Multidrug resistance drives partial EMT to complete EMT: study the network of EMT mediators 楊毅輝1*、陳奇雍2、孟子青３、王正康4# １國所防醫學院醫學系，２國防醫學院生命科學研究所，３國防醫學院生物化學研究所，4中央研究院生物化學研究 Abstract Diverse expression of EMT markers suggested a possible explanation for DR22’s partial EMT status Taxo 10 exhibit higher motility and invasion propensity as well as increased in vivo tumorigenecity Drug resistance commonly exhibited by cancer cells remains an impediment for conventional cancer chemotherapy. Epithelial mesenchymal transition (EMT) has been reported as an essential mechanism in tumor invasion and metastasis. It has been reported that drug resistance may contribute to EMT. In this study, we treated Myc83, a c-Myc-overexpressing mouse mammary carcinoma cell line with EGFR inhibitor PD153035 to develop a drug resistant cell line, DR22. DR22 resisted to PD153035 but sensitive to other chemotherapeutic drugs and radiation exposure while exhibited significantly higher tumorigenicity and proliferation rate. Furthermore, partial EMT was observed in DR22, with lowered motility, compact clustered cell islands and paradoxical expression of EMT markers. We found that diverse expression of Snail and Slug, two EMT mediators, suggested a possible reason for this phenomenon. We then postulated that fully EMT will be triggered in multidrug resistant cell line. We utilized two anticancer drugs -- Paclitaxel, Doxorubicin to establish multidrug resistant cell lines. Two multidrug resistant cell lines, Taxo10 and Doxo10 were generated after 10 times drug selections. Taxo10 expressed markedly augmented mesenchymal markers, displayed fibroblastoid morphology and more aggressive actin rearrangement while Doxo10 remained similar to DR22. The motility and invasiveness were significantly upregulated in Taxo10 but not in Doxo10. We found that one of the possible mechanisms that engendered the discrepancies between two cell lines was VEGF autocrine loop. Our works provided further insights on molecular mechanisms that drive partial EMT to fully EMT and suggested that VEGF autocrine loop as a potential target for anticancer therapies. DR22 showed different levels of Slug and Snail, and transfection of Slug into DR22 is associated with reacquisition of mesenchymal markers. Diverse expression of EMT markers suggested a possible explanation for DR22’s partial EMT status Using wound healing assay and in vitro invasion assay, we further confirmed that the motility and invasion ability in Taxo10 were higher than DR22 and Doxo10. Subcutaneous injection of three cell lines revealed that Taxo10 has the highest in vivo tumorigenicity. VEGF autocrine loop suggested a possible mechanism to the discrepancies of Taxo10 and Doxo10 Epithelial phenotype Fibronectin α-SMA Vimentin E-cadherin Partial EMT Fibronectin α-SMA Vimentin E-cadherin Mesenchymal phenotype Fibronectin α-SMA Vimentin E-cadherin Novel Partial EMT Fibronectin α-SMA Vimentin E-cadherin DR22 was resistant to PD153035 but sensitive to other anti-cancer drugs and radiation exposure We generated a c-Myc overexpressed transgenic mice by MMTV-Myc. Breast cancer was eventually developed. We derived the cells and named it Myc83. Using Myc83, We performed drug selection by PD153035 for 22 times. The cell line after drug selection was named DR22. Multidrug resistance was a driving force for complete EMT The level of p-VEGFR2 was specifically enhanced in Taxo10. Moreover, the secretion of VEGF was also increased in Taxo10. Upstream targets of VEGF were found to be upregulated in Taxo10. These findings suggested that VEGF autocrine loop might contribute to the EMT in Taxo10. Myc83 DR22 Conclusion DR22 was highly resistant to PD153035. However, compared to Myc83, DR22 was obviously more sensitive to other chemotherapeutic drugs and radiation exposure. Novel partial EMT  DR22 Loss of epithelial traits Loss of mesenchymal characteristics Increased EMT mediators network DR22 Taxo10 Doxo10 Reduced We utilized Taxol and Doxorubicin to performed 10 times drug selection on DR22. Multidrug resistant cell lines, Taxo10 and Doxo10, were established. The adhesion between cells was decreased and the number of cell protrusion was increased. EMT was orchestrated by inducer network but not by a single participant. Different participants(such as Snail and slug) might play different role in EMT progression. Their elevation and down regulation at the same time contributed to the partial EMT. DR22 showed higher proliferation rate and tumorigenicity but decreased motility Taxo10, rather than Doxo10, displayed higher level of mesenchymal markers and more intense actin rearrangement Myc83 DR22 Taxo10 Doxo10 DR22 Taxo10 Doxo10 Development of Taxol resistance Upregulation of HIF-1α and p-c-Jun Elevated phosphorylation of FAK and Paxillin Higher VEGF secretion DR22 expressed higher proliferation rate. Moreover, tumorigenicity of DR22 was markedly more aggressive than Myc83. However, wound healing assay showed that the motility of DR22 was decreased. Elevated phosphorylation of VEGFR2 Elongated cell shape Augmented mesenchymal markers Dynamic of actin rearrangement Motility Invasion ability While remained loss of E-cadherin, Taxo10 specifically expressed higher level of mesenchyaml markers and RhoA. Furthermore, filopodia and stress fiber expression was more aggressive in Taxo10, suggesting that the dynamic of actin rearrangement in Taxo10 was greater than DR22 and Doxo10. Discussion Taxo10 exhibited enhanced Focal Adhesion Kinase(FAK) signaling DR22, grew in tightly packed cell cluster, exhibited a novel partial EMT status Role of Snail and Slug in EMT progression. Other EMT inducers involved? Knockdown experiment of VEGF or upstream target  role of VEGF autocrine loop Investigation of the interaction of Snail, Slug and VEGF signaling pathway. The activity of phospho-tyrosine kinase was more robust in Taxo10. Some bands particularly overexpressed in Taxo10 were focused and studied. FAK and Paxillin, which regulate cell invasion and motility, were significantly higher phosphorylated in Taxo10. Along drug selection, DR22 gradually tend to grew in tightly packed cell clusters. The expression of both epithelial marker(E-cadherin) and mesenchymal markers(Vimentin, α-smooth muscle actin and fibronectin) was downregulated.