Study On The Effect And Mechanism Of Chaperone-mediated Autophagy In Breast Cancer Proliferation And Metastasis

Breast cancer, second only to lung cancer, is one of the major malignant tumors thatthreaten the health of women. In recent years, the incidence of breast cancer is increasingyear by year. Each year about1.2million women wordwide suffer from breast cancer.Although the survival rate for breast cancer has increased with the widely use of adjuvantchemotherapy and mammography and other diagnosis and treatment, the long-time survivalrate has not been improved significantly. About40percent of breast cancer patientsreceiving chemotherapy after surgery still died from tumor metastasis, which has become amajor problem for the prevention and treatment of breast cancer. Therefore, elucidating themolecular mechanisms of breast cancer metastasis is of great significance to search for newtargets of breast cancer diagnosis, the improvement of treatment and the prolongation of thepatients’ lives.Autophagy, an evolutionary conserved process in eukaryotic cells, is very important forintracellular material turnover. It has been known that autophagy consists of three forms,including macroautophagy (MA), chaperone-mediated autophagy (CMA) and microautophagy.Recently, the important role of CMA in tumor growth and metastasis has arousedwidespread concern in the medical field. Some studies have reported that CMA is highlyactivated in lung cancer, melanoma, etc., and that it can promote tumor formation andmetastasis of lung cancer cells. Importantly, studies also showed that LAMP2A level wassignificantly higher in the breast cancer tissues than the adjacent healthy tissues by studyingseven breast cancer tissues. Besides, the H2O2simulation tests showed that silent LAMP2Aor overexpressed LAMP2A could inhibit or promote breast cancer cell growth, indicatingthat CMA plays an important role in the development of breast cancer cells. However, therole of CMA in the proliferation of breast cancer cells still requires further elucidation.Especially, there has no reported study on whether CMA has an effect on the metastasis ofbreast cancer cells. In this study, gene silencing via lentivirus infection, transfecting overexpressionplasmid, immunofluorescence, confocal laser, qRT-PCR, western blot and key geneknockout technique were applied, besides, other proliferation and metastasis assays, both invivo and in vitro, including MTT, clony formation, transwell migration and invasion,wound healing, tumorigenicity in nude mice and lung metastasis of breast cancer cellsinjected by mice tail vein were also used to study the effect and mechanism of CMA inbreast cancer proliferation and metastasis.Main contents and results:1. CMA is highly activated in breast cancer tissues, and has a positive correlation withthe clinical stage of breast cancerBreast cancer tissue microarray including70cases of breast cancer and7cases ofnormal breast tissue was used for immunohistochemical analysis. The results showed thatLAMP2A and HSC70was highly expressed in clinical breast cancer samples, andpositively correlated with the clinical stage of breast cancer.2. CMA promotes the proliferation and metastasis of breast cancer in vitro①MDA-MB-231and MDA-MB-468breast cancer cell lines with different LAMP2Alevels were successfully constructed through the infection of LAMP2A shRNA lentiviralexpression vector and transfection of LAMP2A overexpression plasmid, and the above celllines were also validated by western blotting technique.②Immunohistochemical staining revealed that LAMP2A knockdown significantlydown-regulated the CMA activity of MDA-MB-231and MDA-MB-468cells.Instead,LAMP2A overexpression remarkably up-regulated the CMA activity of MDA-MB-231andMDA-MB-468cells.③MTT assay confirmed that down-regulation of LAMP2A expression inMDA-MB-231and MDA-MB-468cells markedly reduced cell proliferation.④Cell survival rate assay with H2O2stimulation showed that down-regulation ofLAMP2A in MDA-MB-231and MDA-MB-468cells significantly reduced the cell survivalrate under oxygen stress.⑤Clony formation assay showed that LAMP2A knockdown remarkably inhibited theclony formation of MDA-MB-231and MDA-MB-468cells. ⑥Transwell migration and invasion assays showed that down-regulation of LAMP2Aexpression in MDA-MB-231and MDA-MB-468cells could significantly inhibit themigration and invasion ability of breast cancer cells. Instead, up-regulation of LAMP2Acould significantly promote the migration and invasion ability of breast cancer cells.⑦Wound-healing assay showed that LAMP2A knockdown significantly reduced themigratory capability of MDA-MB-231breast cancer cells.3. CMA promotes the proliferation and metastasis of breast cancer cells in vivo①Nude mice were inoculated with MDA-MB-231/LAMP2A RNAi andMDA-MB-231/control siRNA breast cancer cell lines. The tumor growth in the siLAMP2Agroup was significantly inhibited.②Nude mice were inoculated with MDA-MB-231/LAMP2A RNAi andMDA-MB-231/control siRNA breast cancer cell lines. More tumor foci from lung werefound in the LAMP2A-implanted mice than the siLAMP2A-implanted mice.③Nude mice were injected with MDA-MB-231/LAMP2A RNAi and MDA-MB-231/control siRNA breast cancer cell lines via the tail vein. LAMP2A knockdown both inMDA-MB-231and MDA-MB-468cells could inhibit the tumor foci formation from lung.4. CMA inhibits the macroautophagy of breast cancer cells①Confocal laser assay showed that suppression of LAMP2A expression inMDA-MB-231cells significantly increased the number of RFP-LC3-Ⅱspot formation. Thesame result was not found in MDA-MB-468cells.②Transmission electron microscope assay showed that knockdown of LAMP2A inMDA-MB-231cells significantly increased macroautophagic vacuoles. The same phenomenonwas not found in MDA-MB-468cells.③Transmission electron microscope assay revealed that inhibiton of LAMP2Aexpression significantly increased macroautophagic vacuoles in the tumor tissue.④Down-regulation of LAMP2A in MDA-MB-231cells significantly increased thelevels of LC3-Ⅱ after stimulated with0h,6h,12h and24h, which was positivelycorrelated with the starvation time. The same result was not found in MDA-MB-468cells.5. CMA promotes the proliferation and metastasis of breast cancer cells by inhibiting ATG5-mediated autophagy pathway①qRT-PCR assay proved that LAMP2A knockdown in MDA-MB-231cells increasedthe ATG5mRNA level.②Western blot assay showed that LAMP2A knockdown up-regulated the proteinlevels of ATG5and LC3-Ⅱ in MDA-MB-231cells, while it did not affect the expressionof the key proteins in mTORC1pathway, such as p-mTOR Ser2448, Rapter, p-P70S6KThr389and p-4EBP1Thr37/46, and the key proteins in mTORC2pathway, such asp-mTOR Ser2481, Rictor and p-Akt Ser473.③H2O2stimulation assay showed that down-regulation of macroautophagyactivity through knockdown of ATG5can reverse the inhibitory effect of LAMP2A RNAion MDA-MB-231cell growth and survival.④Transwell migration assay and wound-healing assay both confirmed that ATG5knockdown in MDA-MB-231cells reverse the inhibitory effect of LAMP2A RNAi onMDA-MB-231cell migration ability.⑤Immunohistochemistry assay showed that tumor tissue from the siLAMP2A miceshowed significantly increasing LC3-II and ATG5expression and decreased expression ofLAMP2A and HSC70.6. Inhibition of CMA can reduce the resistance of breast cancer cells to paclitaxelMTT assay showed that LAMP2A knockdown enhanced the sensitivity ofMDA-MB-231breast cancer cells to paclitaxel.Conclusion: CMA promotes the proliferation and metastasis of breast cancer cells.The underlying mechanism is that CMA could suppress ATG5macroautophagy signalingpathway. This project aims at improving the understanding of the correlation between theproliferation, metastasis and CMA of breast cancer cells in order to provide some newtargets for the diagnosis and treatment of breast cancer.