TMC5 Promotes Breast Cancer Bone Metastasis Through Calcium-Related ERK1/2 Signaling Pathway

Background:Breast cancer is the most common female malignant tumor and has gradually become an important problem threatening global public health.The latest estimates have shown that the number of new cases of breast cancer worldwide in 2020has reached 2.26 million.As a heterogeneous disease,breast cancer has a great difference in prognosis.Bone metastases occur in 65-75%of patients with advanced breast cancer,commonly in the thoracic,lumbar,rib,and hip regions.Bone tissue,as a complex ecological environment,is composed of osteoblasts,osteoclasts and a variety of cytokines.The invasion of breast cancer cells to bone tissue causes a series of bone-related events,which seriously affect the quality of life of patients.At present,the treatment for bone metastasis of breast cancer mainly includes radiotherapy,surgery and combination therapy,but there is still no effective cure.Therefore,the study of effective targets for the prevention and treatment of bone metastasis of breast cancer is crucial to improve the prognosis of patients.Abnormal expression of genes in the transmembrane channel-like protein（TMC）family has been identified in many cancers.Abnormal expression of ion channel proteins can be involved in various tumor development processes,such as abnormal proliferation,angiogenesis,invasion and metastasis.TMC5 is a multiplex transmembrane protein that mediates ion transport.At present,the expression of TMC5 in breast cancer and its relationship with the occurrence and development of breast cancer have not been reported.This study explored the TMC5 expression in breast cancer tissue samples and function in vivo and in vitro experiments in breast cancer,especially its impact on the ability of bone metastases of breast cancer.Our study illustrated TMC5 promoted breast cancer cells of bone metastases and its specific mechanism of action,providing a new therapeutic target for the treatment of bone metastases of breast cancer.Methods:1.The expression of TMC5 in breast cancer tissues and adjacent tissues was tested by Real-time PCR,and its correlation with clinicopathological data was analyzed.2.Lentivirus stable cell lines were constructed to verify the effect of TMC5 on the metastasis of breast cancer in mice.X-ray imaging,mice anatomy,tissue H&E staining and bone TRAP staining were used to detect the metastasis of breast cancer in distant organs in mice.The effect of TMC5 on migration and invasion of breast cancer cells was verified in vitro.3.The effect of TMC5 on the chemotaxis of bone metastasis and colonization ability of bone microenvironment of breast cancer cells was investigated by simulating bone microenvironment,and the effect of TMC5 on osteoclast activation was clarified by TRAP experiment.Western blotting was used to detect the effect of TMC5on the expression level of Cat L in breast cancer cells.4.Fluo-4,a specific calcium probe,was used to study the effect of TMC5 on regulating calcium influx in breast cancer cells,and to explore the effect of Ca²⁺signal on the biological function of TMC5 by using calcium chelating agent.5.Western blotting was applied to explore the effect of ERK1/2phosphorylation on TMC5 biological function.Results:1.The expression level of TMC5 in breast cancer tissues was significantly up-regulated compared with adjacent tissues.Combined with clinicopathological analysis,the high expression of TMC5 in breast cancer tissues was significantly associated with high histological grade（P=0.028）,lymph node metastasis（P=0.045）,and bone metastasis（P=0.022）.2.In vivo experiments demonstrated that overexpression of TMC5 significantly promoted the occurrence of bone metastasis of breast cancer cells,but had no significant effect on lung and liver metastasis.3.In vitro experiments showed that overexpression of TMC5 significantly enhanced the metastatic potential of breast cancer cells,while knockdown of TMC5 significantly inhibited the metastatic potential of breast cancer cells.Western blotting showed that overexpression of TMC5 promoted Snail and Vimentin,and inhibited E-cadherin.4.In the bone microenvironment provided by MC3T3E1 cells,overexpression of TMC5 promoted the chemotactic migration of breast cancer cells to the bone metastasis microenvironment,while knocking down TMC5 inhibited the chemotactic migration of breast cancer cells to the bone metastasis microenvironment.Soft AGAR colony formation experiments showed that overexpression of TMC5 increased the proliferation of breast cancer cells in bone microenvironment,while TMC5 knockdown inhibited the proliferation of breast cancer cells in bone microenvironment.TRAP results showed that overexpression of TMC5promoted osteoclast differentiation,while TMC5 knockdown inhibited osteoclast differentiation.Western blotting results showed that overexpression of TMC5significantly promoted the expression of Cat L,while knockdown of TMC5 significantly inhibited the expression of Cat L.Transwell results showed that Z-FYCHO,as a Cat L specific inhibitor,could significantly reverse the increased migration and invasion of breast cancer cells induced by overexpression of TMC5,and also significantly inhibit the increase in the number of mature osteoclasts induced by overexpression of TMC5.5.Confocal results showed that overexpression of TMC5 enhanced Ca²⁺influx intensity,while TMC5 knockout reduced Ca²⁺influx intensity.Transwell results showed that the use of the intracellular calcium repressor BAPTM/AM eliminated the promoting effect of TMC5 on cell migration and invasion.Western blotting and immunofluorescence showed that BAPTM/AM eliminated Snail nucleus translocation and EMT-related gene expression changes induced by overexpression of TMC5.Western blotting results showed that BAPTM/AM inhibited the promotion effect of TMC5 overexpression on Cat L expression and osteoclast activation.6.Western blotting results showed that overexpression of TMC5 significantly promoted the phosphorylation of ERK1/2 in breast cancer cells.Transwell results showed that the increased migration and invasion ability of breast cancer cells induced by TMC5 overexpression could be reversed by Snail inhibition,while the decreased migration and invasion ability of breast cancer cells induced by Snail inhibition could be reversed by Snail overexpression.At the same time,ERK pathway inhibitor U0126 can eliminate the promotion effect of TMC5overexpression on the migration and invasion of breast cancer cells.Conclusions:1.The expression level of TMC5 in breast cancer tissues was significantly up-regulated compared with adjacent non-cancer tissues.2.Overexpression of TMC5significantly promoted the migration and invasion of MDA-MB-231 and T47D,as well as their tendency to migrate to and colonize in bone microenvironment.3.TMC5promoted bone metastasis of breast cancer by enhancing Ca²⁺influx of breast cancer cells and activating Ca²⁺-ERK1/2-Snail signaling pathway.