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. 2011 Feb 23;32(5):779–785. doi: 10.1093/carcin/bgr037

TRPV1-antagonist AMG9810 promotes mouse skin tumorigenesis through EGFR/Akt signaling

Shengqing Li 1,†, Ann M Bode 1,†, Feng Zhu 1,†, Kangdong Liu 1, Jishuai Zhang 1, Myoung Ok Kim 1, Kanamata Reddy 1, Tatyana Zykova 1, Wei-ya Ma 1, Andria L Carper 1, Alyssa K Langfald 1, Zigang Dong 1,*
PMCID: PMC3086704  PMID: 21349818

Abstract

In addition to capsaicin, a transient receptor potential channel vanilloid subfamily 1 (TRPV1) agonist, two kinds of antagonists against this receptor are used as therapeutic drugs for pain relief. Indeed, a number of small molecule TRPV1 antagonists are currently undergoing Phase I/II clinical trials to determine their effect on relieving chronic inflammatory pain and migraine headache pain. However, we previously reported that the absence of TRPV1 in mice results in a striking increase in skin carcinogenesis, suggesting that chronic blockade of TRPV1 might increase the risk of tumor development. In this study, we found that a typical TRPV1 antagonist, AMG9810, promotes mouse skin tumor development. The topical application of AMG9810 resulted in a significant increase in the expression level of the epidermal growth factor receptor (EGFR) and its downstream Akt/mammalian target of rapamycin (mTOR)-signaling pathway. This increase was not only observed in AMG9810-treated tumor tissue but was also found in skin tissue treated with AMG9810. In telomerase-immortalized primary human keratinocytes, AMG9810 promoted proliferation that was mediated through the EGFR/Akt/mTOR-signaling pathway. In summary, our data suggest that the TRPV1 antagonist, AMG9810, promotes mouse skin tumorigenesis mediated through EGFR/Akt/mTOR signaling. Thus, the application of this compound for pain relief might increase the risk of skin cancer.

Introduction

The transient receptor vanilloid 1 (TRPV1), the archetypal member of the vanilloid TRP family, was initially identified as the capsaicin receptor and is a non-selective cation channel (1). It is activated by numerous stimuli including heat, voltage, vanilloids, lipids, protons and cations (2). TRPV1 has a diverse tissue distribution, including epidermal keratinocytes (3), bladder urothelium (4) and other smooth muscles (5) and immunocytes. Both TRPV1 agonists and antagonists are used as pain relief drugs. The TRPV1 agonist, capsaicin, is widely used for pain relief and is available over the counter and by prescription (6). The pharmacological development of TRPV1 antagonists is based on the belief that endogenous agonists acting on TRPV1 might contribute to certain chronic pain conditions (7). AMG9810, [(E)-3-(4-t-butylphenyl)-N- (2,3-dihydrobenzo[b][1,4]dioxin-6-yl) acrylamide] is a novel TRPV1 antagonist with high selectivity. It is a competitive antagonist of capsaicin activation and blocks all known modes of TRPV1 activation, including activation by protons, heat and endogenous ligands, including anandamide, N-arachidonyl dopamine and oleoyldopamine (8). AMG9810 is effective at preventing capsaicin-induced eye-wiping and reverses hyperalgesia in an in vivo animal model of inflammatory pain (8).

TRPV1 is also expressed in cancer cells, including tongue squamous cell carcinoma (9), prostate carcinoma (9), hepatocellular carcinoma (10) and bladder transitional cell carcinoma (11). High expression of TRPV1 is associated with a better prognosis of patients with hepatocellular carcinoma (10). A progressive loss of TRPV1 expression is found in the urothelium with advanced stage transitional cell carcinoma and is associated with lower cell differentiation (11). These results suggested that TRPV1 might function as a tumor suppressor. We reported that TRPV1 knockout (TRPV1−/−) mice exhibit a marked increase in 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced skin carcinogenesis compared with wild-type TRPV1 (TRPV1+/+) mice (12). The effect was associated with an interaction between TRPV1 and the epidermal growth factor receptor (EGFR), which led to EGFR degradation (12). The MEK/extracellular signal-regulated kinases (ERKs) and PI3-K/Akt-signaling pathways are two primary downstream effectors of EGFR. A critical regulating target of Akt is the protein kinase mammalian target of rapamycin (mTOR), which is important in promoting cellular metabolism, growth, proliferation and tumor formation (13). Many cancers are characterized by aberrant activation of mTOR because of the deregulation of the upstream PI3-K/Akt pathway (14). Preclinical and clinical studies have demonstrated that tumors bearing genetic alterations that activate mTOR are sensitive to pharmacologic inhibition of mTOR (15).

EGFR is overexpressed and activated in many human epithelial cancers and TRPV1 might act as a tumor suppressor by negatively regulating EGFR signaling. This raises the question of whether the chronic blockade of TRPV1 might elevate EGFR signaling and increase the risk of tumorigenesis. Here, we show that topical application of the TRPV1-antagonist AMG9810 alone can act as a carcinogen by promoting mouse skin tumor development. AMG9810 treatment resulted in enhanced EGFR signaling not only in a mouse model but also in cultured human keratinocytes. Thus, the application of TRPV1 antagonists for pain relief might be associated with an increased risk of skin tumorigenesis.

Materials and methods

Reagents and antibodies

Chemical reagents, including CHX, Tris, NaCl and sodium dodecyl sulfate, for molecular biology and buffer preparation were purchased from Sigma–Aldrich (St Louis, MO). Keratinocyte-serum free medium (SFM), bovine pituitary extract (25 mg) and recombinant epidermal growth factor (2.5 μg) were from Invitrogen (Carlsbad, CA). HyClone Dulbecco’s modified Eagle’s medium and F-12 Ham's media were purchased from HyClone Laboratories (Logan, UT). Antibodies for western blot analysis were purchased from Cell Signaling Technology (Beverly, MA).

Cell culture

Telomerase-immortalized primary human keratinocytes 1 (N/TERT1) were cultured at 37°C in a 5% CO2 incubator with 10% fetal bovine serum in keratinocyte-SFM supplemented with 25 mg BPE and 2.5 μg recombinant epidermal growth factor. N/TERT1 cells were split at 80–90% confluence with 0.25% trypsin–ethylenediaminetetraacetic acid and the trypsin reaction was stopped by adding 10 ml Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum. After centrifugation, the cell pellet was gently resuspended in 15 ml keratinocyte-SFM complete medium. N/TERT1 cells were starved 24 h with serum-free F-12 Ham's medium before treatment with AMG9810.

Protein sample preparation and western blotting

Skin tissue samples were weighed and specific lysis buffer (5 ml/g; 20 mM Tris–HCl pH8.0, 150 mM NaCl and protease inhibitor) was added. After homogenization, skin samples were centrifuged (13  000 r.p.m.) at 4°C three times for 5 min each. The supernatant fractions were recovered for protein quantitation using a detergent compatible protein assay kit (Bio-Rad, Hercules, CA). Tumor and cell samples were extracted with RIPA cell lysis buffer. Protein samples were separated on the appropriate percentage sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred polyvinylidine difluoride membranes. The membranes were blocked with a solution of phosphate buffered saline with Tween20 containing 5% non-fat milk. Then the membranes were incubated at 4°C for 16 h in a 1:1000 dilution of the primary antibody. After three washes with phosphate buffered saline with Tween20, the membranes were incubated with a 1:5000 dilution of the corresponding secondary antibody. Proteins were detected with ECL Plus western blotting detection reagents (GE Healthcare, Piscataway, NJ).

Cytotoxicity and cell proliferation assay

To assess cyotoxicity of AMG9810, N/TERT1 cells (2 × 104) in 100 μl of Keratinocyte-SFM complete medium were seeded into each well of a 96-well plate and cultured in a 5% CO2 incubator at 37°C. For proliferation, 1 × 103 N/TERT1 cells were seeded into each well. Cells were treated with different concentrations of AMG9810 and cultured for various periods of time. The CellTiter 96 AQueous One Solution (20 μl; Promega, Madison, WI) was added to each well and then cells were kept in a 37°C, 5% CO2 incubator for 1 h. Absorbance was then measured at 492 and 690 nm with a plate reader (Labsystems Multiskan MS; Analytical Instruments, LLC, Golden Valley, MN).

Two-stage carcinogenesis animal study

For the two-stage carcinogenesis animal study, age- and gender-matched SKH-1 hairless mice were divided into three groups, and all were initiated by topical application of 200 nmol of 7,12-dimethylbenz(a)anthracene (DMBA). Two weeks later, topical application of AMG9810 (1 mg in acetone), TPA (17 nmol in acetone) or vehicle (acetone) was begun and continued twice a week for a total of 28 weeks. Group 1 (n = 30) was treated with vehicle only; group 2 (n = 30) was treated with 1 mg AMG9810 only and group 3 (n = 30) was treated with 17 nmol TPA only. Mice were weighed and tumors counted and measured weekly, beginning when the first measurable tumors (1 mm3) were observed (week 15). At the end of the study, all tumor samples were collected for protein extraction or immediately fixed in 10% neutral buffered Zamboni’s reagent (Newcomer Supply Middleton, WI) and processed for hematoxylin and eosin (H&E) staining and immunostaining.

Acute animal study

For a short-term animal study, age- and gender-matched SKH-1 hairless mice were divided into three groups. Half of the mouse dorsal trunk skin was treated with 1 mg AMG9810 and the other half of the skin was treated with vehicle only and used as a ‘self control’. Group 1 (n = 3) was treated with AMG9810 and vehicle for 0.5 h; group 2 (n = 3) was treated for 1 h and group 3 (n = 3) was treated for 3 h. The mouse skin samples were collected at the indicated times points and weighed for protein extraction or immediately fixed in 10% neutral buffered Zamboni’s reagent and processed for H&E staining and immunostaining. All animal experiments were carried out according to protocols approved by the University of Minnesota Institutional Animal Care and Use Committee.

Immunostaining

Mouse skin and tumor samples were blocked with 5% donkey serum albumin in 600 μl 1× phosphate-buffered saline/0.03% Triton X-100, (pH 6.0) in a humidified chamber for 1 h at room temperature and then were immunostained with antibodies as follows: (i) 1:100 anti-EGFR raised in mouse (Santa Cruz Biotechnology, Santa Cruz, CA) and 1:200 donkey anti-mouse IgG conjugated to Cy5 (Jackson ImmunoResearch Laboratories, West Grove, PA); (ii) 1:100 anti-pEGFR (Tyr1173) raised in goat (Santa Cruz Biotechnology) and donkey anti-goat IgG conjugated to Cy2 (Jackson ImmunoResearch Laboratories); (iii) 1:100 anti-pAkt (Ser473) raised in rabbit (Santa Cruz Biotechnology) and 1:200 donkey anti-rabbit IgG conjugated to Cy3 (Jackson ImmunoResearch Laboratories) and (iv) 1:100 anti-pmTOR (Ser2481) raised in rabbit (Cell Signaling Technology) and 1:200 donkey anti-rabbit IgG conjugated to Dylight 488 (Jackson ImmunoResearch Laboratories). Image stacks were captured (×20) at room temperature using laser scanning confocal microscopy (NIKON Clsi Confocal Spectral Imaging System; NIKON Instruments Co., Melville, NY).

Results

AMG9810 promotes mouse skin tumorigenesis

AMG9810 and its analogs have a similar basic molecular structure, which comprises a central hydrogen-bond acceptor/donor motif flanked by a lipophilic side chain on one side and an aromatic group that incorporates a hydrogen-bond acceptor on the other side (Figure 1A). These compounds incorporate structural features of the class II TRPV1-antagonist capsazepine (Figure 1B), which are different from the molecular structure of the TRPV1-agonist capsaicin (Figure 1C). We previously found that TRPV1 functions as a tumor suppressor in the DMBA/TPA two-stage mouse skin carcinogenesis model, suggesting that chronically deactivating the TRPV1 might increase the risk of tumor development. In the present study, topical application of AMG9810 onto mouse dorsal skin after initiation with DMBA induced more mice-developing papillomas compared with a vehicle-treated control group (Figure 2A). Mice treated with AMG9810 developed significantly more papillomas per mouse compared with vehicle-treated mice (Figure 2B). Tumors from mice treated with AMG9810 also tended to be larger than tumors in vehicle-treated mice (Figure 2C). H&E staining of AMG9810-induced mouse skin papillomas exhibited a high density of squamous cells and less extracellular matrix compared with the TPA- or vehicle-treated group (Figure 2D). Taken together, these results demonstrate that after initiation with DMBA, long-term treatment with AMG9810 promotes mouse skin tumorigenesis.

Fig. 1.

Fig. 1.

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Molecular structure of AMG9810 (A), capsazepine (B) and capsaicin (C).

Fig. 2.

Fig. 2.

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AMG9810 promotes mouse skin tumorigenesis. Age- and gender-matched SKH-1 hairless mice were divided into three groups: AMG9810 treated, TPA treated and vehicle treated. Two weeks after initiation with DMBA (200 nmol), topical application of AMG9810 (1 mg), TPA (17 nmol) or vehicle was initiated and continued twice a week for a total of 28 weeks. The percentage of mice with tumors (A), the average number of tumors per mouse (B) and the average tumor volume (C) were determined weekly. Tumor volume was calculated using the formula: tumor volume (mm3) = (length × width × height × 0.52). At the end of the study, tumor samples were fixed in 10% neutral buffered Zamboni’s reagent and processed for H&E staining. The bar indicates 200 microns (×10). Representative staining is shown from five samples (D).

The EGFR/Akt/mTOR-signaling pathway is activated in AMG9810-induced skin tumors

Our previous results indicated that the TRPV1 interacts with the EGFR, which facilitated EGFR ubiquitination by the ubiquitin ligase Cbl, resulting in subsequent EGFR degradation through the lysosomal pathway (12). Therefore, we hypothesized that desensitization of TRPV1 by AMG9810 treatment might cause an increase in the protein level of EGFR and its downstream-signaling pathway. Immunofluorescence results indicated that the protein level of EGFR in the AMG9810-treated group was substantially higher than either the TPA- or vehicle-treated group (Figure 3A and B). In addition, the phosphorylation level of EGFR (Tyr1173) also increased dramatically compared with the TPA- or vehicle-treated group (Figure 3A and B). Phosphorylation of EGFR can activate Akt/mTOR and MEK/ERKs signaling, which are closely associated with proliferation and cell transformation. Compared with vehicle-treated tumors, AMG9810 induced a 3.3-fold average increase in phosphorylated Akt (Ser473)-positive staining (Figure 3C and D), whereas in TPA-promoted tumors, Akt was unchanged. TPA- and vehicle-treated tumors exhibited similar expression of phosphorylated mTOR (Ser2481). In contrast, mTOR was 1.9-fold higher in the AMG9810-treated tumors. Phosphatase and tensin homolog (PTEN) is a dual protein/lipid phosphatase, which can negatively regulate Akt activity by mediating the degradation of phosphatidylinositol (3,4,5) triphosphate (PIP3), the product of PI3 kinase. PTEN activity can be lost at high frequency by mutation, deletion or promoter methylation silencing in many primary and metastatic human cancers (16). The activity of PTEN is turned off after phosphorylation at its C terminus by the protein kinase caseine kinase (CK2) (17). We found that the expression level of phosphorylated PTEN (Ser380/Thr382/383) was not different in tumors from untreated, TPA-treated or AMG9810-treated mice (supplementary Figure 1 is available at Carcinogenesis Online). In addition, the expression levels of phosphorylated MEK1/2 (Ser218/222) and ERK1/2 (Thr202/Tyr204) were also similar among three groups (Figure 3D, supplementary Figure 2 is available at Carcinogenesis Online). From these data, we can conclude that Akt/mTOR signaling might play the most important role in AMG9810-induced tumorigenesis. We further validated changes in this signaling pathway in skin papillomas by western blotting using specific antibodies. Compared with the vehicle- or TPA-treated group, both the total and phosphorylated levels of Akt and mTOR were increased in the AMG9810-treated group (Figure 3E). Overall, these data indicated that AMG9810-induced tumorigenesis is closely associated with the EGFR/Akt/mTOR-signaling pathway.

Fig. 3.

Fig. 3.

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The EGFR/Akt/mTOR-signaling pathway is activated in AMG9810-induced skin tumors. The tumor samples from mice treated with AMG9810, TPA or vehicle were fixed in 10% neutral buffered Zamboni’s reagent and processed for immunostaining with specific primary antibodies to detect total EGFR (secondary antibody conjugated to Cy5, blue color) and phosphorylated EGFR (Tyr1173, secondary antibody conjugated to Cy2, green color) (A) and for phosphorylated Akt (Ser473, secondary antibody conjugated to Cy3, red color) and phosphorylated mTOR (Ser2481, secondary antibody conjugated to Cy2, green color) (C). Samples were observed by confocal microscopy (NIKON Clsi Confocal Spectral Imaging System; NIKON Instruments Co.). The bar indicates 100 microns (×20). Representative staining is shown from five independent samples. The expression levels of total and phosphorylated EGFR (Tyr1173) (B) and phosphorylated Akt (Ser473) and mTOR (Ser2481) (D) in skin tumors from each of the three groups were quantified using the ImageJ software program (Version 1.42q). Data are presented as means ± standard deviations of values from five independent samples and the asterisk indicates a significant difference (*P < 0.05, **P < 0.01) (D). Tumor samples from each of the three groups were homogenized in RIPA buffer and the supernatant fractions were recovered by centrifugation for protein quantification. The protein samples were subjected to western blot analysis to visualize the total and phosphorylation levels of Akt (Ser473) and mTOR (Ser2481). β-Actin was used to verify equal loading of protein (E).

The Akt/mTOR-signaling pathway is activated by AMG9810 in vivo

To determine whether Akt/mTOR signaling can be activated by AMG9810 treatment without DMBA initiation in vivo, we performed a short-term animal study in which the mouse dorsal trunk skin was treated with AMG9810 (1 mg in acetone) for 0.5, 1 or 3 h. H&E staining showed that the skin tissue from AMG9810-treated mice had similar morphological characteristics as untreated skin, indicating that short-term treatment with AMG9810 had no effect on skin epithelial cells (Figure 4A). Immunofluorescence results indicated that compared with untreated skin, AMG9810 treatment caused a dramatic increase in both phosphorylated Akt (Ser473) and mTOR (Ser2481) (Figure 4B and C). To further confirm these results, we assessed Akt/mTOR-signaling changes by western blotting using protein samples prepared from mouse skin. Compared with untreated skin, the phosphorylation levels of both Akt (Ser473) and mTOR (Ser2481) increased at all three time points after stimulation with AMG9810 (Figure 4D). Taken together, these data demonstrate that AMG9810 can activate the Akt/mTOR-signaling pathway in mouse skin.

Fig. 4.

Fig. 4.

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The Akt/mTOR signal pathway is activated by AMG9810 in vivo. Mouse skin samples were fixed in 10% neutral buffered Zamboni’s reagent and processed for H&E staining. The bar indicates 100 microns (×40). Representative staining is shown from groups treated or not treated with AMG9810 (A). Fixed mouse skin samples from groups treated or not treated with AMG9810 were processed for immunostaining with specific primary antibodies to detect phosphorylation of Akt (Ser473) or mTOR (Ser2481). Samples were observed using a secondary antibody conjugated to Dylight 488 and confocal microscopy (NIKON Clsi Confocal Spectral Imaging System; NIKON Instruments Co.). Bar indicates 100 microns (×20) (B). The phosphorylation expression levels of Akt (Ser473) and mTOR (Ser2481) in skin tumors from each of the two groups were quantified using the ImageJ software program (Version 1.42q). Data are presented as means ± standard deviations of values from five samples. The asterisk (*) indicates a significant difference (P < 0.05), and the asterisk (**) indicates a significant difference (P < 0.01) (C). Proteins from skin samples were extracted by skin tissue specific lysis buffer (20 mM Tris–HCl pH8.0, 150 mM NaCl and protease inhibitors). The total and phosphorylated levels of Akt (Ser473) and mTOR (Ser2481) were assessed by western blot analysis and β-actin was used to verify equal loading of protein (D).

AMG9810 promotes proliferation of human keratinocytes

To determine the effect of AMG9810 on proliferation of human keratinocytes, the cytotoxicity of this compound was first tested in N/TERT1 cells. We found that AMG9810 had no obvious cytotoxic effects on N/TERT1 cells at various concentrations (0–5 μM; Figure 5A). N/TERT1 cells were treated with different concentrations of AMG9810 to determine growth, and results indicated that AMG9810 promoted proliferation of keratinocytes (Figure 5B). We hypothesized that the effect of AMG9810 on growth of N/TERT1 cells might occur through the same signaling pathway as was determined in the in vivo study. Results indicated that the phosphorylation levels of both Akt (Ser473) and mTOR (Ser2481) increased in a time-dependent manner in N/TERT1 cells with AMG9810 treatment (1 μM; Figure 5C). The expression level of EGFR also increased in a time-dependent manner in N/TERT1 cells after treatment with AMG9810 (1 μM; Figure 5D). Overall, these data indicated that AMG9810 also promotes proliferation of N/TERT1 cells through the EGFR/Akt/mTOR-signaling pathway.

Fig. 5.

Fig. 5.

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AMG9810 promotes proliferation of human keratinocytes. N/TERT1 cells (2 × 104 per well of a 96-well plate) were treated with various concentrations of AMG9810 as indicated and cells were subjected to a methyl tetrazsolium salt assay. The absorbance at 490 nm was determined by Labsystem Multiscan MS plate reader at 24 and 48 h after AMG9810 treatment. The data are shown as means ± standard deviations of values from five replicates (A). N/TRT1 cells (1 × 103 per well of a 96-well plate) were treated with various concentrations of AMG9810 as indicated and subjected to an methyl tetrazsolium salt assay. The absorbance at 490 nm was measured by Labsystem Multiscan MS plate reader at different time points as indicated after AMG9810 treatment and the data are shown as means ± standard deviations of values from five replicates (B). The asterisk (*) indicates a significant difference (P < 0.05). N/TERT1 cell lysates were subjected to western blotting using specific antibodies to detect total and phosphorylated Akt (Ser473) and mTOR (Ser2481) (C) and EGFR (D). β-Actin (C) or α-tubulin (D) was used to verify equal loading of protein.

Discussion

Activating the TRPV1 by capsaicin induces Fas/CD95-mediated apoptosis of urothelial cancer cells in an ataxia telangiectasia mutated-dependent manner (18). Capsaicin-induced apoptosis of glioma cells was reported to involve Ca(2+) influx and the mitochondrial pathway, but the observed apoptosis was markedly inhibited by the TRPV1-antagonist, capsazepine (19). In the DMBA/TPA two-stage carcinogenesis model, TRPV1 reportedly exhibited a tumor-suppressing effect in papilloma development through the downregulation of another membrane receptor, the EGFR (12). Thus, inhibiting the function of TRPV1 might increase the risk of tumor development. In this study, we showed that topical application of the TRPV1-antagonist AMG9810 induces skin tumor development after initiation with DMBA.

EGFR function is deregulated in various cancers (20,21) as a result of gene amplification, mutations (resulting in a constitutively active EGFR) or abnormally increased ligand production (22). The EGFR is a receptor tyrosine kinase comprising an extracellular ligand-binding domain, a transmembrane domain and an intracellular tyrosine kinase domain (23). Upon binding their ligands, dynamic conformational changes occur in both the extracellular and intracellular domains of the receptor tyrosine kinases, resulting in the transphosphorylation of tyrosine residues in the C-terminal regulatory domain (23). The tyrosine-phosphorylated motifs of the EGFR recruit various adapters or signaling molecules to transduce signals to downstream pathways (20,21). In this study, after stimulation with AMG9810, the expression level of EGFR increased in both cultured human keratinocytes and in AMG9810-induced skin tumors.

The EGFR activates both the MEK/ERKs and Akt/mTOR-signaling pathways and leads to evasion of apoptosis and increased proliferation, invasion and metastases (24). In this study, Akt/mTOR signaling acts as the primary mediator of AMG9810-induced tumor development as indicated by the markedly higher levels of total and phosphorylated Akt in AMG9810-induced tumors compared with the vehicle-treated group. The Akt/mTOR pathway has been a focus of intense research in recent years because of its substantial role in some of the most fundamental aspects of metabolism, cell growth, proliferation, survival and differentiation at both the cellular and organismal levels (25). This pathway also plays a pivotal role in tumorigenesis. Akt is activated by sequential phosphorylation at Thr308 and Ser473, residues that are located within the activation loop and C-terminal hydrophobic motif, respectively, of Akt. Phosphorylation at Thr308 is induced by 3′phosphoinositide-dependent kinase 1, which requires the binding of PI(3,4,5)P3 to its PH domain and subsequent translocation to the cell membrane for its kinase activity (26). The serine–threonine kinase mTOR exists in two functionally distinct complexes, mTORC1 and mTORC2, in cells. mTORC2 can phosphorylate Akt at Ser473 in vitro and in vivo (27), thereby indicating that mTOR can act as both a substrate and effector of the Akt-signaling pathway. mTORC1 is the downstream target of Akt, comprising mTOR, Raptor, mLST8 and PRAS40, and is sensitive to inhibition by rapamycin (28). mTORC1 promotes protein translation and cell growth by activating p70 ribosomal protein S6 kinase (S6K1) and inactivating eIF4E-binding protein 1 (4E-BP1) (29). Conversely, mTORC2, which is upstream of Akt and insensitive to rapamycin, is composed of mTOR, Rictor, Sin1 and mLST8 (27). The biological function of mTOR in promoting cell growth and tumorigenesis is mediated by phosphorylation of Akt (Ser473). In this study, the Akt/mTOR pathway is activated not only in AMG9810-induced mouse skin tumors and AMG9810-treated skin samples but also in cultured human N/TERT1 keratinocytes.

In future studies, we will determine whether AMG9810 functions as a skin carcinogen by acting through the TRPV1 by using TRPV1 knockout and wild-type mice. Notably, TRPV1 has been validated by genetic deletion and pharmacological inhibition experiments as a target for pain relief. This area of drug development has been moving rapidly forward. Clinical trials with potent small molecule TRPV1 antagonists occurred within less than a decade from the cloning of the TRPV1 (30). The initial reports of the effectiveness of TRPV1 antagonists indicated that they demonstrated pharmacodynamic effects consistent with TRPV1-antagonist activity and anti-hyperalgesic action in humans (31). Thus the question raised is, if TRPV1 antagonists are so effective and unique that other traditional analgesic drugs cannot be substituted for them, how can we avoid the unwanted side effects (e.g. skin cancer)? Because the development of skin tumorigenesis associated with topical application of AMG9810 is linked with activation of the EGFR-signaling pathway, combining inhibitors of EGFR with AMG9810 in long-term topical application might be a good idea. This hypothesis needs further study in additional cellular and mouse models. In conclusion, our data suggest that the TRPV1-antagonist AMG9810 induces skin tumor development through its activation of the EGFR/Akt/mTOR-signaling pathway. Importantly, the idea of inhibiting the biological function of TRPV1 by specific antagonists for pain relief might increase the risk of skin tumorigenesis.

Supplementary material

Supplementary Figures 1 and 2 can be found at http://carcin.oxfordjournals.org/

Funding

The Hormel Foundation and National Institutes of Health (CA111356, CA111536, CA120588, R7CA081064, and ES016548).

Supplementary Material

Supplementary Data
supp_32_5_779__index.html (969B, html)

Acknowledgments

Conflict of Interest Statement: None declared.

Glossary

Abbreviations

EGFR

epidermal growth factor receptor

ERK

extracellular signal-regulated kinase

DMBA

7,12-dimethylbenz(a)anthracene

H&E

hematoxylin and eosin

mTOR

mammalian target of rapamycin

PTEN

phosphatase and tensin homolog

SFM

serum free medium

N/TERT1

telomerase-immortalized primary human keratinocytes 1

TPA

12-O-tetradecanoylphorbol 13-acetate

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supp_32_5_779__index.html (969B, html)
supp_bgr037_Supplemental_Figure_legends.doc (28KB, doc)
supp_bgr037_Sup_Figure_1.tif (4MB, tif)
supp_bgr037_Sup_Figure_2.tif (4.6MB, tif)

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