Okkyung Rho

Deregulated Activity of Akt in Epithelial Basal Cells Induces Spontaneous Tumors and Heightened Sensitivity to Skin Carcinogenesis

Aberrant activation of the phosphoinositide-3-kinase (PI3K)/PTEN/Akt pathway, leading to increased proliferation and decreased apoptosis, has been implicated in several human pathologies including cancer. Our previous data have shown that Akt-mediated signaling is an essential mediator in the mouse skin carcinogenesis system during both the tumor promotion and progression stages. In addition, overexpression of Akt is also able to transform keratinocytes through transcriptional and posttranscriptional processes. Here, we report the consequences of the increased expression of Akt1 (wtAkt) or constitutively active Akt1 (myrAkt) in the basal layer of stratified epithelia using the bovine keratin K5 promoter. These mice display alterations in epidermal proliferation and differentiation. In addition, transgenic mice with the highest levels of Akt expression developed spontaneous epithelial tumors in multiple organs with age. Furthermore, both wtAkt and myrAkt transgenic lines displayed heightened sensitivity to the epidermal proliferative effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and heightened sensitivity to two-stage skin carcinogenesis. Finally, enhanced susceptibility to two-stage carcinogenesis correlated with a more sustained proliferative response following treatment with TPA as well as sustained alterations in Akt downstream signaling pathways and elevations in cell cycle regulatory proteins. Collectively, the data provide direct support for an important role for Akt signaling in epithelial carcinogenesis in vivo, especially during the tumor promotion stage.

Role of PI3K/Akt signaling in insulin‐like growth factor‐1 (IGF‐1) skin tumor promotion

Overexpression of human IGF‐1 with the bovine keratin 5 (BK5) promoter (BK5.IGF‐1 transgenic mice) induces persistent epidermal hyperplasia and leads to spontaneous skin tumor formation. In previous work, PI3K and Akt activities were found to be elevated in the epidermis of BK5.IGF‐1 transgenic mice compared to nontransgenic littermates. In the present study, we examined the importance of the PI3K/Akt signaling pathway in mediating the skin phenotype and the skin tumor promoting action of IGF‐1 in these mice. Western blot analyses with epidermal lysates showed that signaling components downstream of PI3K/Akt were altered in epidermis of BK5.IGF‐1 mice. Increased phosphorylation of GSK‐3 (Ser9/21), TSC2(Thr1462), and mTOR(Ser2448) was observed. In addition, hypophosphorylation and increased protein levels of β‐catenin were observed in the epidermis of BK5.IGF‐1 mice. These data suggested that components downstream of Akt might be affected, including cell cycle machinery in the epidermis of BK5.IGF‐1 mice. Protein levels of cyclins (D1, E, A), E2F1, and E2F4 were all elevated in the epidermis of BK5.IGF‐1 mice. Also, immunoprecipitation experiments demonstrated an increase in cdk4/cyclin D1 and cdk2/cyclin E complex formation, suggesting increased cdk activity in the epidermis of transgenic mice. In further studies, the PI3K inhibitor, LY294002, significantly blocked IGF‐1‐mediated epidermal proliferation and skin tumor promotion in DMBA‐initiated BK5.IGF‐1 mice. In addition, inhibition of PI3K/Akt with LY294002 reversed many of the cell cycle related changes observed in untreated transgenic animals. Collectively, the current results supported the hypothesis that elevated PI3K/Akt activity and subsequent activation of one or more downstream effector pathways contributed significantly to the tumor promoting action of IGF‐1 in the epidermis of BK5.IGF‐1 mice.

Activation of epidermal akt by diverse mouse skin tumor promoters.

Akt is a serine/threonine kinase involved in a variety of cellular responses, including cell proliferation and cell survival. Recent studies from our laboratory suggest that Akt signaling may play an important role in skin tumor promotion. To explore this premise, we examined epidermal Akt activation and signaling in response to chemically diverse skin tumor promoters. Mice received single or multiple applications of 12-O-tetradecanoylphorbol-13-acetate (TPA), okadaic acid, or chrysarobin. All three tumor promoters were able to activate epidermal Akt as early as 1 h after treatment. Activation of Akt following tumor promoter treatment led to enhanced downstream signaling, including hyperphosphorylation of glycogen synthase kinase-3β and Bad. Structure activity studies with phorbol ester analogues revealed that the magnitude of activation paralleled tumor-promoting activity. In cultured primary keratinocytes, TPA treatment also led to activation of Akt. Activation of the epidermal growth factor receptor (EGFR) seemed to underlie the ability of TPA to activate Akt as both PD153035, an inhibitor of EGFR, and GW2974, a dual-specific inhibitor of both EGFR and erbB2, were able to effectively reduce TPA-induced Akt phosphorylation as well as TPA-stimulated EGFR and erbB2 tyrosine phosphorylation in a dose-dependent manner. Furthermore, inhibition of protein kinase C (PKC) activity blocked TPA-stimulated heparin-binding EGF production and EGFR transactivation. Inhibition of PKC also led to a decreased association of Akt with the PP2A catalytic subunit, leading to increased Akt phosphorylation. However, combination of EGFR inhibitor and PKC inhibitor completely abrogated TPA-induced activation of Akt. Collectively, the current results support the hypothesis that elevated Akt activity and subsequent activation of downstream signaling pathways contribute significantly to skin tumor promotion. In addition, signaling through the EGFR via EGFR homodimers or EGFR/erbB2 heterodimers may be the primary event leading to Akt activation during tumor promotion in mouse skin. (Mol Cancer Res 2007;5(12):1342–52)

Altered expression of insulin-like growth factor I and its receptor during multistage carcinogenesis in mouse skin.

We examined the possible role of insulin‐like growth factor‐I (IGF‐I) and IGF‐I receptor (IGF‐Ir) during multistage carcinogenesis in mouse skin. For this purpose, the expression of both IGF‐I and IGF‐Ir was investigated in mouse skin during tumor promoter treatment and in primary papillomas and squamous cell carcinomas (SCCs) obtained from SENCAR mice treated with standard initiation‐promotion regimens. IGF‐I transcripts were not detectable or only weakly detectable in normal SENCAR mouse epidermis by northern or reverse transcription (RT)‐polymerase chain reaction (PCR) analysis, respectively, whereas IGF‐I transcripts (primarily a 7.0‐kb transcript) were readily detected in RNA preparations from the dermis by both northern blot analysis and RT‐PCR analysis. In contrast, IGF‐Ir transcripts were observed in RNA samples from both epidermis and dermis of control SENCAR mice. Single and multiple topical treatments with 3.4 nmol of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) had no effect on dermal or epidermal IGF‐I and IGF‐Ir mRNA levels. In contrast, the levels of IGF‐I transcripts were elevated (2.5‐ to 15‐fold) in a significant number of mouse skin tumors (71% of all tumors examined). Transcripts of 7.0, 2.5, and 1.3 kb were more consistently overexpressed in skin tumors compared with epidermis, whereas the two smaller transcripts were most consistently overexpressed compared with the dermis. The levels of an 11.0‐kb IGF‐Ir transcript were also elevated (2.5‐ to 8‐fold) in some papillomas (20%) and SCCs (55%), but the percentage of tumors exhibiting this property (32% of all tumors examined) was lower than the percentage overexpressing IGF‐I. These data suggest that altered expression of IGF‐I and IGF‐Ir may play a role in multistage carcinogenesis in the mouse skin model. The inability of TPA to induce elevated IGF‐I or IGF‐Ir expression suggests that these changes in skin tumors are coincident with tumor formation and not a direct result of altered epidermal proliferation per se. Altered expression of IGF‐I in a high percentage of papillomas may indicate that IGF‐I has an important role in the development of autonomous growth in these tumors. The higher percentage of SCCs with altered levels of IGF‐Ir mRNA may indicate a role for these changes in the later stages (i.e., tumor progression) of carcinogenesis in this model system.

Alteration of Egr-1 mRNA during multistage carcinogenesis in mouse skin

Immediate early genes, including fos, jun, and early growth response‐1 (Egr‐1), are induced during cellular response to changes in extracellular environment. These immediate early genes are believed to mediate processes of cell growth and differentiation. In particular, Egr‐1 is induced during mitogenic stimulation of a variety of cell types, including fibroblasts, B cells, and epithelial cells. In the present study, we examined Egr‐1 gene expression during multistage carcinogenesis in mouse skin. After a single topical treatment with the tumor promoter 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) to SENCAR mouse skin, Egr‐1 mRNA was induced, and maximal induction was observed at 2 h in both epidermis and dermis. Induction of Egr‐1 mRNA by TPA was inhibited by fluocinolone acetonide, a potent inhibitor of tumor promotion by TPA. Egr‐1 mRNA was present in primary keratinocytes derived from adult SENCAR mice. The keratinocyte cultures were maintained in low Ca2+ medium, and Egr‐1 mRNA levels became significantly elevated after the cultures were switched to high Ca2+ medium. Additionally, a large proportion of primary papillomas and carcinomas generated from SENCAR mice by standard initiation‐promotion regimens exhibited elevated Egr‐1 mRNA compared with normal epidermis. Taken together, these data suggest a possible role of Egr‐1 during multistage carcinogenesis in mouse skin. Mol. Carcinog. 27:247–251, 2000.

Overexpression of a constitutively active form of c-src in skin epidermis increases sensitivity to tumor promotion by 12-O-tetradecanoylphorbol-13-acetate

Transgenic mice were developed to study the role of c‐src in epithelial tumorigenesis through targeted expression of a constitutively active form of murine c‐src (src529). Src529 was targeted to the interfollicular epidermis with the human keratin 1 (HK1) promoter. The skin phenotype of these mice was characterized by exaggerated epidermal hyperplasia and hyperkeratosis within the first week after birth. The severity of this phenotype correlated with overall src kinase activity, both of which subsided with age. Treatment of adult HK1.src529 transgenic mice with the phorbol ester tumor promoter 12‐O‐tetradecanoylphorbol‐13‐acetate resulted in an increase in epidermal hyperplasia and labeling index significantly greater than that seen in nontransgenic littermates. In addition, HK1.src529 transgenic mice developed papillomas earlier and in significantly greater numbers compared with nontransgenic littermates in a standard initiation‐promotion experiment. The data support the hypothesis that activation of c‐src kinase plays a role in skin tumor promotion.

Abstract 2481: Rapamycin is a potent inhibitor of skin tumor promotion by TPA

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The activation of Akt signaling occurs early during skin tumorigenesis. Data from our lab has demonstrated that the phorbol ester TPA leads to activation of Akt as well as several downstream effectors of Akt. Of particular interest is the Akt downstream signaling molecule, mTOR, which is elevated in the epidermis of wild-type mice following treatment with TPA. These studies suggest that activation of mTOR may contribute to skin tumor promotion. To further test this hypothesis, rapamycin, which is an established mTORC1 inhibitor, was used to further explore the role of mTOR signaling in epithelial carcinogenesis and specifically during tumor promotion. In a multiple treatment regimen, rapamycin was applied topically 30 min prior to TPA at doses of 1 umol, 100 nmol and 20 nmol. Rapamycin, in a dose dependent manner inhibited TPA induced mTOR activity as well as several downstream signaling proteins including p70S6KT389 and pS6S240/244 as well as p4EBP1T37/46, the FRAP/mTOR phosphorylation site of translational repressor protein 4EBP1. In addition, the highest dose of rapamycin caused a reduction in Akt-mediated phosphorylation of Akt downstream targets GSK3B as well as PRAS40 as assessed by western blotting using phosphospecific antibodies. These data suggest that multiple treatments with rapamycin at higher doses blocked both mTORC1 and mTORC2, while lower doses primarily affected mTORC1. Multiple treatment experiments evaluating the effects of rapamycin (5-200 nmol/mouse) on epidermal hyperplasia and epidermal labeling index, two short-term markers of tumor promotion by TPA, demonstrated a significant inhibition of both parameters in a dose dependent manner. Using a two-stage chemical skin carcinogenesis model with the standard DMBA/TPA protocol, the ability of rapamycin to block skin tumor promotion by TPA was evaluated. Rapamycin exerted a powerful anti-promoting effect reducing the average number of papillomas per mouse as well as the overall incidence of papillomas. In this regard, even a 5 nmol topical dose of rapamycin given 30 min prior to each TPA treatment produced approximately 50% inhibition of papilloma formation. Moreover, topical application of rapamycin to existing papillomas induced regression and/or inhibited growth. Skin tumors from this study were collected from TPA or TPA + rapamycin treated groups and are being evaluated for expression of proteins involved in cell cycle regulation as well as proteins involved in autophagy and apoptosis. The results of these experiments will be presented. Collectively, the current results indicate that signaling through mTORC1 contributes significantly to the process of skin tumor promotion, and that blocking this pathway may be an effective strategy for prevention of epithelial carcinogenesis. Supported by NIH grant CA37111. 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 2481.

Abstract 2475: Effect of mTOR inhibition by rapamycin on promotion of skin tumors in BK5. Aktwt transgenic mice

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Akt is a serine/threonine kinase involved in a variety of cellular responses including cell survival and proliferation. It has been shown that PI3K/Akt signaling is frequently altered (upregulated) in human cancers. mTOR is one of the Akt downstream signaling pathways that regulates protein synthesis, cell growth and proliferation. mTOR exists in two complexes, mTORC1 and mTORC2. Our laboratory previously reported that tumor promoters including TPA, okadaic acid (OA), and chrysarobin (CHRY) activate epidermal Akt after topical treatment of mouse epidermis and mouse primary cultured keratinocytes. Activation of Akt during tumor promoter treatment leads to enhanced downstream signaling including mTORC1 pathways. BK5. Aktwt transgenic mice where Akt is overexpressed in basal epidermis were significantly more sensitive to two-stage carcinogenesis, showing tumors with a much shorter latency, considerably larger in size, and enhanced progression of papillomas to SCCs. Importantly, Akt transgenic mice showed significant elevations in several downstream signaling pathways both in the absence and presence of TPA, including mTORC1 activity. Collectively, the studies with transgenic mice overexpressing Akt in epidermis (BK5.Aktwt) have suggested that activation of mTORC1 signaling may contribute to the process of skin tumor promotion. In this study, we have examined the ability of rapamycin, a known inhibitor of mTOR, on mTORC1 signaling in relation to its effect on TPA-induced epidermal cell proliferation and skin tumor promotion. Rapamycin given at doses of 1 μmol, 100 nmol, 20 nmol, and 5 nmol 30 min prior to 6.8 nmol TPA treatment inhibited mTORC1 activity in a dose-dependent manner. Notably, in a multiple treatment regimen (4 applications of rapamycin and TPA over a two week period) high doses of rapamycin significantly inhibited TPA-induced Akt phosphorylation at serine 473 as well as mTORC1 signaling in a dose dependent way, suggesting mTORC2 inhibition induced by prolonged high dose rapamycin treatment. The ability of rapamycin (at doses of 100, 20, and 5 nmol per mouse) to inhibit skin tumor promotion by TPA in BK5. Aktwt transgenic mice was compared with wild-type mice. Rapamycin significantly reduced TPA promotion in BK5.Aktwt mice, however, it was less effective compared to similar doses used in wild-type mice. The data demonstrate that rapamycin, although less effective, still retained significant inhibitory activity toward TPA promotion even when Akt and mTOR activities were elevated. These data demonstrate an important role for mTOR signaling in skin tumor promotion of TPA. 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 2475.