The Mechanisms Of ATF3Promoting Skin Cancer Cell Proliferation

Background:Skin cancer or cutaneous carcinoma is the most common and life-risky type ofcarcinoma, categorized as melanoma and non-melanoma skin carcer (squamous cellcarcinoma (SCC) and basal cell carcinoma (BCC) are the two most common subtypes),affecting millions worldwide. The incidence of skin cancer is increasing yearly, making it apre-eminent public health threat, and identification of risk factors is needed to stop thisincreasing trend. Multiple risk factors exist, including endogenous factors (genetic factorsand gene mutations) and exogenous factors (sun exposure, chemical carcinogens and otherenvironmental stress). Early diagnosis and treatment are needed to decrease the number ofdeaths and disfigurations due to skin cancer. Whereas, the precise molecular mechanism ofskin cancer remains unclear, which make it difficult to treat and prevent of the disease.Activating transcription factor3(ATF3) is an ATF/CREB family member, whichcontains the basic region and leucine zipper (bZIP) motif. ATF3is expressed at low levels innormal cells but can be rapidly induced by multiple and diverse extracellular signalsincluding growth factors, cytokines and some genotoxic stress agents. The physiologicalfunction of ATF3has been addressed in several cell lines indicated that ATF3might beinvolved in homeostasis, wound healing, cell adhesion, cancer cell invasion, apoptosis andsignaling pathways. Emerging evidence suggests that ATF3may play an critical role in hostdefence by regulating the delicate balance between proliferative and apoptotic signals thatcontribute to the development of cancer. Interestingly, ATF3has been demonstrated to playdifferent roles in cancer development depending on the cell type and context. For example,over-expression of ATF3protein moderately suppresses cell growth through slowing downprogression from G1/S transition in Hela cells. Conversely, ATF3promotes growthfactor-independent proliferation in chick embryo and enhance serum-induced cellproliferation in rat fibroblasts. Although ATF3has been reported to promote skin tumor formation through suppression of p53-dependent senescence, the precise role of ATF3andthe underlying molecular mechanism in skin cancer remain unclear. Stat3is a member ofSTAT family which may be activated by many extracellular signals. In accordance with itscritical roles in the regulation of cellular processes associated with carcinogenesis, Stat3isconstitutively activated in many epithelial cancers including head and neck, breast, lung,skin, and prostate. Nonetheless there is little known about the relationship between ATF3,p53and Stat3signaling in the skin carcinogenesis.Epigenetic changes is another important mechanism of tumorigenesis, includingderegulated histone acetylation patterns. Histone deacetylase inhibitors (HDACi) which canblock the activity of histone deacetylase (HDAC) have been developed and used for avariety of targeted tumor therapy. HDACi such as trichostatin A (TSA) and butyrate canalter the normal chromatin structure, leading to changes in gene expression and regulation.It has been reported that TSA can cause tumor cell growth inhibition and increasedapoptotic sensitivity of cancer cells, meanwhile as reported, ATF3may interact physicallywith HDACs, but the role of ATF3in TSA-induced inhibition of tumor cell growth,especially the interaction between ATF3and p21, the key target gene of TSA remainsunclear.Aims:First, to confirm the role of ATF3in skin carcinogenesis by detecting its expression inskin cancer tissue, meanwhile find out the relationship between the ATF3protein levels andthe proliferation activity of the cells drived from skin cancer tissue and the controlrespectively; then clarify the interactions between ATF3and p53-Stat3cascade in SCC-13and RTS3b (p53null) cells; determine the role of ATF3in TSA-induced inhibition of tumorcell growth, especially the interaction between ATF3and p21, the key target gene of TSA,finally find out the potential mechanism of ATF3promoting skin cancer development.Methods:1.The expression of ATF3in skin cancer tissue and the protein level of ATF3, Stat3andp53in different cell lines.A total of30surgically resected examples of skin cancer were enrolled, thirty skinspecimens from healthy donors were collected as normal control. The ATF3expression ofall the samples were detected by quantitative real-time PCR (qPCR). Then the3pieces of skin cancer tissues with highest ATF3(C1, C2, C3) and the3pieces of normal skin tissueswith lowest ATF3(N1, N2, N3) were primarily cultured and established as cell lines. Thenthe transcriptional expression of the ATF3and the protein level of ATF3, Stat3and p53inthe different cell lines, as well as in the human squamous carcinoma SCC-13cells andp53-null RTS3b cells, were analyzed by qPCR and Western blot analysis.2. Transcriptional activity of Stat3in SCC-13and p53-null RTS3b cells.The luciferase reporter construct was co-transfected with pRL-TK and over expressingplasmids or vectors into SCC-13or RTS3b cells. After transfection for48h, cells wereharvested for the luciferase reporter assay.3. Influence of ATF3, Stat3and p53, as well as their interaction on the proliferation ofthe diffenent Cell lines.SCC-13or RTS3b cells were transfected with control shRNA plasmids, ATF3shRNAplasmids, pcDNA3vector, pcDNA3-p53, pcDNA3-Stat3C and/or pcDNA3-ATF3plasmidsby using Lipofectamine2000. Otherwise the cell lines were treated with Stat3inhibitorStattic. Then the cell proliferation activity of SCC-13cells and RTS3b cells was determinedby MTT at indicated time points after transfection.4. ATF3interferes activation of p21in HDACi-induced growth inhibition ofepidermoid carcinoma cells.Based on epidermoid carcinoma cell A431, joint use of cell culture，cell transformation,qPCR and WB techniques, the relationship between ATF3that can promote tumor cellgrowth and TSA with the opposite effect was analyzed, meanwhile the interaction betweenATF3and the important target gene p21of TSA in the process of growth inhibition inducedby TSA in epidermoid carcinoma was investigated.Results:1. ATF3was significantly upregulated in skin cancer tissue, while it was maintainedat low level in normal epitheliums；the primary cultured skin cancer cells possessed higherATF3protein level, which was consistent with transcriptional expression differencebetween skin cancer and normal tissues.2. ATF3promotes skin cancer cell proliferation. Among the6lines of primarycultured skin cells, the cells from skin cancer (C1-C3) proliferated much faster than thatfrom the normal skin. The proliferation rate of SCC-13cells was suppressed by ATF3 knockdown (KD); conversely, forced expression of ATF3in SCC-13cells significantlypromoted cell growth; these results demonstrated that ATF3is a positive regulator of skincancer cell proliferation.3. Phosphorylated Stat3level was much higher in skin cancer cells with higher ATF3expression. The results of Luciferase reporter assay combined with cell transfectionrevealed that ATF3was required for Stat3phosphorylation, ATF3overexpressionupregulated Stat3phosphorylation level, furthermore, ATF3positively regulated Stat3transcriptional activity. Meanwhile p53showed lower mRNA level in established skincancer cell lines with higher ATF3expression. Correspondingly, ATF3knockdownupregulated p53expression and ATF3overexpression strongly inhibited p53transcription.4. The constitutive active Stat3form, Stat3C overexpression significantly promotedskin cancer cell growth. Conversely, the Stat3inhibitor Stattic inhibited cell proliferation ofSCC-13cells and the promotion effect of ATF3was prevented by Stattic. Moreover, theproliferation rate of skin cancer cells was suppressed by p53overexpression in SCC-13cells. Whereas, ATF3had no significant effects on the cell proliferation of p53-null RTS3bcells. Collectively, these data demonstrate that ATF3might act trough modulating p53-Stat3signaling cascade to enhance skin cancer cell proliferation.5. We also investigate the potential function of ATF3in promoting cancer cell growthfrom the perspective of epigenetic, the results suggest that ATF3may interfere withTSA-induced epidermoid cancer cell growth inhibition by inhibiting p21expression.Conclusion:In summary, ATF3was upregulated in skin cancer tissues and promoted theproliferation of skin cancer cells. The molecular mechanism of ATF3in skin carcinogenesiswas investigated and we found that ATF3enhanced skin cancer cell growth throughmodulating p53-Stat3signaling activity. This study discovered the functions of ATF3-p53-Stat3signal cascade in skin cancer development and will assist to understand the pathologicmechanism of skin carcinogenesis. ATF3might increase the risk of skin cancer and serve asan important prognostic indicator for the disease，and the ATF3-p53-Stat3signaling maybe apotential target for skin cancer prevention. Furthermore, ATF3promote skin cancer cellproliferation may also have a large number of other unknown mechanisms, as shown inepigenetics, ATF3may interfere with TSA-induced epidermoid cancer cell growthinhibition by inhibiting p21expression.