Matrix Stiffness Regulates Tumor Cell Apoptosis Through Autophagy And Mitochondrial Homeostasis

Breast cancer is one of the most common malignancies in females with high malignancy and metastasis.And the progression of breast cancer is associated with abnormal cross-linking and deposition of collagen,which leads to extracellular matrix(ECM)remodeling and stiffening.Therefore,the mechanics of the environment,in particular substrate stiffness,may play an important role in the development of tumorigenesis.The current understanding of the malignant behavior and biological characteristics of tumor cells is mainly based on chemical signaling.However,whether and how substrate stiffness regulates the biological properties of breast cancer remains unclear.It has been documented that the cellular stress in the tumor microenvironment contributes to aberrant regulation of mitochondrial dynamics.Therefore,this dissertation explored the molecular mechanisms underlying the crosstalk of apoptosis and autophagy as well as mitochondrial dynamics by substrate stiffness in breast cancer cells.In this dissertation,polyacrylamide(PAA)gel substrates with stiffnesses of 10,38 and 57 k Pa were used to simulate the different progression stages of breast cancer,corresponding to the respective relative stiffness of benign,malignant and bone metastasis states of breast cancer,while glass was used as a control substrate to observe the cell status.The results showed that soft substrate could reduce cell viability and enhance the number of trypan blue-positive cells.Moreover,the percentage of apoptosis,the number of autophagosomes and autophagy lysosomes were significantly increased in response to soft substrate.Additionally,inhibition of autophagy moderately enhanced soft substrate-induced cell apoptosis.The above results suggested that substrate stiffness induced apoptosis and autophagy in breast cancer cells and modulated the crosstalk between them.The molecular mechanisms of substrate stiffness-induced apoptosis and protective autophagy in breast cancer cells were further investigated.The results showed that soft substrate remarkably reduced mitochondrial membrane potential and activated the reactive oxygen species/c-Jun N-terminal kinase(ROS/JNK)signaling pathway.Specifically,on the one hand,phosphorylation of JNK induced apoptosis through the mitochondrial pathway.Bcl-2-associated X(Bax)translocated to mitochondria and thus induced the release of mitochondrial cytochrome-C(Cyt-c)into the cytoplasm to promote cell apoptosis on soft substrate.On the other hand,it contributed to the suppression of interaction between B-cell lymphoma-2(Bcl-2)and Beclin-1,and free Beclin-1 formed autophagy initiation complex with autophagy related regulator 14(Atg14)and vacuolar protein sorting 34(Vps34)to induce protective autophagy.These results suggested that matrix stiffness triggered apoptosis and protective autophagy through the ROS/JNK pathway,thereby determining breast cancer cell fate.The above results suggested that ECM softening induced mitochondrial oxidative stress and mitochondria-dependent apoptosis,and protective autophagy,underscoring the importance of soft substrate-mediated mitochondrial damage in breast cancer progression.However,crucial questions regarding how matrix stiffness regulates mitochondrial dysregulation,particularly mitochondrial dynamics and mitophagy,remain to be answered.Accordingly,it was investigated whether and how mitochondrial dynamics and mitophagy were affected by substrate stiffness based on the above work.The findings revealed that soft substrates promoted the recruitment of dynamin-related protein 1(Drp1)to mitochondria and subsequent mitochondrial fission and mitophagy via endoplasmic reticulum-mitochondria(ER-MITO)calcium transport.Specifically,soft substrates triggered IP3R-mediated calcium transport by increasing endoplasmic reticulum calcium release and mitochondrial calcium uptake,and Drp1 was phosphorylated and recruited to mitochondria,which induced abnormal mitochondrial function,mitochondrial fission and phosphatase and tensin homologue-induced putative kinase 1/Parkinson disease kinase(PINK1/Parkin)-mediated mitophagy.Finally,in vivo experiments verified that soft ECM also regulates mitochondrial fission and mitophagy through Drp1 activity in live tumors.Furthermore,mitophagy attenuated soft ECM-induced tumor tissue necrosis,contributing to tumor survival.In summary,substrate stiffness could regulate cell biological characteristics of breast cancer through ROS/JNK pathway and ER-MITO calcium transport.Soft substrates induced mitochondrial apoptosis and protective autophagy through ROS/JNK pathway,as well as further regulated mitochondrial dynamics via ER-MITO calcium transport to promote Drp1-mediated mitochondrial fission and mitophagy,which collectively regulated breast cancer fate.This dissertation elucidated the molecular mechanism by which matrix stiffness regulates the biological characteristics of breast cancer and determines cell fate,which provides inspiration and new targets for the mechanisms of cancer development and clinical cancer therapy.