| [Background and Objective]Microcalcification is a crucial and distinguishable X-ray manifestation of some early-stage breast cancer.It is also one of the most reliable clinical features for evaluating the risk of malignancy,which is associated with tumor aggressive progression and poor prognosis.Further exploration of the key regulatory factors contributing to calcification formation and its role in tumor metastasis microenvironment is of great significance for the diagnosis and subsequent accurate treatment of breast cancer.Metabolic reprogramming,which includes aerobic glycolysis,fat formation process,and amino acid utilization,is a hallmark of tumor progression and cell transformation.Nowadays,some studies suggested that tumor metabolic reprogramming could alter cellular energy utilization and regulate the expression of metastasis-related genes,which subsequently promoting cancer progression.Aerobic glycolysis has now been widely accepted as the metabolic hallmark of cancer.Some studies have shown that the Warburg effect is the main metabolic pathway of the tumor,which is closely related to cancer metastasis.The process of mitochondrial oxidative phosphorylation(OXPHOS)produces a large amount of ATP,which is the main source of energy metabolism in cancer.Besides,emerging research indicated that active mitochondrial function also played a key role in tumorigenesis and development,cancer migration and invasion,cancer stemness,and treatment resistance.Given that the molecular mechanism for the formation of "breast cancer with microcalcification"remained unclear,further study on the potential molecular mechanism related to metastasis and metabolism is needed.Our study involved two main aspects:the first part analyzed the relationship between calcification signs and bone metastasis and other prognostic indicators in breast cancer patients based on clinical data.The second part included the preliminary exploration of the molecular mechanism for metastasis in breast cancer with microcalcification in terms of metabolic reprogramming.We simulated the process of calcification formation in breast cancer using cell osteogenesis induction experiment in vitro.The objectives of our study are:(1)To explore the intrinsic metabolic characteristics and the molecular mechanism of epithelial-mesenchymal transition(EMT)in breast cancer during osteogenic differentiation.(2)To address the key role of mitochondrial oxidative phosphorylation(OXPHOS)in the process of breast cancer metastasis during osteogenic differentiation.(3)To clarify the potential relationship between the OXPHOS process and breast cancer metastasis during osteogenic differentiation,and possibly provide new clues for the future clinical precise targeted therapy.[Materials and Methods]1、The clinical,pathological,and imaging data of 404 breast cancer patients were collected for retrospective analysis.Microcalcification as the influencing factors were divided into two groups.The relationship between microcalcification and bone metastasis was mainly analyzed.The two groups data were compared using two independent sample t-tests,the rate was compared using the χ2 test,and the risk assessment was performed using single-factor and multi-factor logistic regression analysis.2、Using the database(http://www.kmplot.com)to draw the Kaplan Meier survival curve and analyze the relationship between the differential expression of calcification-related genes and prognostic survival(OS and DMFS).3、Cell function experiments in vitro were performed to detect relevant indexes such as calcification,metastasis,and metabolism.Using human breast cancer cell lines,the osteogenic cocktail(OC)model in vitro was established to demonstrate the calcification-exacerbated metastasis.The migration and invasion characteristics were determined by wound healing and transwell experiments.The mRNA and protein expression were identified by qPCR and western blotting.The secretion of related factors in the tumor supernatant was detected by ELISA.The metabolic alteration in breast cancer cells was carried out by Seahorse assay.GraphPad Prism 7.0 software was used for mapping and analysis.[Results]Part Ⅰ1、Clinical data:Breast cancer with microcalcification is more malignant compared with those without microcalcification.Besides,the axillary lymph node involvement,and bone metastasis and other distant metastasis are more common in those with microcalcification.Microcalcification is an independent risk factor for bone metastasis in breast cancer patients.2、Kaplan-Meier plotter analysis:Overexpression of OPN and RUNX2 was negatively correlated with overall prognosis survival(OS)in breast cancer patients.Overexpression of OPN and ALP was significantly associated with short-term DMFS.BSP overexpression was slightly correlated with short-term DMFS.Part Ⅱ1、Breast cancer cells osteogenesis induction in vitro simulates the process of calcification formation in breast cancer.Breast cancer calcification nodules increase with the extension of induction time,and the expression of calcificationrelated indicators correspondingly upregulates.Osteogenic cocktail(OC)can promote the process of calcification formation in breast cancer cells in vitro.2、Breast cancer cells activate the classic TGF-β/Smad signaling pathway and nonclassical MAPK signaling pathway during osteogenic differentiation,which subsequently enhances tumor metastasis and invasion capacity and promotes the EMT progression.3、The alteration of cancer metastasis characteristics during osteogenic differentiation may not depend on glycolysis.4、Tumor metabolic reprogramming,especially mitochondrial metabolic activity,is significantly enhanced during osteogenic differentiation.The alteration of cancer metastasis characteristics may depend more on the process of mitochondrial oxidative phosphorylation(OXPHOS).5、The blockade of OXPHOS activity inhibited the activation of TGFβ and MAPK signaling pathways,and further reversed aggressive cancer phenotypes in breast cancer cells during osteogenic differentiation.[Conclusion]1、Microcalcification is an independent risk factor for bone metastasis in breast cancer.The abnormal expression of certain calcification-related genes(e.g.OPN,RUNX2,ALP)in breast cancer patients is associated with shorter clinical survival(OS or DFMS).2、The OXPHOS complex may act as a potential intermediate bridge between calcification and EMT.It plays a leading role in promoting the metastatic plasticity of breast cancer.The intervention of mitochondrial metabolic reprogramming can inhibit the activation of the TGF-β/Smad and MAPK signaling pathway,and further reverse the EMT progression.Targeting the OXPHOS process may be a key strategy for future metastasis treatment of breast cancer with microcalcification. |
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