Cytoplasmic Expression Of Proliferating Cell Nuclear Antigen Regulates Autophagy Of Breast Cancer Cells

Proliferating cell nuclear antigen (PCNA) is a unique nuclear protein. In thenucleus, three PCNA molecules associate in a head-to-tail fashion to form a slidingring structure encircling the DNA double helix, where it recruits and binds to DNApolymerase and miscellaneous proteins and plays crucial roles in DNA synthesis,damage repair as well as cycle control. Thus, nuclear PCNA was regarded as cellproliferation index. High levels of PCNA expression was detected in various tumorcell nucleus regardless of the source, suggesting the uncontrollability of itsproliferation, which makes nuclear PCNA become the molecular marker for diagnosisand poor prognosis in tumors, as well as a new target for tumor therapy. However,some studies verified that PCNA is not only expressed in the nucleus but also incytoplasm, and interacted with a variety of proteins, which showed more extensivebiological activities. In tumor cells, tumor associated proteins bind to cytoplasmicPCNA, suggests that cytoplasmic PCNA was involved in the pathological process oftumor, but there is still a lack of evidence. The researchers used rapamycin andstarvation treated T cell acute lymphoblastic leukemia cell line and mouse embryonicfibroblast cell line, which can change the expression and subcellular localization ofPCNA. Rapamycin and starvation is the most common autophagy inducer, especiallyin tumor cells, so we speculate that PCNA may be associated with the cell autophagy.Autophagy, a long-life proteins degradation pathway in normal cells, has beendemonstrated to be involved in the process of tumor initiation and development.Autophagy inhibits proliferation of transformation cell, thereby promoting cellsenescence and inhibiting tumor generation. Once tumor formation, tumor cellsenhance autophagy in order to survive in metabolism and treatment stress. Autophagyis a protective mechanism to avoid cell injury. If the stress persists, autophagy is tooexcited, it may mediate tumor cells in non apoptotic cell death, at the same time,inflammatory cytokines are released, mobilizing the tumor associated immune activity. Autophagy has become the focus of cancer research for its complex role in cancerpathogenesis. As the second tumor threaten the world women’s health, breast cancerhas always been of great concern. In breast cancer recently, autophagy has beenconfirmed to be involved in tumorigenesis, progression and metastasis, treatmenttolerance, But the effect and regulation mechanism has not been fully elucidated. Itneeds more experiments to reveal the function and regulatory mechanism ofautophagy in breast cancer, in order to accurately regulate autophagy, therebypromoting tumor treatment.In tumor cells, autophagy can be regulated by a variety of proteins and signalingpathway, including autophagy related proteins, PI3K/Akt/mTOR signal pathway,AMPK pathway. Recent studies have shown that abnormal expression of microRNAand p53in a variety of tumors especially in breast cancer can change the autophagyactivity of tumor cells, suggesting that there may be more molecules or proteinsinvolved in the regulation of autophagy in cancer cells. Based on the previousresearch, we hypothesized that the expression of PCNA in the cytoplasm of breastcancer cells may be involved in regulation of autophagy.In our studies, we choose the human breast cancer SK-BR-3cell line and treat itwith the methods of inducing autophagy in order to confirm the subcellularlocalization of PCNA changes in tumor cells, and associates with autophagy,whereafter we regulate the expression of cytoplasmic PCNA by different means andverify the function of PCNA in regulating autophagy of breast cancer cell, which mayprovide a new research direction for accurate regulation of tumor autophagy.Methods:1. The subcellular localization of PCNA: Breast cancer SK-BR-3cell was treatedwith rapamycin in different time, then immunofluorescence staining was used todetermine the subcellular localization of PCNA, and Western blot assay detected theexpression of PCNA in the cytoplasm. Rapamycin also treated uterine cervix cancerHeLa cell line and lung cancer A549cell line, Western blot assay andimmunofluorescence detected PCNA expression in cytoplasm.2. Effects of rapamycin induced breast cancer SK-BR-3cell autophagy:Rapamycin treated cell for different time, and the cell autophagy was determined bysingle dansyl cadaverine (monodansylcadaverin, MDC) staining, and Beclin1 expression was determined by Western blot assay.3. The effects of other mothods induced autophagy on PCNA expression inbreast cancer cell line SK-BR-3: Starvation and hydrogen peroxide inducedautophagy in SK-BR-3cell, MDC straining detected cell autophagy, Western blotassay detected the level of PCNA and Beclin1protein expression in the cytoplasm.4. Effect of cytoplasmic PCNA on autophagy in breast cancer SK-BR-3cells:Nuclear transport inhibitor leptomycin B and RNAi were used to change theexpression and subcellular localization of PCNA in SK-BR-3cells, then the cellautophagy was determined by MDC staining, and Beclin1expression was determinedby Western blot assay.5. Effect of autophagy inhibition on PCNA expression in breast cancer SK-BR-3cells: SK-BR-3cells was treated with3-MA following by rapamycin, autophagy wasdetermined by MDC staining, Western blot assay detected PCNA and Beclin1expression in the cytoplasm.Results:1. With the prolonging time of rapamycin on SK-BR-3cells, the expression ofPCNA in the cytoplasm was firstly increased and then decreased. PCNA was detectedtranslocation from the nucleus to the cytoplasm in HeLa cell and A549cell whenrapamycin treated them.2. After rapamycin treated SK-BR-3cells, autophagy activity was increased atfirst and then reduced, the expression of autophagy related protein Beclin1alsoincreased and then decreased, the trend is consistent with the cytoplasmic PCNA.3. Starvation and oxidative stress treated SK-BR-3cells, cytoplasmic PCNA,autophagy and Beclin1expression showed a decrease after the first increase.4. Interference PCNA expression by RNAi or inhibition PCNA translocation tothe cytoplasm by Leptomycin B, autophagy induced by rapamycin was inhibited inSK-BR-3cells.5.3-MA pretreatment inhibited the level of autophagy in SK-BR-3cells inducedby rapamycin, but had no influence on the expression of PCNA in cytoplasm.Conclusion:The results of this study showed that PCNA may be located in the cytoplasm oftumor and various stress can change autophagy activity and PCNA localization inbreast cancer cell at the same time. The change of cytoplasmic PCNA regulates autophagy activity and the expression of beclin1in breast cancer cells, suggesting thatthe cytoplasmic expression of PCNA in breast cancer cells was autophagy regulater,and beclin1was the mediater. This study provided a new idea for the study of PCNAand autophagy effect and regulation mechanism in human breast cancer.