The Pilot Study For The EGFR-mediated Chemotaxis Of Cancer Cells

Chemotaxis, a conservative cellular process, represents the capacity to detect an extracellular gradient of chemoattractants and directionally move to the higher concentration site. Metastasis is the major cause of morbidity and mortality in cancer patients. Recent studies reveal a key role of chemotaxis in cancer cell metastasis. It has been reported that epidermal growth factor receptors (EGFR), as an important receptor tyrosine kinase (RTK) on cell surface, have potent chemotactic effects on human breast cancer cells. Currently, it is still unclear about the chemotactic signaling transduction of EGFR downstream. Hence, our research about the mechanism of EGFR-mediated chemotaxis will be of great help for cancer therapy and developing novel drug targets.In this thesis, we focused on the role of lipid rafts in EGFR-mediated chemotaxis of human breast cancer cells. Lipid rafts, organized microdomain on plasma membranes, regulate the activation of many membrane receptors. In the current study, our confocal microscopy results suggested that EGFR co-localized with GM1-positive rafts. Disrupting rafts with methyl-β-cyclodextrin (mβCD) inhibited EGF-induced chemotaxis of human breast cancer cells. Supplementation with cholesterol reversed the inhibitory effects partly. Pretreatment with mβCD also impaired directional migration of cells in an in vitro"wound healing"assay, EGF-induced cell adhesion, actin polymerization, Akt phosphorylation and protein kinase Cζ(PKCζ) translocation. Taken together, our study indicated that integrity of lipid rafts was critical in EGF-induced chemotaxis of human breast cancer cells.High expression of 3'-phosphoinositide-dependent protein kinase-1 (PDK1) has been detected in various invasive cancers. In the current study, we investigated its role in mediating cancer cell chemotaxis and metastasis. Down-regulation of PDK1 expression by small interference RNA (siRNA) severely inhibited chemotaxis of human breast cancer cells. The defects were rescued by expressing a wild type PDK1 plasmid. PDK1-knockdown cells showed impaired EGF-induced actin polymerization and cell adhesion, probably due to a decrease in phosphorylation of LIMK/cofilin and integrinβ1, respectively. Confocal microscopy revealed that EGF induced co-translocation of PDK1 with Akt and PKCζ, regulators of LIMK and integrinβ1. Furthermore, PDK1 depletion dampened EGF-induced phosphorylation and translocation of Akt and PKCζ, suggesting that Akt and PKCζfunctioned downstream of PDK1 in the chemotactic signaling pathway. An in vivo analysis by using a severe combined immunodeficiency (SCID) mouse model showed that PDK1-depleted human breast cancer cells were defective in extravasation to mouse lungs. Taken together, our results clearly indicated that PDK1 played an important role in cancer cell metastasis and it regulated chemotaxis through Akt and PKCζ. Thus, PDK1 can be used as a valuable target for development of novel cancer therapies to block metastasis.In this thesis, we also reported that EGF induced a robust chemotaxis of A549 and H1299 cells, two representative human non-small cell lung cancer (NSCLC) cells. Chelerythrine chloride, an inhibitor of all PKC isozymes, significantly reduced the chemotactic capacity of NSCLC cells while inhibitors of classical or novel PKC isozymes, such as G?6976, calphostine C, or G?6850, showed no effect, which suggested that atypical PKC might be involved in the chemotactic process of NSCLC cells. EGF elicited translocation and phosphorylation of atypical PKCζ, indicating that EGF could activate PKCζ. Treatment with a PKCζspecific inhibitor, a myristoylated pseudosubstrate, blocked chemotaxis in a dose dependent manner, further confirming that atypical PKCζwas required for NSCLC chemotaxis. Mechanistic studies suggested that PKCζwas regulated by phosphatidylinositol 3 kinase (PI3K)/Akt. Furthermore, PKCζmediated chemotaxis by regulating actin polymerization and cell adhesion. Taken together, our study suggested that PKCζwas required by NSCLC cell chemotaxis, thus could be used as a target to develop anti-lung cancer metastasis therapies.The appearance of cancer cells in human peripheral blood of cancer patients is considered as an early indication of dispersion and spreading of primary tumor the original tissue. Therefore, early detection and treatment for tumor metastasis can significantly improve the curative effect of cancer therapy. To identify the tumor cell metastasis as early as possible, the detection methods with high sensitivity would be needed. However, the common clinical assays are difficult to work on it. In the current study, we hope to develop a kit for the detection of human breast cancer metastasis with higher sensitivity and specificity, which can detect 2-5 breast cancer cells from 5ml peripheral blood. Thus, we used the immunomagnetic bead enrichment combined with single-cell RT-PCR in the detection process. In the step of cell enrichment, we selected four specific antibodies whose antigens expressed on the surface of epithelial cells. In the following step of RT-PCR, three genes were amplified as the tumor markers. At present, we had successfully confirmed the feasibility and operability of the combination of bead enrichment and single-cell RT-PCR in our in vitro study system. Furthermore, utilizing this method in in vitro experiments, we could detect about 50 breast cancer cells from 5ml blood and also detect about 30 cells from 1ml blood.Upon activation induced by EGF or chemoattractant SDF-1α, PKCζredistributes from cytosol to plasma membrane. Based on this property, we developed a cell model with a rapid assay for screening inhibitors of ligand-induced PKCζactivation. PKCζgreen fluorescent protein (GFP) was transfected into human breast cancer cells, MDA-MB-231, to establish a stable cell line, PKCζ-GFP/MDA-MB-231. PKCζ-GFP/MDA-MB-231 maintained phenotypes, such as chemotaxis, adhesion, cell migration and PKCζphosphorylation, similar to those of its parental cell line. Therefore it could be used as a representative cancer cell line. We can monitor the actions of PKCζ-GFP so as to indirectly determine that of endogenous PKCζ. Translocation of PKCζ-GFP could be easily and directly recorded by an inverted fluorescence microscope. Inhibitors of translocation of PKCζ-GFP also possibly impaired the chemotaxis. Utilizing this rapid cell model, six potent compounds had been screened out as PKCζinhibitors from the small molecule bank by two-step selection.