Gankyrin Regulates Autophagy And Its Function And Mechanism In The Progression Of Hepatocellular Carcinoma

Background andPurposeAutophagy is extra-conservative cellular function which delivers the cytoplasmicmaterials to lysosome for degradation. It could be divided into three types:macroautophagy, microautophagy and chaperone-mediated autophagy. Macroautophagy isthe major type, so it also called autophagy for short. The principal function of autophagy isgenerating the two membrane structures which sequestered intracellular proteins andcomponents to degrade and recycle these materials in response to environmental stress.This process contributes to basal cellular and tissue homeostasis; its dysfunction can affectpathogenesis, such as neural degenerative diseases, Virus infection, inflammatory boweldisease and cancer. Autophagy may exert a multifactorial influence on the initiation andprogression of cancer. Autophagy provides an anticarcinogenic function in primary cells byeliminating the aberrant protein aggregates and organelles for keeping the homeostasis. Inestablished tumors, however, autophagy may confer a survival advantage on tumor cellsthat are under metabolic stress, as a result of a high proliferation rate and that are underselective pressure from therapeutic intervention.Gankyrin is non-ATPase regulated subunits of26s proteasomes, it includes seven ankyrinrepeats. It is founded that gankyrin is high expression in liver cancer in2000. Gankyrin caninteract with MDM2for p53degradation, unite with CDK4to inhibit the phosphorylationof RB for accelerating cell cycle, cooperate with NF-kb/RelA to inhibit the transcriptionalfunction, improve the Ras-related lung cancer through RhoA/ROCK pathway, promote theinvasion and metastasis of HCC through up-regulation PI3K/AKT/HIF-1alpha signalingpathway, inhibit the degradation of OCT4by WWP2to activate and maintain the functionof tumour-initiating cells, up-regulate HNF-4alpha to affect the dedifferentiation of liver cancer. These results imply the irreplaceable function of gankyrin in the initiation andprogression process of hepatoma.Some research has reported that some oncogenes can affect autophagy to promote theprogression of cancer. Gankyrin can interact with many important molecules to improvethe cancer progression. However, it is not clear whether autophagy contributes to theoncogenic role of gankyrin, especially in nutrient deprivation and drug resistance.Our research focus on regulation of gankyrin in autophagy and its function in HCCprogression, provide scientific evidence for transforming the gankyrin-relatedachievements to clinical application.Method:1. Detect autophagic flux as evidenced by conversion from LC3-Ⅰ to LC3-Ⅱ anddegradation of p62with western blotting, GFP-LC3punctate distribution, withfluorescence microscope and the number of autophagsomes with transmission electronmicroscope in SMMC-7721gankyrin over-expression cells and knockdown of gankyrin inHCC-LM3cells.2. Generate gankyrin transgenic mouse, detect the LC3-Ⅱ formation in the livers ofstarved mice and liver cancers induced with DEN plus DCPOBOP-treatment.3. Check whether gankyrin regulate autophagy through p53.4. Detect the autophagy-related molecules which interact with gankyrin under EBSStreatment. Verify the interaction of gankyrin and Atg7with confocal microscopy. Test thebinding sites of gankyrin and Atg7with gankyrin and Atg7mutants.5. Detect whether gankyrin improved the expression of Atg7.6. Observe the Subcellular localization of gankyrin under EBSS treatment with nuclearplasma separation experiment and confocal microscopy.7. Research the mechanism of gankyrin in nucleus with Luciferase reporter andco-immunoprecipitation experiments.8. Union of nuclear plasma separation and co-immunoprecipitation experiments to studythe relationship between gankyrin and HSF1. 9. detect the expression of gankyrin and Atg7and the relationship in clinical liver cancersamples with immunohistochemistry and western blotting.10. Research whether gankyrin is resistant to sorafenib and whether it rely on its regulationon autophagy.Results:1. Gankyrin promote conversion from LC3-Ⅰ to LC3-Ⅱ, degradation of p62, GFP-LC3punctate distribution and the number of autophagsomes.2. Gankyrin up-regulate LC3-Ⅱ formation in the livers of starved mice and liver cancersinduced with DEN plus DCPOBOP-treatment.3. Gankyrin regulates autophagy independent of p53.4. Gankyrin cannot interact with Beclin1，Atg5，Atg12under EBSS treatment, however,bind to Atg7.5. Gankyrin interact Atg7with the last three ankyrin repeats or C-terminal tail domain.6. Gankyrin upregulates mRNA and protein expression of Atg7.7. Gankyrin interacts with HSF1at the promoter region of Atg7.8. A strong correlation between gankyrin and Atg7was observed in HCC patientspecimens.9. Gankyrin can inhibit the sorafenib-induced cell death, which depends on its regulationof autophagy.Conclusion:Our current study showed that gankyrin regulated Atg7to affect autophagy in two differentmanners. The direct association of gankyrin with Atg7in the cytoplasm occurred earlierupon starvation. Subsequently, gankyrin entered into the nucleus, and bound to Atg7promoter in coordination with HSF1, to transactivate Atg7, leading to sustained autophagy.Remarkably, combination of these two parameters increased prognostic value, stronglysuggesting that the concerted activities of gankyrin and Atg7detected in our study arerecapitulated in clinical patients with HCC. Gankyrin may be a potential molecularclassification of HCC patients who is suitable for sorafenib or chemotherapy in combination with autophagy inhibition.