Matrix Stiffness Modulates Response To Chemotherapeutic Agents In Breast Cancer Cells Via ILK Mediated YAP Activation

Breast cancer has been one of the most common malignant tumors of women.Nowadays chemotherapy is still the major therapy method for breast cancer.However,breast cancer cells tend to develop resistance to chemotherapy during treatment.Therefore,it is of great importance to investigate the mechanisms of chemotherapy resistance.During the occurrence and development of breast cancer,the mammary gland matrix tends to be hardened due to mass deposition of collagen fibers,increasing the stiffness of breast matrix.Hence the breast matrix stiffness has been used as a regular index for the clinical diagnosis of breast cancer.Presently researches on the mechanisms of chemotherapy resistance are mainly conducted through aspects like drug inactivation,Alterations in drug targets,drug efflux,DNA damage repair,Deregulation of apoptosis,etc.But few reports have been found on the influence of matrix stiffness.Therefore,researches in this area will be able to provide a new direction to expound the mechanisms of chemotherapy resistance in breast cancer and provide a new basis for therapeutic agents screening.In this research,to investigate the dependence of chemotherapy resistance of breast cancer cells on the matrix stiffness,polyacrylamide hydrogels with different hardness were prepared in a 2D form to simulate the breast cancer tissue stiffness at different phases.Cells were cultured on the surface of polyacrylamide hydrogels with different stiffness(10 kPa,38 kPa,and 57 kPa),and were then treated with different concentrations of doxorubicin,respectively.After treatment,cell proliferation was assayed by Cell Twitter and ki67 immunofluorescence.It was found that the cell proliferation on 38 kPa stiffness was significantly stronger than those on 10 kPa and 57 kPa.Next,cell apoptosis was investigated via Calcein-PI stain and Western Blot experiments,where the cells on 38 kPa stiffness were found to show higher Bcl-2 / Bax ratio,lower caspase-3 and PARP cutting,and lower apoptotic cell proportion.Next,confocal microscopy and flow cytometry were used to investigate the drug influx of cells.It is found that the drug influx of cells on 38 kPa stiffness was relatively weaker.After that,drug efflux was measured by flow cytometry.The cells on 38 kPa stiffness showed stronger drug efflux and higher expression of P-gp in both mRNA and protein levels,indicating a stronger drug resistance.The molecular mechanism of the stronger drug resistance in 38 kPa stiffness matrix was investigated in the following.It is found from the clinic database that higher expressions of ILK and YAP tend to reduce the survival rate of breast cancer patients.Compared to the general population,patients with breast cancer tend to show higher ILK expression in mRNA,and the ILK mRNA expressions at breast cancer phase IV are also higher than those at phase I.Furthermore,it is also found that there is a strong positive correlation between the expressions of ILK and YAP for both normal and cancer tissues.Based on the information above,the ILK expressions in the matrixes with different stiffness were investigated by using Western Blot,immunofluorescence,RT-PCR,luciferase assay,database analysis,and other testing methods.It is found that for the matrix with 38 kPa stiffness,the ILK expressions in mRNA and protein were both significantly higher,and inhibition of ILK would reduce the drug resistance of cells.Meanwhile,the matrix with 38 kPa stiffness also showed higher expression of p-Merlin,lower expressions of p-MST1,p-LATS1,and p-YAP,and YAP / TAZ relocated from the cytoplasm into the nucleus.These results indicated that YAP was activated in the matrix with 38 kPa stiffness.On the other hand,when the ILK was inhibited,sequestration of YAP in the nucleus would be decreased,and so were the expressions of p-MST1,p-LATS1,and p-YAP.Further experiments showed that YAP gene activity and YAP target gene mRNAs expression were both increased in the matrix with 38 kPa stiffness,indicating a higher YAP functional activation.On the other hand,when the YAP was inhibited,the cell drug resistance was reduced,and P-gp expression was decreased.These results show that YAP activation can promote cell drug resistance.In conclusion,it has found in this research that the drug resistance of breast cancer cells can be modulated by the matrix stiffness via ILK mediated YAP activation.