SOX2, a member of the SRY-related HMG-box (SOX) family, is abnormally expressed in many tumors and associated with cancer stem cell-like properties. Previous reports have shown that SOX2 is a biomarker for cancer stem cells in human bladder cancer (BC), and our most recent study has indicated that the inhibition of SOX2 by anticancer compound ChlA-F attenuates human BC cell invasion. We now investigated the mechanisms through which SOX2 promotes the invasive ability of BC cells. Our studies revealed that SOX2 promoted SKP2 transcription and increased SKP2-accelerated Sp1 protein degradation. As Sp1 is a transcriptionally regulated gene, HUR transcription was thereby attenuated, and, in the absence of HUR, FOXO1 mRNA was degraded fast, which promoted BC cell invasion. In addition, SOX2 promoted BC invasion through the upregulation of nucleolin transcription, which resulted in increased MMP2 mRNA stability and expression. Collectively, our findings show that SOX2 promotes BC invasion through both SKP2-Sp1-HUR-FOXO1 and nucleolin-MMP2 dual axes.
Abstract Fas (CD95)-mediated apoptosis is an essential mechanism for the maintenance of homeostasis, and disruption of this death pathway contributes to many human diseases. The cell survival protein kinase Akt/protein kinase B (PKB) is a known regulator of apoptosis, but its role in Fas-mediated cell death and its regulatory mechanisms are unclear. In this study, we show that stimulation of the Fas receptor by its ligand (FasL) induces rapid phosphorylation of Akt/PKB and a parallel increase in cell apoptosis in epidermal Cl41 cells. Inhibition of PI3K/Akt by dominant-negative overexpression of PI3K (Δp85) and Akt (Akt-T308A/S473A) protects the cells from apoptosis, indicating an unexpected proapoptotic role of PI3K/Akt in the Fas signaling process. Treatment of the cells with pharmacological inhibitors of PI3K, wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-1 (LY294002), similarly inhibits FasL-induced apoptosis and Akt/PKB phosphorylation, indicating that PI3K is an upstream mediator of Akt/PKB and is involved in Fas-mediated cell death. Electron spin resonance studies show that FasL treatment induces rapid generation of reactive oxygen species, and inhibition of ROS by antioxidants effectively inhibits Akt/PKB signaling, suggesting that FasL activation of Akt/PKB is redox sensitive. In cells transfected with dominant-negative PI3K/Akt, Fas expression is down-regulated, but FLIP expression is unaffected. Reporter gene and mRNA expression assays show that FasL activates fas transcriptional activity and this effect is inhibited by PI3K/Akt suppression. Together, our results indicate that the PI3K/Akt, in addition to its normal prosurvival role, also plays an apoptotic role in Fas-mediated cell death through a mechanism that involves transcriptional activation of Fas receptor.
Aspirin is under consideration as a promising chemopreventative agent for human cancers. To study the usefulness of aspirin as a chemopreventative agent for UV-induced human skin cancer, we investigated the effect of aspirin on UVB-induced activator protein-1 (AP-1) activity. In the JB6 cell culture system, aspirin or sodium salicylate (SA) inhibited UVB-induced AP-1 activity in a dose-dependent manner; this inhibitory effect occurred only in cells pretreated with aspirin or SA before UVB irradiation but not cells treated with aspirin or SA after UVB irradiation. Furthermore, these inhibitory effects on UVB-induced AP-1 activity appeared to be mediated through blocking of activation of MAP kinase family members, including extracellular signal-regulated protein kinases, c-Jun N-terminal kinases, and p38. It was not due to absorption of UVB light by aspirin. In the skin of AP-1-luciferase transgenic mice, UVB irradiation induced a rapid increase in AP-1 activity, which reached the peak at 48 h post-UVB irradiation. The topical pretreatment of mouse skin with aspirin markedly blocked the UVB-induced AP-1 transactivation in vivo. These data provide the first evidence that aspirin and SA are inhibitors of UV-induced signal transduction and thus could be used as a chemopreventative agent for skin cancer. Aspirin is under consideration as a promising chemopreventative agent for human cancers. To study the usefulness of aspirin as a chemopreventative agent for UV-induced human skin cancer, we investigated the effect of aspirin on UVB-induced activator protein-1 (AP-1) activity. In the JB6 cell culture system, aspirin or sodium salicylate (SA) inhibited UVB-induced AP-1 activity in a dose-dependent manner; this inhibitory effect occurred only in cells pretreated with aspirin or SA before UVB irradiation but not cells treated with aspirin or SA after UVB irradiation. Furthermore, these inhibitory effects on UVB-induced AP-1 activity appeared to be mediated through blocking of activation of MAP kinase family members, including extracellular signal-regulated protein kinases, c-Jun N-terminal kinases, and p38. It was not due to absorption of UVB light by aspirin. In the skin of AP-1-luciferase transgenic mice, UVB irradiation induced a rapid increase in AP-1 activity, which reached the peak at 48 h post-UVB irradiation. The topical pretreatment of mouse skin with aspirin markedly blocked the UVB-induced AP-1 transactivation in vivo. These data provide the first evidence that aspirin and SA are inhibitors of UV-induced signal transduction and thus could be used as a chemopreventative agent for skin cancer. Acetylsalicylic acid (aspirin) is one of the leading nonsteroidal anti-inflammatory drugs and is widely used as a chemopreventative agent for cancers (1Flower R.J. Bailey J.M. Prostaglandins, Leukotrienes and Lipoxins: Biochemistry, Mechanism of Action and Clinical Applications. Plenum, New York1985: 583-591Crossref Google Scholar, 2Giovannucci E. Egan K.M. Hunter D.J. Stampfer M.J. Colditz G.A. Willett W.C. Speizer F.E. New Engl. J. Med. 1995; 333: 609-614Crossref PubMed Scopus (979) Google Scholar, 3Weissmann G. Sci. Am. 1991; 264: 84-90Crossref PubMed Scopus (232) Google Scholar). It was reported that patients with regular aspirin use had a reduced incidence of lung, colon, and breast cancer and/or a decreased death rate from these cancers (4Rosenberg L. Palmer J.R. Zauber A.G. Warshauer M.E. Stolley P.D. 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Because aspirin is effective in inhibiting the cyclooxygenase (COX) 1The abbreviations used are: COX, cyclooxygenase; AP-1, activator protein-1; Erk, extracellular signal-regulated protein kinase; MAP, mitogen-activated protein; JNK, c-Jun N-terminal kinase; TPA, 12-O-tetradecanoylphorbol-13-acetate; SA, sodium salicylate; FBS, fetal bovine serum; MEM, minimal essential medium. activity, the mechanism of aspirin's action has been explained mainly by its ability to inhibit the synthesis of prostaglandin through targeting COX (9Brooks P.M. Day R.O. New Engl. J. Med. 1991; 324: 1716-1725Crossref PubMed Scopus (813) Google Scholar, 10Meade E.A. Smith W.L. DeWitt D.L. J. Biol. Chem. 1993; 268: 6610-6614Abstract Full Text PDF PubMed Google Scholar, 11Mitchell J.A. Akarasereenont P. Thiemermann C. Flower R.J. Vane J.R. Proc. Natl. Acad. Sci U. S. A. 1993; 90: 11693-11697Crossref PubMed Scopus (1566) Google Scholar, 12Vane J.R. Nat. 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Furthermore, experiments using COX1 or COX2 gene knockout mice showed that these gene-deficient mice still exhibited the same swelling response in the ear to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) as did wild-type mice (17Langenbach R. Morham S.D. Tiano H.F. Loftin C.D. Ghanayem B.I. Chulada P.C. Mahler J.F. Lee C.A. Goulding E.H. Kluckman K.D. Kim H.S. Smithies O. Cell. 1995; 83: 483-492Abstract Full Text PDF PubMed Scopus (1043) Google Scholar, 18Morham S.G. Langenbach R. Loftin C.D. Tiano H.F. Vouloumanos N. Jennette J.C. Mahler J.F. Kluckman K.D. Ledford A. Lee C.A. Smithies O. Cell. 1995; 83: 437-482Abstract Full Text PDF Scopus (1027) Google Scholar, 19DeWitt D. Smith W.L. Cell. 1995; 83: 345-348Abstract Full Text PDF PubMed Scopus (152) Google Scholar). The ear swelling experiment has been used as one of the short term experiments for testing tumor promotion activity. Therefore, the inhibition of COX does not appear to be sufficient to explain all the anti-cancer effects of aspirin. More recently, we demonstrated that aspirin and sodium salicylate (SA) inhibited the transactivation of AP-1 activity in the same dose range as seen for inhibition of tumor promoter-induced cell transformation (20Dong Z. Huang C. Brown R.E. Ma W.-Y. J. Biol. Chem. 1997; 272: 9962-9970Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Considering previous findings from different groups that tumor promoter-induced transcription of AP-1 is required for neoplastic transformation (21Dong Z. Birrer M.J. Watts R.G. Matrisian L.M. Colburn N.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 609-613Crossref PubMed Scopus (369) Google Scholar, 22Domann F.E. Levy J.P. Birrer M.J. Bowden G.T. Cell Growth Differ. 1994; 5: 9-16PubMed Google Scholar, 23Alani R. Brown P. Binetruy B. Dosaker H. Rosenberg R.K. Angel P. Karin M. Birrer M.J. 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Dermatol. 1983; 81: 517-519Abstract Full Text PDF PubMed Scopus (150) Google Scholar). Because transactivation of AP-1 plays a key role in tumor promotion (20Dong Z. Huang C. Brown R.E. Ma W.-Y. J. Biol. Chem. 1997; 272: 9962-9970Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 21Dong Z. Birrer M.J. Watts R.G. Matrisian L.M. Colburn N.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 609-613Crossref PubMed Scopus (369) Google Scholar, 29Huang C. Ma W.-Y. Dawson M.I. Rincon M. Flavell R.A. Dong Z. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5826-5830Crossref PubMed Scopus (205) Google Scholar), in the present study we investigated the effect of aspirin on UV-induced AP-1 activity in both cell cultures and AP-1-luciferase transgenic mice. AP-1-luciferase reporter plasmid stably transfected mouse epidermal JB6 P+1-1 cells were cultured in monolayers at 37 °C, 5% CO2 using Eagle's minimal essential medium containing 5% fetal calf serum, 2 mml-glutamine, and 25 μg of gentamicin/ml. Fetal bovine serum (FBS) was from Life Technologies, Inc.; Eagle's minimal essential medium (MEM) was from Calbiochem (San Diego, CA); luciferase assay substrate was from Promega; aspirin was from Sigma. The phospho-specific antibodies against phosphorylated sites of Erks, p38 kinase, and JNK assay kit were from New England Biolabs. 2X TRE-luciferase reporter transgenic mice were originally established by R. A. Flavell and his co-workers (30Rincon M. Flavell R.A. EMBO J. 1994; 13: 4370-4381Crossref PubMed Scopus (246) Google Scholar). A C57BL/6 male mouse carrying the 2X TRE-luciferase transgene was crossed with DBA/2 (SASCO, Omaha, NE 68101) females (29Huang C. Ma W.-Y. Dawson M.I. Rincon M. Flavell R.A. Dong Z. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5826-5830Crossref PubMed Scopus (205) Google Scholar). The F1 offspring were screened by testing both the basal level and TPA-induced level of luciferase activity for the presence of the AP-1-luciferase reporter gene. Males and females were housed separately in solid bottom polycarbonate cages on ventilated animal racks (∼4–5 mice/cage, individualized by incisions in the ears) in temperature-, humidity-, and light-controlled conditions. Food and water were available ad libitum, and the dorsal skin of the mice was shaved every week during the experiment period. The Animal Facility at the Hormel Institute is accredited by the American Association for Accreditation of Laboratory Animal Care. Confluent monolayers of JB6 P+1-1 cells were trypsinized, and 8 × 103 viable cells suspended in 100 μl of 5% FBS MEM were added to each well of a 96-well plate. Plates were incubated at 37 °C in a humidified atmosphere of 5% CO2. 12–24 h later, cells were starved by culturing them in 0.1% FBS MEM for 24 h prior to exposure to UVB irradiation. The cells were exposed to UVB for AP-1 induction with or without different concentrations of aspirin. After 24 h culture, the cells were extracted with lysis buffer, and luciferase activity was measured using a luminometer (Monolight 2010). The results are expressed as relative AP-1 activity (26Huang C. Schmid P.C. Ma W.-Y. Schmid H.H.O. Dong Z. J. Biol. Chem. 1997; 272: 4187-4194Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Immunoblots for phosphorylation of Erks and p38 kinase were carried out as described by the protocal of New England Biolabs using phospho-specific antibodies against phosphorylated sites of Erks and p38 kinase, respectively. JNK activity was assayed as described by the protocol of New England Biolabs. In brief, JB6 C141 cells were starved for 48 h in 0.1% FBS MEM at 37 °C in a 5% CO2 incubator. The cells were first treated with different concentrations of aspirin for 30 min and then exposed to UVB (4 kJ/m2) in the chamber fitted with a Kodak Kodacel K6808® filter that eliminates all wavelengths below 290 nm followed by culturing for another 20 min. The cells were washed once with ice-cold phosphate-buffered saline and lysed in 300 μl of lysis buffer/sample (20 mm Tris, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton, 2.5 mm sodium pyrophosphate, 1 mmβ-glycerolphosphate, 1 mm Na3VO4, 1 mg/ml leupeptin). The lysates were sonicated and centrifuged, and the supernatant was incubated with 2 μg of N-terminal c-Jun (1–89) fusion protein bound to glutathione-Sepharose beads overnight at 4 °C. The beads were washed twice with 500 ml of lysis buffer with phenylmethylsulfonyl fluoride and twice with 500 μl of kinase buffer (25 mm Tris, pH 7.5, 5 mmβ-glycerolphosphate, 2 mm dithiothreitol, 0.1 mm Na3VO4, 10 mmMgCl2). The kinase reactions were carried out in the presence of 100 μm ATP at 30 °C for 30 min. C-Jun phosphorylation was selectively measured by Western immunoblotting using a chemiluminescent detection system and specific c-Jun antibodies against phosphorylation of c-Jun at Ser63. All the mice were characterized by testing both the basal level and UVB-induced level of luciferase activity. The AP-1-luciferase reporter bearing male and female mice (6–9 weeks old) were randomly divided into four groups, including negative control group (control), UVB control group (UVB), aspirin-treated (10 μmol/mouse) group (experimental group 1), and aspirin-treated (40 μmol/mouse) group (experimental group 2). There were 13–14 mice in each group. 2 weeks after grouping, both the basal level and UVB-induced level of luciferase activities were measured with the skin biopsy tissue obtained by using biopsy punch (1.5 mm) (Acuderm, Inc., Ft. Lauderdale, FL). 2 weeks after the last punch biopsy, five topical doses of aspirin dissolved in 300 μl of acetone were applied to the dorsal skin of the mice over 8 days. The last of the five topical doses of aspirin was given 3 h prior to UVB irradiation. Then the mice were placed in a box and exposed to 10 kJ/m2 of UVB irradiation. 48 h after UVB exposure, the mice were punch biopsied to determine the effect of aspirin on UVB induction of AP-1 transactivation in the epidermis. Negative control mice were treated with acetone alone. The luciferase activity of punch biopsied epidermis was measured as described previously (26Huang C. Schmid P.C. Ma W.-Y. Schmid H.H.O. Dong Z. J. Biol. Chem. 1997; 272: 4187-4194Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 29Huang C. Ma W.-Y. Dawson M.I. Rincon M. Flavell R.A. Dong Z. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5826-5830Crossref PubMed Scopus (205) Google Scholar). The relative AP-1 activity was presented as relative to basal level of luciferase activity of each mouse. The significance of the difference in the AP-1 activity was determined with the Student's t test. The results are expressed as the means ± S.E. To determine whether aspirin and its analogue, SA, have any inhibitory effects on UVB-induced AP-1 activation, we incubated the JB6 cells with these compounds. Both aspirin and SA show marked inhibition of UVB-induced AP-1 activity (Fig. 1 A). This inhibitory effect appears to be dose-dependent (Fig. 1 B). The inhibitory concentration range of aspirin and SA was from 0.25 to 4 mm (Fig. 1 B). To rule out the possibility that the inhibition of AP-1 activity by aspirin and SA is due to their cytotoxic effect on JB6 cells, we observed the influence of aspirin and SA on cell proliferation. JB6 cells were seeded in 96-well plates in the presence of different concentrations of aspirin or SA. After 36 h of culture, 0.5 μCi of [3H]thymidine was added to each well. The cells were harvested 12 h later, and [3H]thymidine incorporation was determined using a liquid scintillation counter. The results indicated that aspirin and SA at doses ranging from 0.25 to 4 mm did not show any cytotoxic effect on JB6 cells (data not shown). To determine when aspirin or SA must be present to inhibit UVB-induced AP-1 activity, the aspirin or SA was added to cell cultures at different time points before or after cell exposure to UVB irradiation. As shown in Fig.2, the AP-1 activity of JB6 cells induced by UV irradiation was markedly inhibited by aspirin or SA when given 1 h prior to UVB irradiation, but there was no inhibitory effect on AP-1 activity observed if the agents were given after UVB irradiation (Fig. 2).Figure 2Requirement of pretreatment of aspirin (Asp) or SA for inhibition of UVB-induced AP-1 activity in JB6 cells. P+1-1 cells suspended in 5% FBS MEM were added to each well of 96-well plates. The cells were cultured and starved as described under "Materials and Methods." Then the cells were treated with aspirin or SA at the time (as indicated) before or after the cells exposure to UVB (2 kJ/m2). 24 h later, the AP-1 activity was determined by luciferase activity assay. The results are presented as relative AP-1 activity.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because normal UVB lamps also generate some UVC light, we compared the AP-1 inductions between normal UVB light and UVB light filtered with a Kodak Kodacel K6808® filter that eliminates all wavelengths below 290 nm. The results are shown in Fig. 3. AP-1 activity could be induced by either UVB light with or without filter (Fig. 3 A), but a higher dose of filtered UVB light is required for similar AP-1 induction as compared with normal UVB light (Fig. 3 A). The similar inhibition of AP-1 activity induced by filtered UVB light by aspirin is also observed as compared with those induced by normal UVB light (Fig. 3 B). It is well known that UV irradiation results in activation of Erks and JNKs as well as p38 kinase (31Kyriakis J.M. Banerjee P. Nikolakaki E. Dai T. Rubie E.A. Ahmad M.F. Avruch J. Woodgett J.R. Nature. 1994; 369: 156-160Crossref PubMed Scopus (2413) Google Scholar, 32Buscher M. Rahmsdorf H.J. Litfin M. Karin M. Herrlich P. Oncogene. 1988; 3: 301-311PubMed Google Scholar, 33Minden A. Lin A. McMahon M. Lange-Carter C. Derijard B. Davis R.J. Johnson G.L. Karin M. Science. 1994; 266: 1719-1723Crossref PubMed Scopus (1011) Google Scholar, 34Price M.A. Cruzalegui F.H. Treisman R. EMBO J. 1996; 15: 6552-6563Crossref PubMed Scopus (301) Google Scholar). Our results indicated that pretreatment of cells with aspirin led to an inhibition of UVB-induced AP-1 activation. Because AP-1 has been identified as a target of MAP kinase family, including Erks, JNKs, and p38 kinase, we analyzed the inhibitory effects of aspirin on UVB-induced JNK activities and phosphorylation of Erks and p38 kinase. We found that pretreatment of cells with aspirin blocked UVB-induced JNK activity (Fig. 4 A) and phosphorylation of Erks and p38 kinase (Figs. 4, B and C). These data suggested that inhibitory effects of aspirin on UVB-induced AP-1 activity are possibly modulated by blocking the activation of the MAP kinase family. To rule out the possibility that the blocking activity of aspirin on UVB-induced signal transduction is due to its absorbance of UVB light, we determined the absorbing wavelengths of aspirin and examined its ability to absorb UVB light between the wavelengths of 290–320 nm, which was used in the present study. We observed that aspirin absorbs UV light in the wavelength range of 210–280 nm (Fig.5), and aspirin did not show significant absorbance of UVB light at concentrations between 0.5–2.0 mm, which is the dose range used for blocking AP-1 activity and MAP kinase activation (Table I). Therefore, we concluded that inhibitory effects of aspirin on UVB-induced signal transduction were not through its absorbance of UVB light.Figure 5Absorbing wavelength of aspirin to UV light. Absorbing wavelength was scanned using Beckman DU®640 spectrophotometer.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IInfluence of aspirin solution on penetrating ability of UVBAspirinUVB1-aUVB wavelength, 290–320 nm.Mean ± S.D.InhibitionGroup 1Group 2Group 3mmμW/cm 2%0.0227228229228.0 ± 0.80.25238236235236.2 ± 1.2−3.50.5232234231232.1 ± 1.2−1.81.0223222224223.0 ± 0.82.22.0200202201201.0 ± 0.811.84.0171170173171.3 ± 1.225.01-a UVB wavelength, 290–320 nm. Open table in a new tab Because the binding of AP-1 protein to DNA does not always result in an induction of transcription, the AP-1 DNA binding activity measured by gel shift assay may not correlate with AP-1 transcriptional activity in some cases (35Herr I. VanDarn H. Angel P. Carcinogenesis. 1994; 15: 1105-1113Crossref PubMed Scopus (85) Google Scholar). To test whether UVB irradiation leads to elevation of AP-1 transcription activity in vivo, we used AP-1-luciferase reporter transgenic mice. UVB (10 kJ/m2) irradiation induced a rapid increase of AP-1 activity in mouse skin within 12 h after exposure to UVB irradiation (Fig.6 A). The maximum induction of AP-1 activity (∼160-fold) was observed at 48 h post-UV irradiation (Fig. 6 A, n = 7), and the activity returned to near basal levels after 10 days (data not shown). The UV dose response study indicated that optimum UVB dose for induction of AP-1 was 10 kJ/m2 (Fig. 6 B,n = 7); this dose for AP-1 induction is consistent with the previous dose used for skin tumor induction (36Gensler H.L. Timmermann B.N. Valcic S. Wachter G.A. Dorr R. Dvorakova K. Alberts D.S. Nutr. Cancer. 1996; 26: 325-335Crossref PubMed Scopus (124) Google Scholar, 37Wang Z.Y. Hong J.Y. Huang M.T. Ruehl K.R. Conney A.H. Yang C.S. Cancer Res. 1992; 52: 1943-1947PubMed Google Scholar, 38Wang Z.Y. Huang M.T. Lou Y.R. Xie J.G. Kenhl K.R. Newmark H.L. Ho C.T. Yang C.S. Conney A.H. Cancer Res. 1994; 54: 3428-3435PubMed Google Scholar). These results demonstrated for the first time that UVB irradiation leads to activation of AP-1 transcription activity in AP-1-luciferase transgenic mice. Our preliminary results from using mouse epidermal cell line JB6 indicated that aspirin inhibits UVB-induced AP-1 activity in vitro. To test this effect in vivo, we first determined the reproducibility of UVB-induced AP-1 activation between two times of UVB exposure in the same group of AP-1-luciferase transgenic mice. The transgenic mice were punch biopsied (1.5 mm) twice on the dorsal skin to determine the basal luciferase activity with a 3-week interval. 1 week after each of these punches, the mice were irradiated with 10 kJ/m2 of UVB, and 48 h later the same mice were punch biopsied to determine the UVB-induced AP-1 activity. The UVB-induced levels of luciferase activity from the two time points in the same group of mice were similar (n = 13, p> 0.05) (Fig. 7 A). These data suggested that induction of AP-1 activity by UVB in the same transgenic mice can be used as a control. Because chemoprevention of cancer is a long term process, the aspirin inhibition of AP-1 induction by UVB irradiation was determined following five applications of aspirin to mouse skin over 8 days. Multiple treatments of mouse dorsal skin with aspirin resulted in significant inhibition of UVB-induced AP-1 activity at both 10- and 40-μmol doses/mouse as compared with either UVB control group mice or experimental control mice (Fig. 7 B) (n = 13, p < 0.05). These results strongly demonstrate that application of aspirin not only blocks UVB-induced AP-1 activity in a cell culture model but also inhibits AP-1 transactivation in vivo. Both epidemiological and clinical studies have indicated that aspirin and related compounds have considerable potential as chemopreventative agents for cancers. Because several lines of evidence have suggested a causal role for AP-1 during tumor promotion, here we investigated the inhibitory effect of aspirin on UVB-induced AP-1 activity in both in vitro and in vivo models. Pretreatment of cells with aspirin significantly blocked the UVB-induced AP-1 transactivation and JNK activation, as well as phosphorylation of Erks and p38 kinases. This inhibition by aspirin was only observed when the aspirin treatment of the cells preceded UVB irradiation but not post-UVB irradiation. The negative results for the absorbance of UV light by aspirin suggested that inhibitory effects of aspirin on UVB-induced signal transduction is due to its pharmacological effects and not due to UVB absorbance. Topical application of aspirin on AP-1 reporter transgenic mice markedly inhibits UVB-induced AP-1 transactivation. Thus, these results, together with previous findings that UVB-induced AP-1 transactivation is a major mediator of development of human skin cancer, indicated that aspirin may serve as a chemopreventative drug for skin cancers induced by UV irradiation. Exposure of mammalian cells to UV irradiation not only causes DNA damage, resulting in either cell death or somatic mutation, but also induces specific cell reactions, including induction of transcriptional activation such as AP-1 and nuclear factor-κB (39Stein B. Rahmsdorf H.J. Steffer A. Litfin M. Herrlich P. Mol. Cell. Biol. 1989; 9: 5169-5181Crossref PubMed Scopus (456) Google Scholar, 40Devary Y. Gottlieb R.A. Lau L.F. Karin M. Mol. Cell. Biol. 1991; 11: 2804-2811Crossref PubMed Scopus (602) Google Scholar, 41Derijard B. Hibi M. Wu I.-H. Barrett T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2955) Google Scholar). The signal transduction pathways leading to transcription factor activation have been extensively studied in the last several years. It is believed that stress-related signals, such as UV light, induce the activation of MAP kinase pathways (Erks, JNKs, and p38). AP-1, consisting of Jun/Fos dimers, is a downstream target of these three MAP kinase members (42Whitmarsh A.J. Davis R.J. J. Mol. Med. 1996; 74: 589-607Crossref PubMed Scopus (1389) Google Scholar). We have reported that the anti-tumor promotion effects of aspirin are mediated by blocking tumor promoter-induced AP-1 activation (20Dong Z. Huang C. Brown R.E. Ma W.-Y. J. Biol. Chem. 1997; 272: 9962-9970Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). We have found that aspirin inhibited UVB-induced AP-1 activity in a dose-dependent manner, and this inhibitory effect appears to be through blocking UVB-induced activation of Erks and JNKs as well as p38 kinase. Interestingly, it appears that the signal transduction pathway leading to activation of AP-1 by UVB irradiation is different from that induced by TPA or epidermal growth factor because although aspirin inhibits UVB signals through MAP kinase-dependent pathway, it blocks TPA- or epidermal growth factor-induced signaling through a MAP kinase-independent pathway (20Dong Z. Huang C. Brown R.E. Ma W.-Y. J. Biol. Chem. 1997; 272: 9962-9970Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). These findings agree with the Schwenger et al. report that SA inhibited tumor necrosis factor-induced Erks activation but did not affect Erk activation by epidermal growth factor under the same condition (43Schwenger P. Skolnik E.Y. Vilcek J. J. Biol. Chem. 1996; 271: 8089-8094Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Solar irradiation has been associated with nonmelanoma skin cancer for centuries (44Matsui M.S. Delco V.A. Mukhtar H. 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Carcinogen. 1992; 6: 221-229Crossref PubMed Scopus (27) Google Scholar), and rescuing the response to tumor promoter in AP-1 activation by introducing wild-type extracellular signal-regulated protein kinase-2 (Erk2) converts tumor promotion-resistant phenotype to tumor promotion-sensitive phenotype. 2C. Huang and Z. Dong, unpublished data. This notion was further supported by our very recent finding that the retinoid SR11302, an AP-1 inhibition-specific retinoid, markedly inhibits the TPA-induced papilloma formation and AP-1 activation in 7,12-dimethylbenz(a)anthracene-initiated AP-1-luciferase transgenic mouse skin, whereas repeated applications of another retinoid, SR11235, a transcriptional activator of retinoic acid response element, does not inhibit papilloma formation and AP-1 activation (26Huang C. Schmid P.C. Ma W.-Y. Schmid H.H.O. Dong Z. J. Biol. Chem. 1997; 272: 4187-4194Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). In light of the role of AP-1 activation in chemical- and photo-carcinogenesis, we investigated the possible usefulness of aspirin in chemoprevention of UV-induced skin cancer by determining its inhibitory effect on UVB-induced AP-1 activity in both a cell culture model and in an AP-1-luciferase transgenic mouse model. The results indicated that pretreatment of cells and mouse skin with aspirin blocks UV-induced AP-1 transactivation effectively in both models. It is suggested that aspirin may be used as a chemoprevention agent for nonmelanoma skin cancers. We thank Dr. Harald H. O. Schmid for critical reading, Dr. Richard A. Flavell and Dr. Mercedes Rincon for the generous gift of the C57BL/6 AP-1-luciferase transgenic mice, Ruth Morton for helping with the animal care, and Carmen Hotson for secretarial assistance.
Abstract Nickel has been proved to be a human carcinogen. Hypoxia induced factor-1α (HIF-1α) plays a key role in the pathological processes of cancers. Previous studies have shown that nickel can exert its carcinogenic effects through the induction of HIF-1α accumulation. However, the molecular mechanisms have not been well understood. The c-Jun N-terminal kinase 1 (JNK1) is implicated in several physiological processes, including proliferation, apoptosis and differentiation, which are thought to be involved in several cancer development. JNK1 is also known to regulate the level of Hsp90/Hsp70. Our present study was conducted to investigate the role of JNK1 in the accumulation of HIF-1α induced by nickel. In current studies, we demonstrated that JNK1 is required for such an Hsp-dependent HIF-1α regulation. HIF-1α stabilization was impaired in JNK1−/− cells, and could be rescued by reconstitutional expression of JNK1 in either hypoxia or chemical-mimicked hypoxia conditions. Accordingly, HIF-1-dependent transcriptional activity and its downstream gene's expression were dramatically reduced in JNK1-deficient cells. We further disclosed that in JNK1−/− cells the low expression level of Hsp90/Hsp70 proteins affected the protective roles of these chaperones in maintaining newly synthesized HIF-1α stabilization, and forced expression of Hsp90 or Hsp70 in JNK1−/− cells showed a notable increase in HIF-1α stability compared with that of parental cells. Furthermore, our studies found that defective HDAC6 expression and subsequently increased Hsp90 acetylation could account for the reduction of Hsp90 chaperon activity in JNK1−/− cells. Our studies for the first time disclose a novel function of JNK1 in the modulation of HIF-1ααstabilization under nickel mimicked chemical hypoxia conditions through regulation of Hsp90/Hsp70 expression as well as HDAC6-mediated Hsp90 acetylation modification. This novel function may contribute to JNK1-mediated carcinogenic effects in response to nickel exposure. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4373.
Inorganic arsenic is an environmental human carcinogen, and has been shown to act as a co-carcinogen with solar ultraviolet (UV) radiation in mouse skin tumor induction even at low concentrations. However, the precise mechanism of its co-carcinogenic action is largely unknown. Apoptosis plays an essential role as a protective mechanism against neoplastic development in the organism by eliminating genetically damaged cells. Thus, suppression of apoptosis is thought to contribute to carcinogenesis. It is known that cyclooxygenase-2 (COX-2) can promote carcinogenesis by inhibiting cell apoptosis under stress conditions; and our current studies investigated the potential contribution of COX-2 to the inhibitory effect of arsenite in UV-induced cell apoptosis in mouse epidermal Cl41 cells. We found that treatment of cells with low concentration (5 μM) arsenite attenuated cellular apoptosis upon UVB radiation accompanied with a coinductive effect on COX-2 expression and nuclear factor-κB (NFκB) transactivation. Our results also showed that the COX-2 induction by arsenite and UVB depended on an NFκB pathway because COX-2 co-induction could be attenuated in either p65-deficient or p50-deficient cells. Moreover, UVB-induced cell apoptosis could be dramatically reduced by the introduction of exogenous COX-2 expression, whereas the inhibitory effect of arsenite on UVB-induced cell apoptosis could be impaired in COX-2 knockdown C141 cells. Our results indicated that COX-2 mediated the anti-apoptotic effect of arsenite in UVB radiation through an NFκB-dependent pathway. Given the importance of apoptosis evasion during carcinogenesis, we anticipated that COX-2 induction might be at least partially responsible for the co-carcinogenic effect of arsenite on UVB-induced skin carcinogenesis.
ABSTRACTBladder cancer (BC) is the most expensive cancer to manage on a per-patient basis, costing about $4 billion in total healthcare expenditure per annum in America alone. Therefore, identifying a natural compound for prevention of BC is of tremendous importance for managing this disease. Previous studies have identified isorhapontigenin (ISO) as having an 85% preventive effect against invasive BC formation induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). The results showed here that ISO treatment inhibited EGF-induced cell transformation of human urothelial cells through induction of tumor suppressor p27 transcription secondary to activation of an E2F1-dependentpathway.ISOtreatmentrenderedcellsresistanttoEGF-induced anchorage-independent growth concurrent with p27 protein induction in both UROtsa and SV-HUC-1 cells. ISO inhibition of EGF-induced cell transformation could be completely reversed by knockdown of p27, indicating that this protein was essential for the noted ISO inhibitory action. Mechanistic studies revealed that ISO treatment resulted in increased expression of E2F1, which in turn bound to its binding site in p27 promoter and initiated p27 transcription. The E2F1 induction was due to the elevation of its translation caused by ISO-induced miR-205 downregulation. Consistently, miR-205 was found to be overexpressed in human BCs, and ectopic expression of miR-205 mitigated ISO inhibitory effects against EGF-induced outcomes. Collectively, the results here demonstrate that ISO exhibits its preventive effect on EGF-induced human urothelial cell transformation by induction of p27 through a miR-205/E2F1 axis. This is distinct from what has been described for the therapeutic effects of ISO on human BC cells.KEYWORDS: ISOp27urothelial cellsbladder cancertransformation Disclosure statementNo potential conflict of interest was reported by the author(s).Ethical standardsEthical approval: All specimens were obtained with appropriate informed consent from the patients and the Medical Ethics Committee of China. And the comprehensive information of tissues were described in Materials and Methods.Author contributionsMH, XH, JX and JS acquisition and analysis of the data, drafting the manuscript; ZT, JW, HC, XW and PS, acquisition of data, critically reviewed the article; HY and JS analysis and interpretation of the data, critically reviewed the article; and CH conception and design, analysis and interpretation of the data, drafting and revising the manuscript. All the authors have read and approved the final manuscript.Data availabilityThe authors declare that all data supporting the findings of this study are available with the article or from the corresponding author upon reasonable request.Supplementary materialSupplemental data for this article can be accessed online at https://doi.org/10.1080/15384101.2022.2074623Additional informationFundingThis work was partially supported by grants from Natural Science Foundation of China (NSFC81773391 to C.H.); Oujiang Research Project (OJQD2022006 to C.H.); Natural Science Foundation of Ningbo (202003N4205 to J.S.); the Research Foundation of Ningbo Institute of Life and Health Industry, University of Chinese Academy of Sciences (research fund of ningbo institute of life and health industry, university of Chinese academy of sciences 2020YJY0209 to J.S.); the National Natural Science Foundation of China (82003199 to J.S.).
The X-linked inhibitor of apoptosis protein (XIAP) contains three N-terminal BIR domains that mediate anti-apoptosis and one C-terminal RING finger domain whose function(s) are not fully defined. Here we show that the RING domain of XIAP strongly inhibits the expression of p63α, a known tumor suppressor. XIAP knockdown in urothelial cells or RING deletion in knockin mice markedly upregulates p63α expression. This RING-mediated p63α downregulation is critical for the malignant transformation of normal urothelial cells following EGF treatment. We further show that the RING domain promotes Sp1-mediated transcription of miR-4295 which targets the 3'UTR of p63α mRNA and consequently inhibits p63α translation. Our results reveal a previously unknown function of the RING of XIAP in promoting miR-4295 transcription, thereby reducing p63α translation and enhancing urothelial transformation. Our data offer novel insights into the multifunctional effects of the XIAP RING domain on urothelial tumorigenesis and the potential for targeting this frequently overexpressed protein as a therapeutic alternative.
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