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PKBΑ Isoform Signaling Oncogene (2013) 32, 3254–3262 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc ORIGINAL ARTICLE The mTOR inhibitor rapamycin opposes carcinogenic changes to epidermal Akt1/PKBa isoform signaling K Sully1, O Akinduro1, MP Philpott1, AS Naeem2, CA Harwood1, VE Reeve3, RF O’Shaughnessy2 and C Byrne1 Epidermal squamous cell carcinoma (SCC) is the most aggressive non-melanoma skin cancer and is dramatically increased in patients undergoing immunosuppression following solid organ transplantation, contributing substantially to morbidity and mortality. Recent clinical studies show that use of the mammalian target of rapamycin (mTOR) inhibitor rapamycin as a post- transplantation immunosuppressive significantly reduces SCC occurrence compared with other immunosuppressives, though the mechanism is not fully understood. We show that rapamycin selectively upregulates epidermal Akt1, while failing to upregulate epidermal Akt2. Rapamycin increases epidermal Akt1 phosphorylation via inhibition of the mTOR complex 1-dependent regulation of insulin receptor substrate-1. Epidermal Akt1 is commonly downregulated in SCC while Akt2 is upregulated. We now demonstrate similar Akt1 downregulation and Akt2 upregulation by ultraviolet (UV) radiation, the most important skin carcinogen. Hence, rapamycin’s upregulation of Akt1 signaling could potentially oppose the effects of UV radiation and/or tumor-associated changes on Akt1 signaling. We show in skin culture that rapamycin does enhance restoration of Akt1 phosphorylation in skin recovering from UV radiation, suggesting a mechanism for rapamycin’s antitumor activity in epidermis in spite of its efficient immunosuppressive properties. Oncogene (2013) 32, 3254–3262; doi:10.1038/onc.2012.338; published online 13 August 2012 Keywords: mTOR; rapamycin; epidermis; Akt/PKB; squamous cell carcinoma; ultraviolet radiation INTRODUCTION Rapamycin inhibits mTORC1 downstream of the PI3K/Akt path- 22 A serious problem associated with long-term immunosuppression way (reviewed in Zoncu et al. ). However, in many cells negative following solid organ transplantation is development of skin feedback loops exist between mTORC1 signaling and the receptor cancer, in particular squamous cell carcinoma (SCC) which shows accessory protein, insulin receptor substrate-1 (IRS-1) causing 23–25 dramatically increased incidence.1 Clinical studies2,3 show that the rapamycin-mediated upregulation of Akt signaling. For mammalian target of rapamycin (mTOR) inhibitor rapamycin example, mTORC1 can directly phosphorylate IRS-1 at S636/639, substantially reduces cancer incidence, with findings confirmed in interrupting the docking of IRS-1 to PI3K and downregulating the 26 mouse.4,5 The rapamycin-mediated anticancer mechanism is not PI3K/Akt pathway in response to growth factor stimulation. In fully understood but is probably partly via inhibition of tumor tumors where this mTORC1-dependent negative feedback loop is 27,28 vascularization (Guba et al.,6 reviewed by Geissler and Schlitt7). active, rapamycin treatment can result in poor clinical outcome. Epidermal SCC is associated with changes in Akt (Protein It is unclear whether mTORC1-dependent negative feedback 29 kinase B) signaling.8,9 Akt1 and Akt2 isoforms are expressed in loops result in epidermal Akt activation. In epidermis, Akt1 and epidermis.10–12 Akt2, associated with less differentiated Akt2 signaling is spatially separated (Figure 3) and the two keratinocytes, is upregulated in SCC.13 In contrast, Akt1, active in isoforms appear to have different functions, leading to the 12,13,30 highly differentiated keratinocytes,12 is downregulated in likelihood of independent regulation. The two isoforms 13 response to high-risk human cutaneous papillomavirus early behave in opposite ways during SCC progression. Rapamycin- gene expression and in SCC, suggesting a tumor-suppressor role mediated upregulation of epidermal Akt signaling may, therefore, for Akt1.13 have different carcinogenic effects depending on which of the Akt is activated by translocation to the plasma membrane and two isoforms is affected. We therefore sought to find the effects of phosphorylation at two sites. Akt is recruited to the plasma rapamycin on epidermal Akt signaling. membrane by phosphatidylinositol-(3,4,5)triphosphate, produced by phosphatidylinositol 3-kinase (PI3K).14,15 Akt is then phosphory- lated in the activation loop, at T308, by phosphoinositide- RESULTS dependent kinase 1, and also in the hydrophobic pocket, Rapamycin increases Akt phosphorylation in keratinocytes at S473, predominantly by mTOR complex 2 (mTORC2), resulting Rapamycin causes a dose-dependent increase in Akt phosphory- 16,17 in full activation of Akt. Akt activates mTOR complex 1 lation at both S473 and T308 within 1 h of treatment and at (mTORC1), the second mTOR complex, via the tuberous rapamycin concentrations as low as 1 nM in rat epidermal keratino- sclerosis complex18,19 and also via the proline-rich Akt substrate cytes (REKs; Baden and Kubilus31) (Figure 1a; Supplementary 40 kDa.20,21 Figure S1a). The efficacy of rapamcyin action is shown by efficient 1Centre for Cutaneous Research, Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, UK; 2Immunbiology and Dermatology, Institute of Child Health, UCL, London, UK and 3Faculty of Veterinary Science, University of Sydney, Sydney, NSW, Australia. Correspondence: Professor C Byrne, Centre for Cutaneous Research, Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London E1 2AT, UK. E-mail: [email protected] Received 18 October 2011; revised 17 May 2012; accepted 24 June 2012; published online 13 August 2012 mTOR inhibition and epidermal Akt/PKB signaling K Sully et al 3255 0 10 20 30 60 Rap (min) 24 Duration (hr) due to signaling events upstream of PI3K in keratinocytes, which is compatible with rapamycin-induced changes to IRS-1. pAkt S473 - + Rapamycin It was then asked if rapamycin-induced Akt phosphorylation pAkt S473 was due to regulation of keratinocyte IRS proteins. Rapamycin pAkt T308 increased IRS-1 protein levels from 4–6 h to at least 8 h after drug treatment (Figure 2b). IRS-2 demonstrated little change in protein Total Akt level, however, rapamycin increased the electro-mobility of both Total Akt IRS-1 and IRS-2 (marked by *). This mobility change has been pS6 S240 reported to be due to changes in IRS tyrosine and serine/ 37 pS6 S240 /244 threonine phosphorylation. Rapamycin had little effect on IRS-1 /244 and IRS-2 transcript levels (Supplementary Figure S2), supporting Actin the idea that increased IRS-1 protein is due to post-transcriptional Total S6 changes. Previous reports show that IRS-1 is degraded by the 26S proteasome, which can be inhibited by the specific proteasome 1.5 37,38 pAkt S473 pAkt T308 inhibitor lactacystin. To find the consequences of IRS-1 protein accumulation, changes in IRS-1 protein levels were investigated 1.0 following treatment with either rapamycin or lactacystin (Figure 2c). Lactacystin increased IRS-1 and also Akt phosphoryla- tion, though less markedly than rapamycin, demonstrating that 0.5 increased IRS-1 increases Akt serine phosphorylation in keratinocytes. To show that rapamycin increases the stability of the IRS-1 0.0 protein, keratinocytes were treated with rapamycin and lactacystin pAkt relative to total Akt (au) pAkt relative 0406020 in the presence of the translational inhibitor, cycloheximide Time (min) (Figure 2d). Cycloheximide treatment gradually decreased IRS-1 Figure 1. Rapamycin increases epidermal Akt phosphorylation. levels as the protein pool was degraded. Both rapamycin and (a) Akt (S473 and T308) and S6 phosphorylation after 10 nM lactacystin maintained IRS-1 levels in the presence of cyclohex- rapamycin treatment of REKs for 0–60 min. Decrease in S6 imide, confirming that the rapamycin-mediated increase in IRS-1 phosphorylation at S240/244 demonstrates the efficacy of rapamy- was due to increased protein stability. cin activity. (b) Quantification of phosphorylation relative to total The effect of rapamycin on IRS-I phosphorylation was examined. Akt showing similar kinetics of S473 and T308 phosphorylation. IRS-1 becomes phosphorylated at multiple tyrosine residues (c) Long-term (24 h) rapamycin treatment maintains epidermal Akt within its C-terminus by activated receptor tyrosine kinases.39 phosphorylation in REKs. These phosphorylated tyrosine sites interact with SH2 domains of downstream substrates to increase signaling. For example, Y612 and Y632 are the major docking sites for PI3K (Figure 2e; Esposito downregulation of S6 phosphorylation at serine 240/244 (S240/ et al.40). Rapamycin treatment increases tyrosine phosphorylation 244; Figures 1a and c; Supplementary Figures S1a–c, plus later of IRS-1 in keratinocytes (Figures 2e and g), demonstrating that figures). There is little difference between the kinetics rapamycin promotes IRS-1 signaling. In contrast, phosphorylation of phosphorylation at S473 and T308 (Figure 1b), suggesting a of serine residues in the C-terminus of IRS-1 can inhibit IRS-1 common activation mechanism. Rapamycin elevates Akt phos- signaling in a number of ways, including disruption of the docking phorylation for at least 24 h (Figure 1c). of PI3K (reviewed by Boura-Halfon and Zick41). IRS-1 is directly Rapamycin also induces Akt phosphorylation in human primary phosphorylated at S636/639 by mTORC1, which lies close to the keratinocytes
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