The Role Of Axl In The Invasion And Chemosensitivity Of Breast Carcinoma And Its Clinical Significance

Objective: To investigate the mechanism of Axl promoting tumor metastasis and chemoresistance in human breast carcinoma cell lines. To study their relevance with PI3K/Akt Signaling Pathway and Slug, and analysis the correlation between the clinicopathologic characteristics and expression of Axl protein in breast carcinoma. We support the possibility that Axl is a novel regulator of tumor metastasis and chemosensitivity in breast cancer and a promising target for breast cancer therapy.Methods: The mRNA and protein expression levels of Axl in MDA-MB-231 and MCF-7 cells lines were evaluated by Real-Time PCR and western blot analysis and their diversity was clarified. We analyze the possible role and mechanism of Axl in the tumor invasion and chemosensitivity of breast carcinoma.(1) We silenced the expression level of Axl in MDA-MB-231 cells by RNAi assay, and examined the different expression levels of Axl. We further explore the invasion ability of MDA-MB-231 cells by in vitro cell invasion assays.(2) Five-week-old female athymic nude mice were obtained from Animal Facility of Dalian Medical University, and were provided with sterilized food and water. Approximately 1(107 cells were injected subcutaneously into the right flank of each nude mouse, respectively. Once bearing palpable tumors(about 3 weeks after tumor cell inoculation), tumor-bearing mice were randomly divided into control and treatment groups(n=6 animals per group). The treatment groups received 30 mg/kg 5-FU i.p.(intraperitoneal) three times per week for 3 weeks, and the control groups received physiological saline alone. Mice were sacrificed and their tumors were isolated and weighed. Immunohistochemical study was used to analyse the the expression level of Axl in mice tumor tissues.(3) After the silence of Axl expression level, we study the expression and activity of the PI3K/Akt pathway and Slug gene. To further determine the potential involvement of PI3K/AKT signaling and survey whether the invasion and metastasis process were regulated by signal transduction gene. We applied LY294002, Akt shRNA, and SB415286, selective antagonist of the PI3 K, Akt, and GSK3%, respectively.(4) we knockdown Axl in MDA-MB-231 cells and subsequently overexpress PI3K/Akt pathway associated genes, to detect whether overexpression of PI3K/Akt pathway could rescue the cells from suppressed invasion and chemoresistance caused by Axl knockdown. Knockdown PI3K/Akt pathway associated genes and then knockdown Axl, to detect whether Axl knockdown could affect invasion and chemosensitivity when PI3K/Akt pathway was overinhibited. Axl was PI3K/Akt signaling pathway dependent. These two experiments further verified if the function of Axl was PI3K/Akt signaling pathway dependent.(5) MDA-MB-231 cells were transfected with Slug sh RNA or control shRNA, and we assessed the invasion property and chemosensitivity. Breast cancer and transitional tissues(3 cm from the tumor edge) were collected from the same 101 patients who underwent surgical resections from July 2010 to May 2012 at the Second Affiliated Hospital of Dalian Medical University. We analyzed the Correlation between the clinicopathologic characteristics and expression of Axl protein in breast carcinoma.Results: MDA-MB-231 cells have a higher Axl expression compared with the MCF-7 cells on both mRNA and protein levels; Knockdown of Axl alters cell invasion ability and chemoresistance of MDA-MB-231 cells in vitro and in vivo; Knockdown of endogenous Axl resulted in the increased expression of epithelial maker-E-cadherin and the reduced expression of various mesenchymal makers, namely N-cadherin, Snail, and Slug; Silence of Axl inhibited the expression and activity of the PI3K/Akt pathway; The inhibition of PI3K/Akt pathway alters cell invasion ability and chemoresistance; The function of Axl was PI3K/Akt signaling pathway dependent; Slug expression is essential for the chemosensitivity and invasion-promoting activity of Gas6/Axl signaling; No significant evidence indicated Axl has relationship with age, and distant metastasis in breast cancer patients(P>0.05), While the protein expression of Axl was closely correlated with histological grade, lymph node metastasis, and clinical stage in patients with breast cancer(P<0.05).Conclusion:(1) Axl played an important role in association with breast cancer cells invasion and chemoresistance via modulating the PI3K/Akt/GSK3% signaling pathway, and it is transcriptionally regulated by Slug.(2) The elevated expression of Axl was not only shown in breast cancer tissue compared with corresponding noncancerous tissues but also closely associated with histological grade, lymph node metastasis, and clinical stage in patients with breast cancer.(3) These findings suggest that Axl can regulate tumor Invasion and chemosensitivity of breast carcinoma, and it may serve as a new prognostic marker and therapeutic target for treating breast carcinoma. What is more, through the research of the detailed mechanism of Axl in breast carcinoma, we can reduce the invasion and chemosensitivity of breast tumor by targeting Axl or its related signaling pathways.