Study On The Effect Of PI3K/AKT Signaling Pathway On Invasion And Meastasis Of Nasopharyngeal Carcinoma

[Background] Nasopharyngeal carcinoma (NPC) is one of the most common carcinomas in Guangdong, Guangxi and Sichuan province in Southern China, while rare in most parts of the world. NPC is a highly malignant cancer which often invasives adjacent regions and metastasizes to regional lymph nodes and distant organs. Although many NPC patients may undergo radiation therapy for possibly cure and new strategies have improved the survival for patients with metastasis, 30%-40% NPC patients die from local recurrence and distant metastasis. Comparing the most other head and neck carcinomas, NPC shows a poor prognosis consequently. A better understanding of signaling pathways contributing to NPC survival and apoptosis will provide targets for new therapeutic agents. The properties of invasiveness and, even more so, meatastasis more unequivocally identify a neoplasm as malignant than any of the other neoplastic attributes. Approximately 30% of newly diagnosed patients with solid tumors present with clinically evident meatastasis. An additional 20% have occurred metastases at the time of diagnosis. The spread of tumors is a complex process involving a series of sequential steps, which is affected by either host-related or many tumor-related factors. Many signalings play essential roles in tumor development and homeostasis. Evidence from several laboratories indicates that phosphatidylinositol-3 OH kinase (PI3K)/AKT signaling pathway is constitutively activated in various cancers, including gastric, renal cell, ovarian, and lung cancers, and plays a critical role in tumor formation. The PI3K/AKT signaling pathway has been shown to contribute to cancer survival, apoptosis, and regulating a variety of cellular processes. In particular, AKT serine/threonine kinase is believed to play a critical role in controlling the balance between cell survival and apoptosis. Previous studies had shown that phosphatidylinositol 3,4,5-trisphosphate(PIP3) generated by PI3K acts as a lipid second messenger essential for the translocation of protein kinase B (AKT) to the plasma membrane. AKT is phosphorylated at two sites, the T308 in kinase domain and S473 in regulatory tail. Phosphorylation at T308 and S473 is essential for maximal AKT activation. Phosphorylated AKT regulates the function of a broad array of intracellular proteins involve in fundamental processes including cell proliferation, cell death, cell motility/adhesion, cell transformation, neovascularization, and the inhibition of apoptosis. PIP3 levels and AKT activation are regulated by the action of phosphatase and tensin homologue deleted from chromosome 10(PTEN). The AKT survival pathway is regulated negatively by PTEN lipid phosphatase, which selectively dephosphorylates the 3'site on polyphosphoiositides produced by PI3K. Alterations of the PI3K/AKT pathway in human carcinomas have been reported. Many studies demonstrated that PI3K/AKT pathway is constitutively activated in various cancers. There is now convincing evidence that the alterations of the PI3K/AKT pathway is related not only to tumor progression but also to human resistance to radiation and systemic therapies.Epithelial cell-cell junctions provide tissue integrity and promote cell polarity. The adherens junctions play a pivotal role in regulating the activity of the entire complex, and the major adhesion molecules in the adherens junctions are cadherins. Cadherins mediate cell-cell adhesion through their extracellular domains and connect to the actin cytoskeleton by associating with catenins through their cytosolic domain. Loss of intercellular adhesion is a hallmark of migratory cells. E-cadherin is expressed by most normal epithelial tissues, thereby forming the central role of the epithelial adherens junction. Many epithelium-derived tumors have lost E-cadherin partially or completely as they progress toward malignancy. Provious work has revealed that E-cadherin protein prominently associated with tumor invasion, metastatic dissemination, and poor patient prognosis. During invasion and metastasis, first step is tumor cell lost cell-cell adhesion, gain mobility, and leave the site of the primary tumor to invada adjacent tissues and intravasation. E-cadherin loss ostensibly promotes meastasis by enabling the first step of the imetastatic cascade. The significance of E-cadherin for metastasis has been shown in a variety of in vitro and in vivo models. However, most studies have proved both both strong anti-invasive and antimetastatic roles for E-cadherin. Very recently, evidence from several laboratories indicates that metastasis of many epithelium-derived tumors is close correlation with epithelial-to-mesnchymal transition (EMT).EMT is characterized as loss epithelial morphology and acquisition of mesnchymal characteristics. Loss of E-cadherin expression seems to be heavily involved in EMT, and E-cadherin is therefore emerging as one of the epithelial phenotype. Among the mechanisms largely associated with the metastatic conversion of epithelial cells and the EMT, the loss of E-cadherin-mediated cell adhesion is most prominent. During the EMT, cell-cell junctions are disupted, the actin cytoskeleton is extensively reorganized and the cells acquire increased migratory characteristics. It has been reported previously that loss of E-cadherin protein provokes an EMT, attended by motility, invasiveness, and resistance to apoptosis. Most recently, many studies have reported that cancer cells could acquire the mesnchymal to epithelial ransition (MErT) in order to adapt the microenvironments and re-expression of E-cadherin being a critical indicator of MErT. However, the precise mechanism and biological or clinical importance of the MErT in canrcinomas have been little known in in vitro and in vivo.Previous work has revealed that several signaling pathways involved in EMT or MErT. Much of the meeting highlighted signaling pathways that regulate or mediate the EMT or MErT, focusing both refinement and extension of known pathways, but also on the discovery of new regulators and novel pathways.It has been reported recently that AKT has been shown to repress transcription of the E-cadherin gene. This transcriptional repression induces responses leading to the conversion of epithelial cell into invasive mesnchymal cells. Some studies shown that the relationship of AKT to the three metastasis operatives:EMT, resistance to apoptosis and angiogenesis.LY294002 (2-4-morpholinyl-8-phenlchromone) is a special chemical inhibitor of PI3K, which has been used extensively to study the role of PI3K/AKT pathway in normal and transformed cells. Inactivation of PI3K using LY294002 has been demonstrated to lead to the dephosphorylation of AKT at both T308 and S473, consequently inducing specific G1 arrest in cell growth and finally to cell apoptosis. The inhibitors of PI3K also have antitumor activity in vitro and in vivo in a variety of tumor types, and it is possible that cells expressing constitutively active AKT become dependent on its survival-promoting effects. Although these results have been observed in many human cancers, the role of LY294002 in human nasopharyngeal carcinoma has not been well documented yet.To evaluate the significance of AKT phosphorylation in apoptosis, EMT, and metastasis of human nasopharyngeal carcinoma, we investigated the role of AKT phosphorylation and the effect of LY294002 in vitro and in vivo. Our goal was to examine that the PI3K/AKT pathway might be a new therapeutic target on clinic treatment for nasopharyngeal carcinoma patients.The thesis composed of two parts. The specific aim of the first study was to investigate and compare the expression of AKT, pAKT, and E-cadherin in chronic nasopharyngitis and nasopharyngeal carcinoma tissues. To detecte and compare the expression of AKT, pAKT, E-cadherin, Vimentin, and SMA in lymph node metastasis group, lymph node negative group, some primary NPC tissues and their matched lymph-node metastases, analyse their relationship respectively. The secondly research was aimed to explore the relationship and its mechanisms of the PI3K/AKT pathway on apoptosis, EMT or MErT, and metastasis in CNE-2Z (undifferentiated nonkeratinizing NPC cell line) cells. To observe the LY294002 which is specific inhibitor of PI3K/AKT pathway has effects on apoptosis, EMT or MErT, and metastasis of CNE-2Z.[Methods] The first part was based on the data of 130 patients with undifferentiated nonkeratinizing nasopharyngeal carcinoma which were analyzed at the department of pathology, the Affiliated hospital and the Affiliated Gaozhou hospital of Guangdong Medical College. Immunohistochemical(IHC) SP method was used to examine the expression of AKT, pAKT, E-cadherin, Vimentin, and SMA in 20 chronic nasopharyngitis,130 undifferentiated nonkeratinizing nasopharyngeal carcinoma tissues,23 primary NPC tissues and their matched lymph-node metastases. Positive expression of AKT, p-AKT, Vimentin, and SMA locates in the cytoplasm. Positive of expression E-cadherin shows in the cell membrane. The mean percentage of positive tumor cells was determined from five areas at highpower field (×400). The percentage of positive cells was by two experienced pathologists blinded to the detection. A minimum of 1000 tumor cells were counted.In the second part of investigation, After CNE-2Z cells were treated with a special chemical inhibitor of PI3K/AKT, LY294002, MTT assay was used to identify the effects of the LY294002 on growth and proliferation, IHC and Western blot analysis were used to detecte the expression of AKT, pAKT, E-cadherin, Vimentin and SMA, RT-PCR was used to detect the expression of E-cadherin mRNA. Moreover, ELISA was adopted to examine the contents of pAKT in CNE-2Z cells treated with LY294002, Cell Migration Assay and Matrigel Invasion Assay in vitro was tested in CNE-2Z cells. Apoptosis was measured by propidium iodide staining and analyzed with an EPICS flow cytometer. The second part was about the tumor xenograft experiments. Treated mice were monitored any signs. Body weight and tumors size were measured twice a week. Tumor size was measured using calipers and tumor volume was calculated (volume=long axis×short axis2). IHC or/and Western blot analysis were used to detecte the expression of AKT, pAKT, E-cadherin, caspase-9, Ki67, Vimentin, and SMA, in CNE-2Z which was treated with the LY294002. TUNEL assay was carried out according to the protocol of the ApopTag Peroxidase in situ apoptosis detection kit. The growth index (GI) and the apoptosis index (AI) were calculated by counting the Ki67-and TUNEL-positive cells in a total of 1000 tumor cells observed from more than representative highpower fields, respectively. Immunohistochemical results were evaluated independently. Finally, all organs of mices were indentied the metastasis.[Results]1. The positive expression rates of AKT, pAKT (ser473), and E-cadherin were 30%(6/20),25%(6/20),100%(20/20,77.7%(101/130),68.5%(89/130), and 75.4%(98/130) in chronic nasopharyngitis and NPC respectively with significant difference (P<0.05;P<0.01). The positive expression rates of pAKT, E-cadherin,Vimentin, and SMA in NPC which are lymp node metastasis, lymph node negative groups with significant difference (P<0.05; P<0.01). Comparing the primary NPC tissues with their matched lymph-node metastases, the positive expression of E-cadherin and Vimentin were reduced with significant difference (P <0.05)2. In NPC with lymph node metastasis group, the expression of pAKT (ser473) increased with the reduced expression of E-cadherin (r=-0.368, P<0.01), the expression of Vimentin and SMA were increased with the reduced expression of E-cadherin (r=-0.448, P=0.001, P<0.01; r=-0.738, P<0.01 respectively). In NPC without lymph node metastasis group, the expression of AKT and pAKT (ser473) were increased with the reduced expression of E-cadherin (r=-0.294, P<0.05; r=-0.494, P<0.01, respectively), the expression of Vimentin and SMA was increased with the reduced expression of E-cadherin (r=-0.356, P<0.01; r=-0.334, P <0.01, respectively)3. Activation of PI3K/AKT signaling pathway in NPC induced epithelial-mesnchymal transition by reduced the expression of E-cadherin protein and the increased activation of Vimentin and SMA. 4. As shown by the results of MTT assay and flow cytometry analysis, when the CNE-2Z cells were cultured in medium containing different concentrations of LY294002 for 24h and 48h, cell proliferation was remarkably decreased in a dose-dependent manner. And the growth and proliferation of CNE-2Z cells were inhibited significantly by LY294002 compared with the control group, which was in dose dependent manner. When caspase-9 specific inhibitor, ZVAD, was added, the apoptosis was absorbance decreased at 48h.5. The results of immunocytochemistry(ICC) were shown that a remarkable decrease of pAKT (ser473) protein and increase of E-cadherin protein were occurred in cells treatted with LY294002.6. With the increasing concentrations (0-75μM) of LY294002, the levels of pAKT were decreased by using ELISA assay.7. Administration of LY294002 could increase the expression of E-cadherin mRNA in CNE-2Z by RT-PCR.8. Results of Matrigel Invasion Assay were shown that treatment the CNE-2Z cells with LY294002 could inhibit the invasion and migration.9. Results of Western blot analysis and IHC were revealed that the remarkable decrease of pAKT (ser473) protein and increase of E-cadherin protein were examined in cells treated with LY294002. The caspase-9 activity in CNE-2Z cells was up-regulated by LY294002, while the level of caspase-9 was not changed after using ZVAD.10. Treatment the mice with LY294002 (50mg/kg,75 mg/kg) significantly reduced the mean NPC tumor burden as compared with the control group (control versus LY29400250mg/kg,75 mg/kg; P<0.001). Treatment with 10mg/kg or 25 mg/kg LY294002 was less effective in decreasing tumor burden. Mean NPC tumor burden was remarkably decreased in mice treated with LY294002 by a dose-dependented manner, whereas the mean body weight was no obvious difference between control and treated groups (control versus LY294002 10mg/kg,25mg/kg, 50mg/kg, and 75 mg/kg; P>0.05) 11. Results of the histological changes showed that tumor cells in LY294002-treated mice were suffered more necrosis than those of control group. And the number of proliferation cells in mice treated with LY29400 showed significant reduction in a dose-dependent manner, with significant difference (P< 0.05; P< 0.01, respectively). TUNEL-positive cells in mice treated with LY294002 were significantly increased in a dose-dependent fashion, with significant difference (P<0.01). AKT phosphorylation (S473) in xenograft tumor was inhibited by LY294002 in a time- and dose-dependent manner. The expression of Vimentin and SMA was inhibited by LY294002. Total AKT protein level was not difference between control and treated groups. The levels of caspase-9 were increased than those of vehicle controls. The results of Western blot and IHC in tumor tissues also demonstrated similar trends of total AKT, phosphorylated AKT (S473) and caspase-9 with those in vitro.12. All organs of mices were detected and examined. Three of four mices were found micromatastasis of lung in control group, one of four mices was found micromatastasis of lung in LY294002 treated groups (10mg/kg and 25mg/kg) respectively, no any matastasis was found in LY294002 treated groups (50mg/kg and 75mg/kg) respectively.[Conclusions]1. The positive expression rates of pAKT, E-cadherin,Vimentin, and SMA in NPC with or without lymp node metastasis are significant difference (P<0.05, P< 0.01, respectively). Comparing the primary NPC tissues with their matched lymph-node metastases, the positive expression of E-cadherin and Vimentin are significant difference (P<0.05)2. LY294002, the special chemical inhibitor of PI3K/AKT signaling pathway, could inhibit the growth and proliferation of the CNE-2Z cells effectively and induce its apoptosis via caspase-9 pathway.3. Activation of PI3K/AKT signaling pathway could induce CNE-2Z cells epithelial-mesnchymal transition by reduce of E-cadherin adhere and increase of invasion and migration of tumors cells.4. Administration of LY294002 could induce CNE-2Z cells mesnchymal-epithelial transition by up-expression of E-cadherin and inhibition the invasion and metastasis in vivo.5. These results in vivo and in vitro suggest that therapeutic inhibition of PI3K/AKT may be a useful new strategy to control tumor cell invasion and metastasis.