treatment.Conclusion: The EGF or Gβγinduced activation of AKT and ERK is independent of Smo in esophageal cancer cell lines. The PI3K/AKT axis plays a critical role in EGF-, Gβγ- and Shh-induced Hh signaling in esophageal cancer cell lines. PI3K/MAPK and Shh signaling pathways synergize during esophageal cancer cell proliferation and inhibiting apoptosis. The translocation of Gli1 from cytosol to nucleus after EGF stimulation was also dependent on AKT activation but not ERK activation.PartⅢ: Relationship between Shh, Gli1, p-AKT or p-ERK expression and clinicopathological features as well as prognosis in ESCC patientsObjective: To explore the relationship between Shh, Gli1, p-AKT or p-ERK expression and clinicopathological features as well as prognosis in ESCC patients.Methods: Patients who underwent esophageal cancer surgery were investigated the survival situation following up by way of telephone or letter. The distribution feature of the immunohistochemistry expression of Shh, Gli1, p-AKT and p-ERK in ESCC were explored by statistical methods, and compared with the clinical characteristics in ESCC such as age, sex, tumor size, tumor location, lymph node metastasis, tumor invasion depth, tumor differentiation, clinical TNM stage and postoperative survival.Results: The expression of Shh in ESCC were associated with lymph node metastasis (p = 0.027) and a poor prognosis (log-rank test; p = 0.017), and Gli1 expression was associated with lymph node metastasis (p = 0.043) and a poor prognosis (log-rank test; p = 0.005). The expression of p-AKT was associated with depth of invasion (p = 0.008) and a poor prognosis (log-rank test; p = 0.001).Conclusion: The expression of Shh or Gli1 was positively correlated with lymph node metastasis in ESCC, whereas p-AKT expression was positively correlated with the depth of invasion in ESCC, and both were associated with poor prognosis. Joint inhibition of these pathways may be more effective in the treatment strategy of ESCC with activation Shh and PI3K/AKT pathways.