ADA-07 Suppresses Solar Ultraviolet-induced Skin Carcinogenesis By Directly Inhibiting TOPK

Skin cancer is one of the most frequently diagnosed malignancies in the United States,and its incidence has been increasing at an astonishing rate over the past few decades,exceeding the number of all other human cancers combined.Cumulative exposure to solar ultraviolet(SUV)irradiation is regarded as the major etiologic factor in the development of skin cancer.The SUV spectrum can be divided into 3 subtypes according to wavelength and include UVA(320-400 nm),UVB(280-320 nm)and UVC(200-280 nm).Although UVC is filtered out by stratospheric ozone,UVA and UVB each has strong carcinogenic effects on the skin,which can lead to DNA damage,erythema,sunburn,immunosuppression,and,eventually,skin cancer.Statistically,nearly 90% of NMSC is estimated to be caused by chronic exposure to SUV.The mitogen-activated protein kinases(MAPKs)are serine/threonine protein kinases that are strongly activated by SUV irradiation,and are essential in the regulation of fundamental cellular processes,such as proliferation,differentiation,and apoptosis.T-LAK cell-originated protein kinase(TOPK)is a member of the MAPKK family and is highly expressed in many cancers.TOPK is an upstream activator of ERKs,p38 and JNKs and has been identified as an oncogenic protein that is involved in various cellular functions,such as DNA damage,neoplastic transformation and inflammation.Moreover,an accumulation of data provides evidence that the inhibition of TOPK might be useful in cancer chemoprevention and treatment.However,very few effective TOPK inhibitors have been discovered.In the current study,ADA-07,a novel TOPK inhibitor,was synthesized and characterized.Pull-down assay,ATP competition and in vitro kinase assay data revealed that ADA-07 interacted with TOPK at the ATP-binding pocket and inhibited its kinase activity.Western blot analysis showed that ADA-07 suppressed SUV-induced phosphorylation of ERK1/2,p38,and JNKs,and subsequently inhibited AP-1 activity in skin cancer cells.Importantly,topical treatment with ADA-07 dramatically attenuated tumor incidence,multiplicity,and volume in SKH-1 hairless mice exposed to chronic SUV.Our findings suggest that ADA-07 is a promising chemopreventive or potential therapeutic agent against SUV-induced skin carcinogenesis that acts by specifically targeting TOPK.Chapter Ⅰ ADA-07 inhibits proliferation of NMSC cellsMethods 1.Identifying a novel compound that can prevent SUV-induced skin cancer by directly targeting TOPK,and use MTS assay to determine whether ADA-07 exerted any cytotoxic effects against nomal skin cells.2.The anchorage-independent growth assay were performed to assess the effect of ADA-07 on cell transformation by using EGF treated JB6 P+ cells.3.The crystal violet staining assay were performed to assess the effect of ADA-07 on cell proliferation by using NMSC cells.Results 1.ADA-07 has no cytotoxicity against normal skin cells ADA-07 was synthesized in-house by Dr.Kanamata Reddy.The MTS result showed that ADA-07 had no cytotoxicity at concentrations less than 10 μM.2.ADA-07 inhibits anchorage-independent growth of EGF treated JB6 P+ cells.Soft agar assay indicated that colony formation of JB6 P+ cells was attenuated after treatment with different concentrations of ADA-07.3.ADA-07 inhibits cell proliferation of human NMSC cells.The crystal violet staining assay results showed that ADA-07 strongly decreased SCC12,SCC13,and A431 cell proliferation in a dose-dependent manner.Chapter Ⅱ ADA-07 directly suppresses TOPK kinase activityMethods 1.Homology modeling and subsequent molecular docking were conducted to determine whether ADA-07 binds to TOPK.2.ATP competition assay with ADA-07-conjugated SepharoseTM 4B beads were conducted to futher determin the binding ability of ADA-07 with TOPK.3.Pull-down assay was performed using ADA-07-conjugated beads and Ha Ca T cell lysates.4.An in vitro TOPK or MEK1 kinase activity assays were conducted to determine the inhibiting efficiency of ADA-07.Results 1.ADA-07 binds TOPK at the ATP-binding pocket The binding model indicated that ADA-07 formed interactions within the ATPbinding pocket of TOPK and two potential hydrogen bonds were formed with the hinge residues Gly118 and Gly119 of TOPK and ADA-07.2.ADA-07 binds TOPK at the ATP-binding pocket,competing with ATP ATP competition assay showed that the binding ability of ADA-07 with TOPK was altered in the presence of ATP.3.ADA-07 binds to either TOPK or MEK1/2 ex vivo Pull-down assay indicated that both TOPK and MEK1/2 were detected in the ADA-07-conjugated beads group but not in the beads only group.4.ADA-07 inhibited TOPK kinase activity but not MEK1 activity An in vitro TOPK or MEK1 kinase activity assay were conducted with increasing concentrations of ADA-07.The results indicated that ADA-07 effectively inhibited TOPK kinase activity but not MEK1 activity.Chapter Ⅲ ADA-07 attenuates SUV-induced TOPK downstream signalingMethods 1.To examine the effect of ADA-07 on the SUV-induced TOPK signaling pathway in Ha Ca T and JB6 P+ cells by Western blot.2.To examine TOPK downstream signaling in SCC12 and A431 skin cancer cells after treatment with ADA-07 by Western blot.3.To examine phosphorylation or total c-Jun protein levels in Ha Ca T,JB6 P+,SCC12 and A431 cells by Western blot.4.To examine the effect of ADA-07 on SUV-induced transactivation of AP-1 by luciferase reporter assay.Also,the AP-1 activity was measured using the combination of ADA-07 and knockdown of TOPK.Results 1.Western blot results showed that the level of phosphorylation of ERK1/2,p38,and JNKs in Ha Ca T and JB6 P+ cells exposed to SUV(60 k J/m2)was markedly suppressed after treatment with different concentrations of ADA-07.2.Western blot results showed that the level of phosphorylation of ERK1/2,p38,and JNKs was blocked by ADA-07 in SCC12 and A431 cells dose-dependently.3.Western blot results showed that the level of phosphorylation of c-Jun in Ha Ca T and JB6 P+ cells exposed to SUV(60 k J/m2)was blocked by ADA-07,and the level of phosphorylation of c-Jun in SCC12 and A431 cells was markedly suppressed by ADA-07 in a dose-dependent manner.4.To examine the effect of ADA-07 on SUV-induced transactivation of AP-1,we exposed JB6 P+ cells stably transfected with an AP-1 luciferase reporter plasmid to ADA-07 and SUV.The data showed that ADA-07 inhibited SUV-induced transactivation of AP-1 dose-dependently.Moreover,we also measured the AP-1 activity using the combination of ADA-07 and knockdown of TOPK.The data indicated that the AP-1 activity was suppressed after knockdown of TOPK in A431 cell line.More specifically,in the control group,the AP-1 activity was inhibited by ADA-07 in a dose-dependent manner.However,in the TOPK knockdown group,the Ap-1 activity was only slightly affected by ADA-07 at the highest concentration(5 μM),which confirmed that ADA-07 suppresses AP-1 activity by directly targeting TOPK.Chapter Ⅳ ADA-07 suppresses SUV-induced skin carcinogenesis in SKH-1 hairless mice(early-stage prevention model)Methods 1.To establish a SUV-induced mouse skin tumorigenesis model(early-stage prevention mode),and examine the chemopreventive effect of ADA-07 in vivo.2.To evaluate the histopathological differences and PCNA levels between each group by using H&E staining and immunohistochemical.3.To analyse the effect of ADA-07 on mouse skin by western blot assay.Results 1.Topical application of ADA-07 on mouse dorsal skin resulted in a total inhibition of SUV-induced papilloma formation in the early-stage prevention group.2.H&E Staining indicated that,in the vehicle groups,atypia or dysplasia of the keratinocytes can be seen in the basal layers of the epidermis,and most of them accompanied by parakeratosis.Some slides shows that irregular sheets or islands of atypical squamous cells invade the dermis,and Keratin pearls also can be seen clearly.However,only SUV-induced epidermal thickness can be seen in the ADA-07-treated groups.In addition,IHC data showed that the expression of PCNA was decreased in the ADA-07-treated groups.In a word,ADA-07 dramatically decreased epidermal thickness and inhibited the formation of SCCs.3.Western blot analysis of mouse skin showed that phosphorylation of ERK1/2,p38,and JNKs induced by SUV was dramatically suppressed in the ADA-07-treated groups.Chapter Ⅴ ADA-07 suppresses SUV-induced skin carcinogenesis in SKH-1 hairless mice(late-stage prevention model)Methods 1.To establish a SUV-induced mouse skin tumorigenesis model(late-stage prevention mode),and examine the chemopreventive effect of ADA-07 in vivo.2.To evaluate the histopathological differences and PCNA levels between each group by using H&E staining and immunohistochemical.3.To analyse the effect of ADA-07 on mouse skin by western blot assayResults 1.The late-stage prevention model indicated that compared with the vehicle-treated group,although some papilloma were observed in the late-stage prevention study at 28 weeks after SUV exposure,results indicated that topical treatment with 0.1 or 1 mg ADA-07 significantly reduced tumor volume and multiplicity.In short,a substantial suppression of SUV-induced tumor incidence in the late-stage prevention group.2.H&E Staining results indicated that both AKs and SCCs were induced by SUV exposure in the late-stage prevention model.However,treatment with 0.1 or 1 mg ADA-07 dramatically decreased epidermal thickness and inhibited the formation of SCCs.In addition,IHC data showed that the expression of PCNA was decreased in the ADA-07-treated groups.3.Western blot analysis of mouse skin showed that phosphorylation of ERK1/2,p38,and JNKs induced by SUV was dramatically suppressed in the ADA-07-treated groups.Conclusions 1.The novel compound,ADA-07,is a potent TOPK inhibitor that effectively suppresses SUV-induced activation of MAPKs signal transduction resulting in reduced SUV-induced skin carcinogenesis.2.Pull-down assay,ATP competition and in vitro kinase assay data revealed that ADA-07 interacted with TOPK at the ATP-binding pocket and inhibited its kinase activity,thereby suppressing ERK1/2,p38 and JNKs activation.3.SUV-induced mouse skin tumorigenesis model,including early-stage prevention model and late-stage prevention model was established succesfully.The data support the idea that cumulative length of SUV exposure is a risk factor for SUVinduced NMSC and highlights the fact that ADA-07 might ultimately decrease skin carcinogenesis through its direct inhibition of TOPK.Therefore,ADA-07 is a promising chemopreventive and potential therapeutic agent that might be applied to human skin malignancies.