Autophagic Degradation Of Oct4 Promotes Differentiation Of Cancer Stem Cells And It’s The Molecular Mechanism

Background:Oct4 is a homeodomain transcription factor of the POU family, which is critically involved in the multipotency and self-renewal of undifferentiated embryonic stem cells, germline stem cell and embryonic carcinoma. In addition, Oct4 is associated with reprogramming and maintaining a stem cell “stemness”. The research has been reported that Oct4 is degraded by UPS(ubiquitin-proteasome system), and WWP2 can promote degradation of Oct4. According to the recent reported paper, however, autophagy can regulate homeostasis of pluripotency-associated proteins(Oct4, Sox2, Klf4, c-Myc) in h ESCs, but its underlying molecular mechanism has not been elucidated.Autophagy is a highly conserved intracellular pathway that mediates the degradation of protein and organelles in lysosomes. There are three pathways of autophagy: Microautophagy, Microautophagy and Chaperone-mediated autophagy(CMA). In CMA, the heat shock cognate protein of 70 k Da(hsc70) recognizes proteins with a pentapeptide motif and(KRFERQ) delivers them to the lysosome surface for binding to lysosome-associated membrane protein 2A(LAMP-2a). But more importantly, whether Oct4 can be degraded by CMA have not been reported.Some researchs are reported that Oct4 can be phosphorylated by AKT and ERK to regulate pluripotent of stem cells and promote its self-renewal capacity, which make it potential that Oct4 can have epigenetic modifications. Thus, Oct4 can be degraded by phosphorylated modification there is a theoretical basis that Oct4 can be degraded by autophagic degradation with phosphorylated modification.Objective:This study focus on the ideas that whether Oct4 can be degraded by CMA, and that CMA targeting Oct4 promotes differentiation of cancer stem cells and it’s the molecular mechanism.Methods:FACS(Fluorescence Activated Cell Sorter) was used to sort SP cells. Western blot analysis was performed to detect the protein levels of Oct4(“stemness” Marker) from SP cells and n SP cells. q RT-PCR analysis was used to detect the m RNA level of Oct4. Western blot analysis of Oct4 in CNE-2-S18 cells by knockdown of LAMP2 A, ERK and Hsc70. Western blot analysis of Oct4 in CNE-2-S18 by Nuclear/Cytosol Fractionntion Kit. Stability analysis of Oct4 in CNE-2-S18 by CHX test. Co-immunoprecipitation and Immunofluorescence were used to determine interaction of Oct4, Hsc70 and LAMP1. Sequence alignment and crystal structure analysis of Oct4 protein by Vector NTI and 3D-Mol software. Interaction of WT-Oct4-SQ and MT-Oct4-AA was detected by site-directed mutagenesis experiment.Results:1. Oct4 is high level expression of cancer stem-like SP cells in CNE-2-S18 cellsUsing FACS assay, we found that the percentage of cancer stem-like SP cells was high in CNE-2-S18, and that the expression of Oct4 in SP cells was higher than in n SP cells, which would imply that there are a large number of cancer stem cells in CNE-2-S18 cells.2. Blockage of lysosomal degradation leads to accumulation of Oct4 protein.The accumulation of Oct4 depends on the lysosomal degradation pathway in CNE-2-S18 untreated(CTRL) or treated with Bafilomycin A1(Baf A1),3-methyladenine(3MA),leupetin(Leu) or MG132. But this accumulation did not depend on levels of m RNA.3. Blockage of CMA flux increases accumulation of Oct4 protein.Due to previous experiment that macroautophagy inhibitor 3-MA did not cause the significant accumulation of Oct4 protein, So we speculated that the accumulation of Oct4 protein was likely to be another selective autophagy-CMA. What’s more, we found that the accumulation of Oct4 was significant by knockdown of LAMP2 A and Hsc70, which speculated that blockage of CMA flux could increase accumulation of Oct4 protein.4. Blockage of CMA flux promotes Oct4 protein nuclear- cytoplasm translocation and interaction with Hsc70.Because Oct4 protein executed biological functions in the nucleus, we had a hypothesis that Oct4 might occur nuclear- cytoplasm translocation in CMA. We found that the accumulation of Oct4 was significant in cytoplasm in CNE-2-S18 treated with lysosome blockers(Baf A1,CQ,Leu) by Nuclear/Cytosol Fractionntion Kit. In addition, long-time starvation would increase the instability of Oct4 protein in CNE-2-S18 by CHX test, and this instability was likely to result from degradation of Oct4 by CMA.5. KVFSQ motif from Oct4 is potential target sequence of CMA.Oct4 protein have a potential KFERQ motif by bioinformatics analysis. We found that the KVFSQ pentapeptide motif was relatively conservative in different species and POU family, especially FSQ sequence in a row. KVFSQ lied between alpha helix and beta fold, which made it easy to change protein spatial structure by analysis of crystal band model. Meanwhile, KVFSQ was located in the internal space as a whole by analysis of protein crystal ball model. Also we found that Ser180 from Oct4 might be phosphorylated to simulate KFERQ motif by software prediction.6. ERK regulates in degradation of Oct4 by CMA.We found that MT-Oct4(KVFAA) could not interact with Hsc70, and proved that KVFSQ was necessary recognition motif by Hsc70. ERK as an regulator degradation of Oct4 by CMA by kinase inhibitor screening test and si RNA test.7. Degradation of Oct4 protein by CMA promotes the differentiation of CSC.We found that the CMA could promote translation from SP cells into n SP cells with long-time starvation. However, the blockage of CMA could promote translation from n SP cells into SP cells. As a result, a hypothesis was put forward that Oct4 protein was through CMA to regulate the differentiation of cancer stem cells.Conclusions:1. CMA targets degradation of Oct4 under the condition of long-time starvation, which can regulatethe quality control of Oct4.2. KVFSQ from Oct4 can be phosphorylated by ERK as an activator degradation of Oct4 by CMA to simulate KFERQ motif by software prediction and necessary recognition motif by Hsc70.3. Degradation of Oct4 protein by CMA promotes the differentiation of CSC.