Process Of Tumor Promotion Research Articles

Noninvasive evaluation of hemodynamics and light scattering property during two-stage mouse cutaneous carcinogenesis based on multispectral diffuse reflectance images at isosbestic wavelengths of hemoglobin.

.We investigate a multispectral imaging method to evaluate spatiotemporal changes in both cutaneous hemoglobin concentration and light scattering parameter in mouse skin through diffuse reflectance spectroscopy using the reflectance images acquired at isosbestic wavelengths of hemoglobin (420, 450, 500, and 585 nm). In the proposed approach, Monte Carlo simulation-based empirical formulas are introduced to extract the scattering power representing the wavelength dependence of light scattering spectrum of skin tissue, as well as the total hemoglobin concentration in dermal vasculatures. The use of isosbestic wavelengths of hemoglobin enables the values of and to be estimated independently of the oxygenation of hemoglobin. Experiments using in vivo mice two-stage chemical carcinogenesis model are performed to confirm the feasibility of the proposed method for evaluating the changes in cutaneous vasculatures and tissue morphology during tumor initiation, promotion, and progression processes. The experimental results reveal that the changes in scattering power of back skin are significantly reduced and followed by the increase in total hemoglobin concentration in the carcinogenesis mice group, which indicates morphological changes in skin tissue such as edema and cell swelling caused by tumor promotion and successive angiogenesis along with tumor progression. The results suggest that the potential of the present method to detect cutaneous carcinogenesis in an early stage and monitor physiological changes during promotion and progression process of nonmelanoma tumors.

Abstract 4617: Targeting mTORC1 by overexpression of PRAS40(T246A) in basal keratinocytes suppresses proliferation and migration of keratinocyte stem cells and skin inflammation during tumor promotion

Abstract Considerable evidence indicates a crucial role for Akt and mTORC1 signaling in cancer. Previously, we discovered that the Akt and mTORC1 signaling pathways play an important role in epithelial multistage carcinogenesis using the mouse skin model, especially during the tumor promotion stage. The proline-rich Akt substrate of 40 kDa (PRAS40), a subunit of mTORC1 was originally identified as a novel Akt substrate through Akt-mediated phosphorylation at Thr246 and this phosphorylation is critical for mTORC1 activity. Moreover, PRAS40 has been identified to negatively regulate mTORC1 signaling. In this study, transgenic mice were generated that overexpress a mutant form of PRAS40 [PRAS40(T246A)] using the bovine keratin 5 (BK5) promoter to further examine the importance of mTORC1 signaling in epithelial carcinogenesis. BK5.PRAS40(T246A) transgenic mice did not have a discernable gross skin phenotype. However, these mice were significantly less sensitive to TPA-induced epidermal hyperproliferation and hyperplasia. Overexpression of PRAS40(T246A) in the epidermis led to significant suppression of skin tumor development and significant reductions in tumor weight and tumor size compared to wild-type mice. Immunoprecipitation analysis of epidermal protein lysates from BK5.PRAS40(T246A) mice showed that PRAS40(T246A) remained bound to raptor even after TPA treatment. BK5.PRAS40(T246A) mice also displayed reduced mTORC1 signaling, reduced levels of cell cycle proteins and increased autophagy signaling as well as the skin inflammatory response in the epidermis following treatment with TPA. Notably, TPA-induced proliferation and migration of bulge-region label retaining cells were also significantly inhibited in BK5.PRAS40(T246A) mice. In addition, expression of PRAS40(T246A) in basal keratinocytes significantly inhibited keratinocyte migration both in vivo and in vitro and led to delayed wound healing. Decreased migration and impaired wound healing may be attributed to altered expression of EMT markers as identified in PRAS40(T246A) epidermal cells. The current data using this novel mouse model provides further evidence that mTORC1 specific signaling in keratinocytes contributes significantly to the process of skin tumor promotion by regulating proliferation and migration of keratinocyte stem cells. In addition, the data demonstrate an important role for mTORC1 specific signaling in keratinocytes in regulating skin inflammation. Collectively, using this novel mouse model where mTORC1 signaling is selectively suppressed in keratinocytes has provided further evidence that this signaling pathway regulates several important mechanistic events involved in the process of skin tumor promotion. Research supported by NIH Grants CA129409, CA037111, and American Cancer Research Center and Foundation Citation Format: Okkyung Rho, Jaya Srivastava, Jiyoon Cho, John DiGiovanni. Targeting mTORC1 by overexpression of PRAS40(T246A) in basal keratinocytes suppresses proliferation and migration of keratinocyte stem cells and skin inflammation during tumor promotion. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4617.

Prolonged overexpression of Wnt10b induces epidermal keratinocyte transformation through activating EGF pathway.

Wnt10b is a signaling protein regulating skin development and homeostasis, and the expression of Wnt10b is restricted to epidermal keratinocytes in embryonic and postnatal skin. Recent studies indicate an elevated expression of Wnt10b in skin tumors. However, how Wnt10b regulates skin tumorigenesis remains largely unknown. Here we report that continuous expression of Wnt10b mediates transformation of epidermal keratinocytes through activating genes involved in EGF/MAPK signaling pathways. We first established a prolonged Wnt10b overexpression system in JB6P- cells to represent the elevated Wnt10b expression level in skin keratinocytes. Through expression assays and observations under phase-contrast microscopy, prolonged expression of Wnt10b activated Wnt/β-catenin pathway and induced morphological changes of cells showing longer protrusions and multilayer growth, indicating early-stage cell transformation. Wnt10b also increased cellular proliferation and migration according to BrdU incorporation and cell mobility assays. Furthermore, multi-doses of AdWnt10b treatment to JB6P- cells induced colony formation, stronger invasive ability in transwell system, and anchorage-independent growth in agar gel. In molecular level, AdWnt10b treatment induced increased transcriptional expressions of Egf, downstream Mapk pathway factors, and MMPs. Administration of Wnt antagonist DKK1 blocked the tumor promotion process induced by Wnt10b. Taken together, these findings clearly demonstrate that Wnt10b promotes epidermal keratinocyte transformation through induced Egf pathway.

Effects of extremely low-frequency electromagnetic fields (ELF-EMF) exposure on B6C3F1 mice

Long-term exposure study was conducted to investigate the effects of extremely low-frequency electromagnetic field on the tumor promotion process and fertility. Ten pregnant C57BL/6NCrj mice were exposed to 50 Hz field 500 mG for 1 week (12 h per day), and 24 male and 42 female B6C3F1mice born from them were further exposed up to 15.5 months. As a control group, 10 pregnant mice were bred without exposure, and 30 produced male and 32 female mice were observed without exposure for the same period. Mean body weights of exposed groups of male and female mice were decreased significantly than those of the control groups. In exposed mice, there was no increased incidence of liver and lung tumor. In female mice, the incidence of chronic myeloid leukemia [3/42 (7%)] in the exposed group was significantly greater than in the control group. The size of seminiferous tubules in the EMF exposed groups were significantly less than the control group. These data support the hypothesis that long-term exposure of 50 Hz magnetic fields is a significant risk factor for neoplastic development and fertility in mice.

Abstract 5357: Targeting mTORC1 suppresses proliferation of keratinocyte stem cells and inhibits skin tumor promotion in mice

Abstract Considerable evidence indicates a crucial role of Akt and mTORC1 signaling in cancer growth and progression. Previously, we discovered that Akt and mTORC1 signaling pathways play an important role in epithelial multistage carcinogenesis using the mouse skin model, especially during the tumor promotion stage. Targeting mTORC1 with rapamycin effectively blocked mTORC1 downstream signaling, epidermal hyperproliferation induced by TPA in a dose-dependent manner in both wild-type and BK5.AktWT transgenic mice. Rapamycin effectively inhibited TPA skin tumor promotion in TPA-hypersensitive BK5.AktWT transgenic mice although higher doses were required to achieve similar levels of inhibition compared to wild-type mice. Elevated Akt expression led to a significant expansion of the label retaining cells (LRCs) as well as a significant increase in K15 expressing cells in the hair follicle of transgenic mice that coincided with expression of phospho-Akt, phospho-S6K and phospho-PRAS40 compared to wild-type mice. Furthermore, TPA-mediated proliferation and migration of LRCs to the interfollicular epidermis from the hair follicle were significantly inhibited by rapamycin. PRAS40 was originally identified as a novel Akt substrate through Akt-mediated phosphorylation at Thr246. Recently, PRAS40 has been identified as a negative regulator when bound to mTORC1. We have used the bovine keratin 5 (BK5) promoter to generate transgenic mice that overexpress a mutant form of PRAS40 (PRAS40T246A) to further examine the importance of mTORC1 signaling in epithelial carcinogenesis. BK5.PRAS40T246A transgenic mice were significantly less sensitive to TPA-induced epidermal hyperproliferation and hyperplasia measured by both epidermal thickness and labeling index. Overexpression of mutant PRAS40T246A in the epidermis led to a significant suppression of skin tumor development by 63% as well as reductions in tumor weight and tumor size compared to wild-type mice. Immunoprecipitation analysis showed that PRAS40T246A in BK5.PRAS40T246A mice remained bound to raptor even after TPA treatment. In addition, BK5.PRAS40T246A mice displayed reduced TPA-induced mTORC1 signaling and cell cycle progression, but increased autophagy signaling and elevated apoptotic cells in the epidermis following TPA treatment. Furthermore, TPA-induced proliferation and migration of LRCs was also effectively inhibited in BK5.PRAS40T246A mice. Collectively, the current data demonstrate that mTORC1 downstream signaling pathways contribute significantly to the process of skin tumor promotion leading to a potential target for chemoprevention of cancer. Supported by CA037111 and CA0129409. Citation Format: Okkyung Rho, Jiyoon Cho, John DiGiovanni. Targeting mTORC1 suppresses proliferation of keratinocyte stem cells and inhibits skin tumor promotion in mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5357. doi:10.1158/1538-7445.AM2014-5357

Impact of mTORC1 inhibition on keratinocyte proliferation during skin tumor promotion in wild‐type and BK5.AktWT mice

In this study, we examined the impact of rapamycin on mTORC1 signaling during 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced keratinocyte proliferation and skin tumor promotion in both wild-type (FVB/N) and BK5.Akt(WT) mice. TPA activated mTORC1 signaling in a time-dependent manner in cultured primary mouse keratinocytes and a mouse keratinocyte cell line. Early activation (15-30 min) of mTORC1 signaling induced by TPA was mediated in part by PKC activation, whereas later activation (2-4 h) was mediated by activation of EGFR and Akt. BK5.Akt(WT) transgenic mice, where Akt1 is overexpressed in basal epidermis, are highly sensitive to TPA-induced epidermal proliferation and two-stage skin carcinogenesis. Targeting mTORC1 with rapamycin effectively inhibited TPA-induced epidermal hyperplasia and hyperproliferation as well as tumor promotion in a dose-dependent manner in both wild-type and BK5.Akt(WT) mice. A significant expansion (∼threefold) of the label retaining cell (LRC) population per hair follicle was observed in BK5.Akt(WT) mice compared to FVB/N mice. There was also a significant increase in K15 expressing cells in the hair follicle of transgenic mice that coincided with expression of phospho-Akt, phospho-S6K, and phospho-PRAS40, suggesting an important role of mTORC1 signaling in bulge-region keratinocyte stem cell (KSC) homeostasis. After 2 weeks of TPA treatment, LRCs had moved upward into the interfollicular epidermis from the bulge region of both wild-type and BK5.Akt(WT) mice. TPA-mediated LRC proliferation and migration was significantly inhibited by rapamycin. Collectively, the current data indicate that signaling through mTORC1 contributes significantly to the process of skin tumor promotion through effects on proliferation of the target cells for tumor development.

Abstract 1302: Targeting mTORC1 by epidermal overexpression of PRAS40(T246A) suppresses TPA-induced epidermal hyperplasia during tumor promotion.

Abstract Akt activation is an important event during the early stages of skin tumor promotion by diverse tumor promoting stimuli. Transgenic mice overexpressing either wild-type Akt1 (Aktwt) or a constitutively active form of Akt1 (Aktmyr) exhibit dramatically enhanced susceptibility to two-stage skin carcinogenesis and skin tumor promotion. Several downstream Akt targets are modulated (i.e., increased phosphorylation) in keratinocytes during tumor promotion, including the mammalian target of rapamycin (mTORC1). Recent experiments show that inhibiting mTORC1 by using topical rapamycin in both wild-type and BK5.Aktwt transgenic mice can effectively block TPA-induced activation of mTORC1 signaling, epidermal hyperproliferation and skin tumor promotion in a dose-dependent manner. Treatment with rapamycin also inhibited TPA-induced proliferation and migration of labeling retaining cells (LRCs) of the bulge region from the hair follicles to upper epidermal compartments. To further examine the importance of mTORC1 signaling in epithelial carcinogenesis, we generated BK5.PRAS40mt (PRAS40T246A) mice using the BK5 vector that targets expression to the basal layer of epidermis. PRAS40 was originally identified as a novel Akt substrate with a phosphorylation site at Thr246 and acts as a negative regulator when bound to mTORC1. PRAS40T246A expressed in BK5.PRAS40mt mice remained bound to raptor even after TPA treatment as determined by immunoprecipitation analysis. BK5.PRAS40mt mice displayed reduced phosphorylation of both S6K (T389) and 4EBP1 (S65) in epidermis following treatment with TPA. Furthermore, these transgenic mice were significantly less sensitive to TPA-induced epidermal proliferation (measured by both epidermal thickness and labeling index) compared to nontransgenic siblings. TPA-mediated migration and proliferation of LRCs was also inhibited in BK5.PRAS40mt mice. BK5.PRAS40mt mice also expressed lower levels of cell cycle proteins in epidermis after TPA treatment. Collectively, the current data further support the hypothesis that downstream signaling pathways through mTORC1 contribute significantly to the process of skin tumor promotion through effects on proliferation. Supported by CA37111 and 129409. Citation Format: Okkyung Rho, John DiGiovanni. Targeting mTORC1 by epidermal overexpression of PRAS40(T246A) suppresses TPA-induced epidermal hyperplasia during tumor promotion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1302. doi:10.1158/1538-7445.AM2013-1302

Peracetylated (−)-epigallocatechin-3-gallate (AcEGCG) potently prevents skin carcinogenesis by suppressing the PKD1-dependent signaling pathway in CD34 + skin stem cells and skin tumors

During the process of skin tumor promotion, expression of the cutaneous cancer stem cell (CSC) marker CD34(+) is required for stem cell activation and tumor formation. A previous study has shown that activation of protein kinase D1 (PKD1) is involved in epidermal tumor promotion; however, the signals that regulate CSCs in skin carcinogenesis have not been characterized. This study was designed to investigate the chemopreventive potential of peracetylated (-)-epigallocatechin-3-gallate (AcEGCG) on 7,12-dimethylbenz[a]-anthracene (DMBA)-initiated and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted skin tumorigenesis in ICR mice and to elucidate the possible mechanisms involved in the inhibitory action of PKD1 on CSCs. We demonstrated that topical application of AcEGCG before TPA treatment can be more effective than EGCG in reducing DMBA/TPA-induced tumor incidence and multiplicity. Notably, AcEGCG not only inhibited the expression of p53, p21, c-Myc, cyclin B, p-CDK1 and Cdc25A but also restored the activation of extracellular signal-regulated kinase 1/2 (ERK1/2), which decreased DMBA/TPA-induced increases in tumor proliferation and mitotic index. To clarify the role of PKD1 in cell proliferation and tumorigenesis, we studied the expression and activation of PKD1 in CD34(+) skin stem cells and skin tumors. We found that PKD1 was strongly expressed in CD34(+) cells and that pretreatment with AcEGCG markedly inhibited PKD1 activation and CD34(+) expression. More importantly, pretreatment with AcEGCG remarkably suppressed nuclear factor-kappaB, cyclic adenosine 3',5'-monophosphate-responsive element-binding protein (CREB) and CCAAT-enhancer-binding protein (C/EBPs) activation by inhibiting the phosphorylation of c-Jun-N-terminal kinase 1/2, p38 and phosphatidylinositol 3-kinase (PI3K)/Akt and by attenuating downstream target gene expression, including inducible nitric oxide synthase, cyclooxygenase-2, ornithine decarboxylase and vascular endothelial growth factor. Moreover, this is the first study to demonstrate that AcEGCG is a CD34(+) and PKD1 inhibitor in the multistage mouse skin carcinogenesis model. Overall, our results powerfully suggest that AcEGCG could be developed into a novel chemopreventive agent and that PKD1 may be a preventive and therapeutic target for skin cancer in clinical settings.

Tumor promoting effects of cyanobacterial extracts are potentiated by anthropogenic contaminants – Evidence from in vitro study

Inhibition of gap junctional intercellular communication (GJIC) is affiliated with tumor promotion process and it has been employed as an in vitro biomarker for evaluation of tumor promoting effects of chemicals. In the present study we investigated combined effects of anthropogenic environmental contaminants 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB 153) and fluoranthene, cyanotoxins microcystin-LR and cylindrospermopsin, and extracts of laboratory cultures of cyanobacteria Aphanizomenon gracile and Cylindrospermopsis raciborskii, on GJIC in the rat liver epithelial cell line WB-F344. Binary mixtures of PCB 153 with fluoranthene and the mixtures of the two cyanobacterial strains elicited simple additive effects on GJIC after 30min exposure, whereas microcystin-LR and cylindrospermopsin neither inhibited GJIC nor altered effects of PCB 153 or fluoranthene. However, synergistic effects were observed in the cells exposed to binary mixtures of anthropogenic contaminants (PCB 153 or fluoranthene) and cyanobacterial extracts. The synergistic effects were especially pronounced after prolonged (6–24h) co-exposure to fluoranthene and A. gracile extract, when mixture caused nearly complete GJIC inhibition, while none of the individual components caused any downregulation of GJIC at the same concentration and exposure time. The effects of cyanobacterial extracts were independent of microcystin-LR or cylindrospermopsin, which were not detected in cyanobacterial biomass. It provides further evidence on the presence of unknown tumor promoting metabolites in cyanobacteria. Clear potentiation of the GJIC inhibition observed in the mixtures of two anthropogenic contaminants and cyanobacteria highlight the importance of combined toxic effects of chemicals in complex environmental mixtures.

Abstract 2559: Targeting mTORC1 may provide selectivity for inhibiting proliferation of hair follicle stem/progenitor cells during tumor promotion

Abstract Activation of Akt occurs during tumor promoter treatment of mouse skin and plays an important role in skin tumor promotion through multiple downstream pathways, including mTORC1. In this study, we examined the impact of rapamycin on mTORC1 signaling in relation to its effect on TPA-induced epidermal proliferation and skin tumor promotion. TPA activates mTORC1 signaling in a time-dependent manner in cultured primary mouse keratinocytes and an immortalized mouse keratinocyte cell line. Early activation (15-30 min) of mTORC1 signaling induced by TPA was mediated in part, by PKC activation, whereas the later activation (2-4 hr) of mTORC1 was mediated by activation of EGFR and Akt. BK5.AktWT transgenic mice where Akt1 is overexpressed in basal epidermis were significantly more sensitive to TPA-induced epidermal proliferation and two-stage skin carcinogenesis. Targeting mTORC1 effectively inhibited TPA-induced epidermal hyperplasia as well as tumor promotion in epidermis of both wild-type and BK5.AktWT mice in a dose-dependent manner. We also examined the effect of rapamycin on the proliferation and migration of bulge-region KSCs from the hair follicles induced by TPA. Ten-day old mice were injected with BrdU every 12h over a 48h time period and then 70 days later mice were treated with acetone, TPA or rapamycin and TPA. Seventy days after the last injection of BrdU, only the label retaining cells (LRCs) were seen in the bulge region of the hair follicles. BK5.Akt1WT transgenic mice, which overexpress Akt1 in the K5 compartment of mouse epidermis (which includes bulge region KSCs) led to significant expansion of LRCs (∼3-fold) per hair follicle compared with wild-type FVB mice. There was also a significant expansion of the K15 expressing cell population, especially in the hair follicle that coincided with expression of phospho-Akt. In addition, a significant increase in phospho-S6K and phospho-PRAS40 was seen in the hair follicles, suggesting an important role of mTORC1 signaling in the alterations seen in terms of LRCs and K15 expressing cells. After two weeks of TPA treatment, LRCs were reduced in the bulge region and moved upward into the interfollicular epidermis. Treatment with rapamycin inhibited the migration and proliferation of the LRCs from the hair follicles to upper epidermal compartments induced by TPA. Furthermore, LRCs remained in the bulge region of hair follicles. Since the bulge-region KSCs are considered to be the key target population for tumor development in the two-stage skin carcinogenesis model, these data indicate that signaling through mTORC1 contributes significantly to the process of skin tumor promotion through effects on proliferation of multiple keratinocyte populations including bulge-region KSCs. Thus, blocking this pathway may be an effective strategy for prevention of epithelial carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2559. doi:1538-7445.AM2012-2559

Rapamycin Is a Potent Inhibitor of Skin Tumor Promotion by 12-O-Tetradecanoylphorbol-13-Acetate

Aberrant activation of phosphoinositide-3-kinase (PI3K)/Akt signaling has been implicated in the development and progression of multiple human cancers. During the process of skin tumor promotion induced by treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), activation of epidermal Akt occurs as well as several downstream effectors of Akt, including the activation of mTORC1. Rapamycin, an established mTORC1 inhibitor, was used to further explore the role of mTORC1 signaling in epithelial carcinogenesis, specifically during the tumor promotion stage. Rapamycin blocked TPA-induced activation of mTORC1 as well as several downstream targets. In addition, TPA-induced epidermal hyperproliferation and hyperplasia were inhibited in a dose-dependent manner with topical rapamycin treatments. Immunohistochemical analyses of the skin from mice in this multiple treatment experiment revealed that rapamycin also significantly decreased the number of infiltrating macrophages, T cells, neutrophils, and mast cells seen in the dermis following TPA treatment. Using a two-stage skin carcinogenesis protocol with 7,12-dimethylbenz(a)anthracene (DMBA) as initiator and TPA as the promoter, rapamycin (5-200 nmol per mouse given topically 30 minutes prior to TPA) exerted a powerful antipromoting effect, reducing both tumor incidence and tumor multiplicity. Moreover, topical application of rapamycin to existing papillomas induced regression and/or inhibited further growth. Overall, the data indicate that rapamycin is a potent inhibitor of skin tumor promotion and suggest that signaling through mTORC1 contributes significantly to the process of skin tumor promotion. The data also suggest that blocking this pathway either alone or in combination with other agents targeting additional pathways may be an effective strategy for prevention of epithelial carcinogenesis.

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

Abstract 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.

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

Abstract 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.

Involvement of glycogen synthase kinase-3β signaling and aberrant nucleocytoplasmic localization of retinoblastoma protein in tumor promotion in a rat two-stage thyroid carcinogenesis model

To investigate the cell cycle kinetics during the tumor promotion process induced by hypothyroidism in a rat model of thyroid follicular cell carcinogenesis, immunohistochemical analysis of cell cycle molecules and related signaling molecules was performed in conjunction with analysis of cell proliferation activity in an initiation-promotion model. Male F344 rats were injected with N-bis(2-hydroxypropyl)nitrosamine, and one week later treated with 6-propyl-2-thiouracil (PTU) at 12ppm in the drinking water for 4, 10 or 15 weeks. At each time point, proliferative lesions increased the expression of cyclin A, cyclin D, cyclin E and cyclin-dependent kinase (Cdk)-2, in association with the development of lesion stages from the early focal hyperplasia to the late carcinoma, while a subpopulation of proliferative lesions showed decreased numbers of both cell division cycle-2- and Ki-67-positive cells at week 15 compared with that at week 10, suggesting a reduced promoting effect of serum thyroid-stimulating hormone in the sensitive cellular population after long-term exposure to PTU. On the other hand, increased immunolocalization of phosphorylated and inactive glycogen synthase kinase (GSK)-3β was observed in a subpopulation of proliferative lesions, in parallel with the cyclins and Cdk2. Nuclear immunoreactivity of phosphorylated and inactive retinoblastoma (Rb) protein was also increased in association with lesion development, with carcinomas showing increased cytoplasmic localization. The results suggest that proliferative lesions activate the cell cycle machinery following tumor promotion via a regulatory mechanism involving inactivation of GSK3β and Rb protein, the latter signaling mechanism involving its aberrant nucleocytoplasmic transport for the acquisition of a malignant phenotype.

Crosstalk between PTEN/Akt2 and TGFβ signaling involving EGF receptor down‐regulation during the tumor promotion process from the early stage in a rat two‐stage hepatocarcinogenesis model

The present study investigated the involvement of signaling of phosphatase and tensin homolog deleted on chromosome 10 (PTEN)/protein kinase B (Akt) and transforming growth factor-beta (TGFbeta) as well as receptor tyrosine kinases in the tumor promotion processes in a two-stage hepatocarcinogenesis model using male F344 rats. The cellular localization of related molecules was examined in liver cell foci expressing glutathione S-transferase placental form (GST-P) at the early stage of tumor promotion by fenbendazole (FB), piperonyl butoxide, or thioacetamide. Distribution in the liver cell foci and neoplastic lesions positive for GST-P was also examined at the later stage of FB promotion. In contrast to the initiation-alone cases, subpopulations of GST-P-positive foci induced by promotion for 6 weeks, regardless of the promoting chemicals used, enhanced down-regulation of PTEN and up-regulation of phosphorylated (active) Akt2 and phosphorylated substrate(s) of Akt-kinase activity. Also, up-regulation of TGFbeta receptor I and down-regulation of epidermal growth factor receptor (EGFR) were enhanced in the subpopulation of GST-P-positive foci in all promoted cases. A similar pattern of cellular distribution of these molecules was also observed in the neoplastic lesions at the late stage. These results suggest a crosstalk between Akt2 and TGFbeta signaling that involves a mechanism requiring EGFR down-regulation during the entire tumor promotion process starting from the early stage. In particular, a shift in subcellular localization of phosphorylated substrate(s) of Akt from the cell membrane in liver cell foci to the cytoplasm in carcinomas was observed, suggesting an alteration of the function or activity of the corresponding molecule(s).

Inhibition of Gap Junctional Intercellular Communication by the Green Tea Polyphenol (−)-Epigallocatechin Gallate in Normal Rat Liver Epithelial Cells

(-)-Epigallocatechin gallate (EGCG), a polyphenolic compound found in green tea, is a promising chemopreventive agent against cancer due to its strong antiproliferative effects on cancer cells; however, its possible toxicity and carcinogenicity must be investigated before EGCG can be used as a dietary supplement for chemoprevention. The inhibition of gap junctional intercellular communication (GJIC) is strongly associated with carcinogenesis, particularly the tumor promotion process; thus, we investigated the effects of EGCG on GJIC in WB-F344 normal rat liver epithelial (RLE) cells. EGCG, but not (-)-epicatechin (EC), another polyphenol found in green tea, inhibited GJIC in a dose-dependent and reversible manner in RLE cells. EGCG also induced the phosphorylation of connexin 43 (Cx43), a major regulator of GJIC. The phosphorylation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) was also observed in EGCG-treated RLE cells. The inhibition of GJIC and phosphorylation of Cx43 and ERK1/2 by EGCG were completely blocked by U0126, a pharmacological inhibitor of mitogen-activated protein kinase/ERK kinase. EGCG generated a larger amount of hydrogen peroxide than EC in a dose-dependent manner. Furthermore, catalase partially inhibited the EGCG-induced inhibition of GJIC and the phosphorylation of Cx43 and ERK1/2. These results indicated that EGCG inhibited GJIC mainly due to its prooxidant activity.

Cellular distributions of molecules with altered expression specific to the tumor promotion process from the early stage in a rat two-stage hepatocarcinogenesis model

A global gene expression profiling specific to the early process of tumor promotion by fenbendazole (FB) or phenobarbital (PB) in a rat two-stage hepatocarcinogenesis model revealed 33 genes to show altered expression in common with both chemicals. The immunohistochemical distribution of transferrin receptor (Tfrc), nuclear receptor subfamily 0, group B, member 2 (Nr0b2) and minichromosome maintenance deficient 6 (MCM6), included in the altered expression profile, were therefore examined in FB- and PB-induced proliferative lesions at both early and late stages of tumor promotion. In addition, immunoexpression of transforming growth factor beta receptor (TGFbetaR) I, TGFbetaRII, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and phosphorylated phosphatase and tensin homolog deleted on chromosome 10 (pPTEN) was also examined. In the early stage, most hepatocellular foci positive for glutathione S-transferase placental form (GST-P) showed co-expression of TGFbetaRI and lack of PTEN and pPTEN, some GST-P-positive foci co-expressing Tfrc and Nr0b2. In the late stage, selective expression of TGFbetaRI, but not TGFbetaRII, was also observed in many adenomas and carcinomas consistently expressing GST-P. Nr0b2 was variably expressed in the proliferative lesions, irrespective of the carcinogenic stage. Like the GST-P-positive foci, adenomas and carcinomas consistently lacked PTEN and pPTEN. Expression of Tfrc and MCM6 was increased in parallel with the carcinogenic stage. In conclusion, loss of PTEN and dysregulation of transforming growth factor beta signaling can be considered to be involved in rat hepatocarcinogenesis from early stages. Selective expression of Tfrc in proliferative lesions suggests an involvement of changes in iron homeostasis during the process of tumor promotion/progression driven by FB or PB.

Cancer chemoprevention by phytochemicals: potential molecular targets, biomarkers and animal models

Recent studies have strongly indicated that certain daily-consumed dietary phytochemicals could have cancer protective effects against transgenic mice cancer models and cancers mediated by carcinogens, irradiations and carcinogenic metabolites derived from exogenous or endogenous sources. The cancer-protective effects elicited by these dietary compounds are believed to be due at least in part to the induction of cellular defense systems including the detoxifying and antioxidant enzymes system, as well as the inhibition of anti-inflammatory and anti-cell growth signaling pathways culminating in cell cycle arrest and/or celldeath. In this review, we summarize the potential mechanisms including the modulation of nuclear factor kappaB (NF-kappaB), cyclooxygenases-2 (COX-2), activator protein-1 (AP-1), mitogen-activated protein kinases (MAPKs) and the induction of phase II cellular detoxifying and antioxidant enzymes mediated mainly by the antioxidant response elements (ARE) within the promoter regions of these genes through nuclear factor-erythroid 2-related factor 2 (Nrf2), a member of the Cap "n" collar (CNC) family of the basic region-leucine zipper transcription factor. In addition, we also review several animal models of carcinogenesis and cancer chemopreventive efficacy studies of these animal models using dietary chemopreventive compounds. Finally, we discuss the cellular signaling cascades mediated by Nrf2, NF-kappaB, AP-1, MAPKs and COX-2, which have been considered to play pivotal roles in tumor initiation, promotion and progression processes, and could be promising molecular targets for the design of drugs targeting cancer prevention and therapy.

The Possible Mechanism of Tetrachlorohydroquinone-Induced Apoptosis and Tumor Promotion in NIH3T3 Cells

ISEE-109 Objective: Pentachlorophenol (PCP) and its salt are used extensively as biocide and wood preservative. Because of improper disposal, PCP has become an environmental pollutant and is considered to be ubiquitous. Tetrachlorohydroquinone (TCHQ) is the major toxic metabolite of PCP. Recently, we found that TCHQ was a tumor promoter in a mouse skin carcinogenesis model. However, the detail mechanism of TCHQ-induced tumor promotion is still not clear. In this study, we intended to discover the role of Bcl-2 in TCHQ-induced apoptosis and its related possible signaling pathways in NIH3T3 cells. Material and Methods: DNA fragmentation assay was used to characterize apoptosis in NIH3T3 cells. Flow cytometry analysis was performed to quantify apoptotic cells and intracellular ROS levels. Western blotting analysis was applied to investigate the molecular basis for the effects of TCHQ in NIH3T3 cells. Results: Over-expression of Bcl-2 effectively suppresses TCHQ-induced apoptosis in NIH3T3 cells, as evidenced by morphologic changes and DNA fragmentation. Although production of reactive oxygen species (ROS) contributes to TCHQ-induced apoptosis, Bcl-2 fails to attenuate TCHQ-elicited increase of intracellular ROS level. In addition, over-expressed Bcl-2 provides only partial protection against TCHQ-induced cellular DNA damage. Interestingly, TCHQ induced a significant up-regulation of Bcl-2 expression, and over-expressed Bcl-2 can inhibit TCHQ-induced apoptosis. We also found that TCHQ increased ATM protein expression, but did not induce downstream p53 accumulation and Bax up-regulation. In addition, TCHQ induced expression of both phospho-p38 and phospho-JNK, which belong to MAPK cascade pathway. Conclusions: These data indicate that Bcl-2 prevents TCHQ-induced apoptosis at the downstream of oxidative damage events. The possible signaling pathway of TCHQ-induced apoptosis may relate to MAPK pathway instead of ATM-dependent pathway. Taken together, we suggest that chronic exposure of TCHQ could facilitate the process of tumor promotion through induction of Bcl-2 expression and subsequent apoptosis inhibition.

Dietary Modulation of the Multistage, Multimechanisms of Human Carcinogenesis: Effects on Initiated Stem Cells and Cell–Cell Communication

: Diet can influence the risk to cancer in both negative and positive ways. Worldwide, more than 10 million persons develop cancer annually. Diet could prevent many cancers. Carcinogenesis is a multistage, multimechanism process, consisting of "initiation," "promotion," and "progression" phases. Although diet could affect each phase, an efficacious strategy for dietary chemoprevention would be intervention during the promotion phase. The tumor-promotion process requires sustained exposure to agents that stimulate the growth and inhibition of apoptosis of initiated cells in the absence of antipromoters. Chronic inflammation has been associated with the promotion process. The mechanism affecting the promotion process appears to be the inhibition of cell–cell communication between normal and initiated cells. Most, if not all, tumor-promoting agents and conditions, reversibly, inhibit cell–cell communication, whereas antipromoters, antioxidants, and anti-inflammatory agents have been shown to ameliorate the effects of tumor promoters on cell–cell communication. Additionally, adult stem cells are hypothesized to be the target cells for initiating the carcinogenic process. A new paradigm has been presented that postulates the first function of the carcinogenic process is to block the "mortalization" of a normal, "immortal" adult stem cell rather than the induction of "immortalization" of a normal mortal cell.