The Function And Mechanism Of GSK3β On The Inhibition Of Autophagy In Cutaneous Squamous Cell Carcinoma

Squamous cell carcinoma (SCC), a kind of common malignant tumor indermatology, proliferates quickly and metastasizes easily in short period anddamages patients’ health severely. Many conventional treatments for SCC areonly directed to symptom and we do not have effective method for overcomingSCC as other malignant tumors. Because the pathogenesis of SCC is not clearand the harzard is serious, it has become a hot spot in order to explore novel,effective targets at home and abroad.Glycogen synthase kinase3β (GSK3β) is a highly conserved andmulti-faceted serine/threonine kinase commonly found in eukaryotic cells. Itparticipates in multiple cell signaling pathways and its functions involved inembryonic development, proliferation, differentiation, and stem cells survivalhave been well known. Recent studies have shown that GSK3β is a key regulator of cellular fate in complex signaling systems. Our previous studiesindicated that GSK3β played an important inhibitive role in the transformationof keratincytes from normal to carcinoma cells. However, the mechanism hasnot been very clear. Autophagy, an important protective and defensive process,is responsible for the degradation of damaged and dysfunctional cellularorganelles and aggregated protein. The autophagy is associated with manydiseases, such as immunity disorders, inflammation, cancer and so on. UV,especially UVB, is one of the major risk factors for SCC. Many papers reportedthat UV irradiation could induce autophagy, and lithium, a GSK3β inhibitor,can protect other cells from autophagy. Then what’s the effect of GSK3β onautophagy in the process of SCC and what’s the mechanism? In our study, wechoose SCC, precancerous lesion actinic keratosis and normal mousekeratinocytes as research objectives, and detected the expression andphosphorylation situation of GSK3β protein, then observed the effect ofdifferent pCDNA3/GSK3β plasmids on cell proliferation and investigated themechanism, in order to develop a new target for effective therapeuticintervention of SCC.Objectives:We choose SCC, precancerous lesion actinic keratosis and normal mousekeratinocytes as research objectives, and the followings are investigatedthrough tissues, cells and molecular level experiments:1. To observe the relationship between GSK3β and the initiation,progression of squamous cell carcinoma;2. To observe the role of GSK3β on cell proliferation under UVB-inducedcondition;3. To investigate the mechanism of biological function of GSK3β in UVB-induced JB6cells.Contents and Methods:1. Expression of GSK3β was examined by immunohistochemical techniqueand western blot in squamous cell carcinoma and precancerous lesionactinic keratosis cases to evaluate the relationship between GSK3β andsquamous cell carcinoma;2. Normal mouse epidermal cells, JB6, were transfected withpCDNA3/GSK3β-WT, pCDNA3/GSK3β-S9A and pCDNA3/GSK3β-K85Rby Lipofectin, the expression levels of V5protein were examined bywestern blot to identify the transfection efficiency;3. MTT assay was used to detect the effect of GSK3β and autophagy on thecell survival in UVB-induced JB6;4. Western blot and transient transfection with GFP-LC3were used to detectthe change of the level of autophagy, the effect and mechanism ofGSK3β on autophagy in UVB-induced JB6.Results:1. The level of GSK3β was lower in Squamous cell carcinoma tissues thanthat in normal skin tissues, and moreover the level was lowest in actinickeratosis. But the expression of p-GSK3βSer9was much higher in precancerand carcinoma than that in normal skin tissues and the expression in actinickeratosis was highest; The difference of the p-GSK3βTyr216expression wasno significant;2. On UVB irradiation, the activation of GSK3β (GSK3β-WT andGSK3β-S9A cell lines) decreased the proliferation, while the inactivation ofGSK3β (JB6pretreated with lithium and GSK3β-K85R cell lines) increasedthe proliferation under UVB-induced condition; 3. The inhibition of autophagy could increase the cell death, while theactivation of autophagy could decrease the death in UVB-induced JB6cells;4. After JB6cells were pretreated with lithium, the expression ofpAMPKThr172protein was positively associated with treating time and theexpressions of PKR, AKT and PERK protein were no different;5. Under the UVB irradiation, the expressions of autophagy-associated proteinLC3, Beclin1and p-GSK3βSer9protein had a positive correlation withtreating time, while the expression of p-GSK3βTyr216protein was negativelyassociated with treating time.Conclusion1. The activation of GSK3β can be inhibited in SCC, which indicates that theinhibition of GSK3β is closely correlated with the pathogenesis ofsquamous cell carcinoma;.2. To inhibit the activation of GSK3β can increase the cell survival, but toincrease the actication of GSK3β can inhibit the cell survival, whichindicates that GSK3β plays a negative regulative role in the proliferation ofcells;3. To inhibit the activation of GSK3β can increase the expression ofpAMPKThr172protein and autophagy, which shows that GSK3β could inhibitthe activation of LKB1-AMPK-mTOR pathway and decrease the level ofautophagy.