JWA Deficiency Suppresses Dimethylbenz[a]Anthracene-phorbol Ester Induced Skin Papillomas Via Inactivation Of MAPK Pathway In Mice

Endogenous and exogenous sources cause oxidative stress induced DNA damage in living organisms by a variety of mechanisms. The resulting DNA lesions are mutagenic, unless repaired, lead to a variety of mutations and consequently to genome instability, which is a hallmark of cancer. Oxidative stress induced DNA damage is repaired in living cells by different pathways that involve a large number of proteins. Unrepaired and accumulated DNA lesions may lead to disease processes including carcinogenesis. While mutations or deletions occur in DNA repair genes, destabilizing the DNA repair system. In general, defects in DNA repair are associated with cancer.Mitogen-activated protein kinase (MAPK) cascades are key signaling pathways involved in the regulation of normal cell proliferation, survival and differentiation. Aberrant regulation of MAPK cascades contribute to cancer and other human diseases. In particular, the extracellular signal-regulated kinase (ERK) MAPK pathway has been the subject of intense research scrutiny leading to the development of pharmacologic inhibitors for the treatment of cancer. ERK is a downstream component of an evolutionarily conserved signaling module that is activated by the Raf serine/threonine kinases. Raf activates the MAPK/ERK kinase (MEK)1/2dual-specificity protein kinases, which then activate ERK1/2. The mutational activation of Raf in human cancers supports the important role of this pathway in human oncogenesis. Additionally, the Raf-MEK-ERK pathway is a key downstream effector of the Ras small GTPase, the most frequently mutated oncogene in human cancers. Thus, the Ras-Raf-MEK-ERK signaling network has been the subject of intense research and pharmaceutical scrutiny to identify novel target-based approaches for cancer treatment.JWA, a gene known for its role in environmental response, had been found to be upregulated under different stress conditions. Recently, it was found to be involved in oxidative stress induced DNA repair. After exposure to oxidative stress, JWA was translocated into the nucleus and co-localized with XRCC1and LIG3. In recent study, we found JWA was also involved in DNA double strand break (DSB) repair induced by VP16and CPT. On the other hand, JWA, a newly identified novel microtubule associated protein, was demonstrated to be indispensable for the rearrangement of actin cytoskeleton and activation of MAPK cascades. We further verified that JWA regulated melanoma metastasis via integrin signaling.Objective:To investigate the role and the underlying mechanisms of JWA as a DNA repair protein and the regulation of MAPK cascades in vivo and to provide the evidence of the significant role of JWA in skin carcinogenesis.Methods:Cre-Loxp mediated recombination was used to establish conditional conventional JWA knockout mice. PCR, RT-PCR and followed sequencing confirmation and immunoblotting were used to genotype the mice at the DNA, RNA and protein level, respectively. DMBA/TPA was administrated to induce the mice skin papillomas for investigating the effect of carcinogenesis in JWA knockout mice. Western blot and immunohistochemistry were used to detect the proliferation of mice papillomas and skin. We compared the expressions of protein of MAPK cascades in two genotypes mice. The primary keratinocytes was isolated to verify the result above; Real-time PCR, Western blot were used to identify the downstream transcriptional factor of MAKP pathway; the neutral comet assay was used to detect DNA damage and and immufluorescense assay was used to detect DNA repair protein translocations during the process of DNA damage and repair..Result:1. Conditional conventional JWA knockout mice were established by Cre-Loxp mediated recombination. PCR, RT-PCR, sequencing techniques and immunoblotting were confirmed that the exon2of JWA gene was completely deleted in JWAΔ2/Δ2mouse.2. JWA deficiency attenuates the development of mouse skin papillomas. Skin papilloma was induced by DMBA/TPA treatment in both JWA+/+and JWAΔ2/Δ2mouse skin. In the present study, the first papilloma was observed after8weeks of TPA treatment in JWA+/+mouse and appeared2weeks later in JWAΔ2/Δ2mouse than in JWA++mouse. The ratio of tumor induction in JWA+/+mice was significantly higher than in JWAΔ2/Δ2mice (P<0.001). There were significantly fewer number and smaller sizes of papillomas occurred in JWAΔ2/Δ2mice than in JWA+/+mice (P<0.05and P<0.01, respectively). Skin specimens with H&E staining confirmed that the papillomas were with no significant difference between both genotypes mice.3. JWA deficiency inhibits DMBA/TPA induced cell proliferation. Data showed the expressions of mRNA (P<0.01), protein, and the amount of PCNA-positive cells in papillomas and skin tissues were significantly higher in JWA+/+than in JWAΔ2/Δ2mice (P<0.05). Similar result obtained from the expression of Ki67in mouse papillomas.4. JWA deficiency blocks TPA-mediated phosphorylations of MAPKs. To determine whether JWA deficiency attenuated papilloma formation was due to inactivation of MAPKs in mice. Both papillomas and skin tissues nearby was extracted for Western blot analysis. As a result, compared to the JWA+/+mice, less activation of p-MEK and p-ERK in JWAΔ2/Δ2mice was found, although the total expressions of MEK and ERK were unaffected. Interestingly, both phosphorylation and expression of JNK and P38proteins were also unaffected in papillomas of both JWA+/+and JWAΔ2/Δ2mice. We got the similar results from primary keratinocytes isolated from both genotypes of JWA.5. JWA regulates transcription factor Elkl via MEK/ERK pathway. We investigated that if the role of JWA on PCNA was mediated by any transcription factors. As a result, compared to JWA+/+mice, expressions of Elk1at both mRNA (P <0.05) and protein levels were significantly down regulated in JWAΔ2/Δ2mouse papillomas and skin tissues. Similar results were obtained from keratinocytes and mouse embryonic fibroblasts.6. JWA deficiency enhances DMBA-induced DNA damage. The neutral comet assay used to detect DNA double strand breaks (DSBs) of keratinocytes induced by DMBA treatment. As a result, DMBA induced more DSBs in JWAΔ2/Δ2keratinocytes than in JWA+/+cells (P<0.01). Consistent with the result from neutral comet assay, JWAΔ2/Δ2keratinocytes had more y-H2AX positive foci than in JWA+/+cells after DMBA exposure (P<0.05).In summary, we demonstrate for the first time that JWA deficiency enhances DNA damage in epidermal cells induced by DMBA, however, suppresses TPA-induced MEK-ERK and transcriptional factor Elkl activation, cell proliferation, and formation of skin papillomas. These data has further illustrated the role of JWA in DNA damage repair and regulation of MAPK cascades.