Study On The Mechanism Of TGF-?1/Smad And PIKKs/Akt Signaling Pathway In UV-Induced Progression Of Actinic Keratosis To Squamous Cell Carcinoma

Part 1.Ultravoilet-induced SKH-1 mouse model of actinic keratosis and squamous cell carcinomaObjective The PIKKs/Akt signaling pathway may play an important role in the pathogenesis of UV-induced actinic keratosis(AK)and the progression of AK to squamous cell carcinoma(SCC).This study was to investigate the activation of DNA-PKcs in PIKKs/Akt signaling pathway in Ultravoilet(UV)-induced AK and SCC,as well as the progression between them.Methods 1.43 SKH-1 hairless mice were randomly divided into experimental group 1(26),experimental group 2(7)and control group(10).The experimental groups used the SUV-1000 solar simulator to simulate daylight-irradiated SKH-1 hairless mouse,and in the 1st to 2 weeks the UVA and UVB mixed sub-erythemal dose(90%MED)irradiation was used.As the experiment progressed,mouse skin gradually thicken and the tolerance to ultraviolet rays increased,and the dose of ultraviolet radiation was also continuously adjusted.The UW dose increased to 1 MED at the third week and then increased by 12.5%MED every week,and then increased to 260 mJ/cm2 at the eighth week.After that,the dose was maintained.The control group did not do any treatment.The experimental group 1 maintained the irradiation for 24 weeks,and the experimental group 2 maintained the irradiation for 16 weeks.The mice were sacrificed at the 12th,18th,24th,and 28th weeks for pathological examination.2.Mice were tested for skin physiological functions at weeks 2,6,12,16,20,24,and 28.3.At the 28th week,different skin lesions on the back of the same mouse were taken for histopathological examination and immunohistochemical detection.Results 1.After 12 weeks,mice skin in the experimental group were thickened on the dorsum of their backs,deepening of the dermis and raised in the dorsal skin of the mice,and their skin elasticity decreased.From the 17th week onwards,pimples with diameter?1 mm gradually appeared on the back skin of experimental group 1 and experimental group 2 mice,and then the number and volume of pimples on the back of both groups increased.After 28 weeks irradiation,tumor formation rate was 100%.There was no tumor formation in the control mice.Histopathology showed:The skin of mice in the experimental group showed epidermal hyperkeratosis,acanthosis,and no atypical cells at the 12th week.After 18 weeks,the skin showed acanthosis,and the cells under the epidermis were slightly atypical,and these manifestations have the pathological features of AK;At 24 weeks,the spinous layer was markedly hypertrophic and the epidermic cells were moderately-atypia,and some of the skin showed full-thickness carcinoma in situ;After 28 weeks,100%of the skia lesions in mice showed subepithelial cell atypia,nucleus gathered in heaps,acanthosis,atypical hyperplasia with SCC;SCC models were observed in both experimental group 1 and group 2 at 28 weeks.Mice in the experimental group 1 had more back rashes and decreased elasticity;2.The skin physiological function test showed that compared with the control group,the PH and erythema levels of the skin in the UV irradiation group were different at the second week.Skin water content in the stratum corneum,transdermal water loss,and skin melanin content differ in the 12th week of UV radiation;3.In the process of UV-induced SCC,there were photoaging,AK,SCC in situ and invasive SCC.DNA-PKcs and p-DNA-PKcs(T2609)were localized in cytoplasm and nucleus,and DNA-PKcs was upregulated in epidermis,while there was no significant difference of p-DNA-PKcs(T2609).Conclusions 1.Successful preparation of mouse AK-like and SCC models;2.Elucidation of UV-induced SCC development is a gradual process associated with the cumulative effect of UV doses;3.It was clarified that during the process of UV-irradiation SKH-1 hairless mice constructing a skin SCC model,there are processes such as skin barrier damage,DNA damage and repair.Part 2.Bioinformatics analysis and skin tissue level verificationObjective TGF-pl/Smad signaling pathway inhibits tumor cell growth in early stage,but promotes tumor invasion and metastasis in advanced tumors,and its mechanism in human skin AK and SCC has not been reported.PIKKs/Akt signaling pathway plays an important role in tumorigenesis,but no studies have been conducted on the role of AK and SCC.This study investigated the mechanism of TGF-?1/Smad and PIKKs/Akt signaling pathway in AK and SCC at the tissue level,and clarified the mechanism of the occurrence and transformation of AK and SCC.Methods 1.We used the websites of http://scangeo.dartmouth.edu/ScanGEO/and http://www.cbioportal.org/to analyze the expression microarray-based sequencing articles and cancer genomics to find differences of TGF-?1/Smad and PIKKs/Akt signaling pathway in expression profile.2.Normal non-exposed skin,normal exposed skin,AK and SCC lesions were collected,and Western blotting was performed to detect the expression of key proteins in the two signal pathway in the four groups of skin tissue samples.Results Compared with normal skin and AK skin tissue,bioinformatics analysis of normal skin,AK,and SCC tissue microarray sequencing showed that Smad3 and TGF-?1 expression increased in SCC,and TGF-?RI expression was downregulated.In AK and SCC tissues,DNA-PKcs are elevated and mTOR is downregulated.Western blotting of skin tissues showed that the overall differences within the group were statistically significant:TGF-?RI,p-Smad3(S423/425);in the intragroup comparison,there were statistical differences:p-Smad3(S423/425)and Smad4 were positively correlated,and TGF-?1 and Smad4 were related.The expression of TGF-?R? in the normal exposed skin,AK and SCC at the exposure site was decreased compared with that in the non-exposed skin.The expression of DNA-PKcs and p-DNA PKcs(T2609)-H2AX(S139)was positively correlated.Conclusions There is a difference in the expression of TGF-?1/Smad and PIKKs/Akt signaling pathway between AK and SCC,suggesting that these two signaling pathway are involved in the occurrence and development of cutaneous SCC.p-H2AX(S139)(y-H2AX)can reflect p-DNA PKcs(T2609)level and DNA damage repair.Part 3.Study on cell biological and molecular changes of different keratinocytes affected by inhibitors of TGF-?1/Smad and PIKKs/Akt signal pathwayObjective A previous study found differences in the expression of TGF-pl/Smad and PIKKs/Akt signaling pathway in AK and SCC tissues,suggesting that these two signaling pathway are involved in the occurrence and development of cutaneous SCC.This study investigated the mechanism of TGF-?l/Smad and PIKKs/Akt signaling pathway in different keratinocytes and clarified the mechanism of UV-induced progression of AK-to-SCC.Methods We cultured HaCaT,UV-HaCaT and SCL-1 cells,respectively.The inhibitors of TGF-?R I/?-LY2109761,Smad3-SIS3,DNA-PK-NU7026,Akt-Perifosine were selected to intervene PIKKs/Akt and TGF-?1/Smad signal pathway in three kinds of keratinocytes.1.After inhibitors-treated for 48h,cell viability,proliferation,cell tumorigenicity,and apoptosis were measured by CCK-8 assay,EdU assay,colony formation assay,and flow cytometry.2.Western blotting was used to detect the expression of related proteins and its phosphorylation levels after interference with cell signaling pathway.Results HaCaT,UV-HaCaT and SCL-1 cells were treated with NU7026,Perifosine,LY2109761 and SIS3,respectively:1.The results of CCK-8 showed that the cell viability decreased with the increase of the action concentration,and in a concentration-dependent manner.2.The cells were stained by HE staining and observed under a microscope.The cells in the control group were flat polygonal,homogeneous and highly refractive,in good growth state and intercalated with each other.After the cells were treated by inhibitors,the cells were shrunken and rounded,cell gap widened and scattered;the number of clonal cells was significantly reduced.3.EdU results:NU7026,Perifosine and SIS3 had inhibitory effects on the proliferation of three types of cells.UV-activated HaCaT cells promoted cell proliferation,while LY2109761 had an inhibitory effect on proliferation of UV-HaCaT cells,but had no significant effect on HaCaT and SCL-1.4.Flow cytometry results showed that Perifosine could inhibit the late apoptosis of HaCaT cells;LY2109761 had no obvious effect on the early apoptosis of HaCaT cells,but inhibited the late apoptosis of HaCaT cells.In addition,the rest of inhibitors promoted apoptosis,and UV inhibited HaCaT cells apoptosis.5.Western blotting analysis of the interaction of TGF-?1/Smad with PIKKs/Akt signaling pathway revealed that NU7026 inhibited the phosphorylation of Akt in SCL-1 cells,the phosphorylation of Smad2 in UV-HaCaT cells was decreased,and the phosphorylation of Smad3 in HaCaT cells was increased.Perifosine had no obvious effect on y-H2AX in three types of cells,while the expression of Smad2 in HaCaT cells was decreased,and Smad2 and phosphorylated Smad2 were decreased in UV-HaCaT cells,and Smad2 was also down-regulated in SCL-1 cells.After LY2109761 treatment,Akt expression in UV-HaCaT cells was down-regulated,and Akt phosphorylation was inhibited in SCL-1 cells,while y,H2AX increased in SCL-1 expression.Although SIS3 didn't affect the phosphorylation of Akt,it promoted the expression of total Akt.In UV-HaCaT and SCL-1 cells,the level of p-S6(S235/236)decreased.Conclusions This study demonstrated that PIKKs/Akt signaling pathway plays a cancer-promoting role in UV-induced AK and SCC development while TGF-?1/Smad signaling pathway has different effect on the cellular biology of keratinocytes at different stages:inhibits cell growth of normal keratinocytes and plays a cancer promoting role in tumor cells.The interaction between TGF-?1/Smad and PIKKs/Akt signaling pathway was clarified.TGF-?1/Smad inhibited the tumorigenesis activity of PIKKs/Akt in normal cells,thereby inhibiting malignant transformation;PIKKs/Akt was activated in tumor cells.The cancer-promoting activity promotes the proliferation and activity of cancer cells.DNA-PKcs/Akt inhibits TGF-?1/Smad in normal cells and plays a tumor suppressive role.In tumor cells,it promotes the activation of TGF-?1/Smad signaling pathway,thus promoting the further development of cancer cells.Part 4.Studies on the mechanism of TGF-?1/Smad and PIKKs/Akt signaling pathway on the infiltration and metastasis of SCCObjective EMT can increase the invasion and metastasis of cancer cells and play an important role in the occurrence and development of SCC.DNA-PKcs is involved in the development and progression of multiple malignant tumor.This study wants to investigate the mechanism of DNA-PKcs in the infiltration and metastasis of SCC,and whether TGF-?1/Smad and PIKKs/Akt signaling pathway are involved in the EMT process of SCC.Methods In this study,we used cell biology methods-scratch wound healing assay and Transwell invasion and migration assays to detect the effect of DNA-PKcs and TGF-?1/Smad signaling pathway on cell invasion and migration.RT-PCR was used to explore EMT related gene expression level,immunofluorescence and Western blotting were used to study the EMT and TGF-?1/Smad and PIKKs/Akt signaling pathway related protein expression,which demonstrates the mechanism of invasion and metastasis in SCC.To explore the possible mechanism of TGF-?1/Smad and PIKKs/Akt signaling pathway in SCC tumor cell infiltration and metastasis.Results The expression level of DNA-PKcs was significantly upregulated in human SCC tissues and SCL-1 cells.The DNA-PK inhibitor NU7026 and transfected with siRNA were used to realize the interference of DNA-PKcs,and the scratch wound healing assay,Transwell invasion and migration assays indicated that the ability of cell invasion and migration was decreased.Meanwhile,EMT-related proteins were detected,the expression level of E-cadherin was increased,while Vimentin was decreased.TGF-?1 can induce EMT of SCL-1 cells,promote the invasion and metastasis of SCC,phosphorylate DNA-PKcs and activate it.In addition,Western blotting showed that TGF-?1 promotes the phosphorylation of Smad2/3,while interfering DNA-PKcs can downregulate the p-Smad3 in SCL-1 cells.Interestingly,simultaneously inhibiting DNA-PKcs and TGF-?1/Smad pathway can significantly inhibit cell invasion and migration,while E-cadherin expression was decreased.Conclusion DNA-PKcs can promote the EMT and induce migration and invasion of SCC cells through the TGF-(31/Smad pathway.The interaction between TGF-?1/Smad and the PIKKs/Akt signaling pathway is involved in the UV-induced transformation from AK to SCC.In addition,besides TGF-?1/Smad pathway,DNA-PKcs may participate in other cellular signaling pathway to regulate EMT.