Dietary energy balance modulates signaling through the Akt/mammalian target of rapamycin pathways in multiple epithelial tissues.

The prevalence of obesity, an established risk factor for several types of cancer, has increased steadily over the past several decades in the United States. New targets and strategies for offsetting the effect of obesity on cancer risk are urgently needed. In the present study, we examined the effect of dietary energy balance manipulation on steady-state signaling in multiple epithelial tissues, with a focus on the Akt and mammalian target of rapamycin (mTOR) pathways. For these experiments, male FVB/N and C57BL/6 and female ICR mice were maintained on a control (10 kcal% fat) diet, a diet-induced obesity (DIO; 60 kcal% fat) regimen, or a 30% calorie restriction (CR) regimen for 15 to 17 weeks. Relative to the control group, the DIO regimen increased, whereas CR decreased, circulating insulin-like growth factor-I (IGF-I) as has previously been reported. Western blot analyses showed that the DIO regimen enhanced, whereas CR inhibited, activation of Akt and mTOR, regardless of epithelial tissue or genetic background. In contrast, activation of AMP-activated protein kinase was modulated by dietary energy balance manipulation in the liver but not in the epidermis or dorsolateral prostate. Western blot analyses of epidermal extracts taken from ICR mice also revealed reduced activation of both the IGF-I receptor and epidermal growth factor receptor in CR mice, compared with control mice or mice maintained on the DIO regimen. Taken together, these novel findings suggest that dietary energy balance modulates signaling through cell-surface receptors (i.e., IGF-I receptor and epidermal growth factor receptor), affecting activation of multiple downstream pathways including Akt and mTOR, thus providing important dietary and pharmacologic targets for disrupting the obesity-cancer link.

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.

Therapeutic effect of rapamycin on gallbladder cancer in a transgenic mouse model.

The macrolide fungicide rapamycin has shown significant antiproliferative action toward a variety of tumor types. In this study, we used BK5.erbB2 transgenic mice as an animal model to examine the therapeutic effect of rapamycin as a potential treatment for gallbladder cancer. Homozygous BK5.erbB2 mice overexpressing the wild-type rat erbB2 gene in basal epithelial cells of the gallbladder have an approximately 70% incidence of gallbladder adenocarcinoma by 2 to 3 months of age. Groups of mice ( approximately 2-3 months of age) were treated with rapamycin by i.p. injection (once daily for 14 days) and then sacrificed 24 h after the last treatment. Rapamycin significantly reduced the incidence and severity of gallbladder carcinoma in BK5.erbB2 mice in a dose-dependent manner. Tumors responsive to treatment exhibited a higher number of apoptotic cells. Furthermore, rapamycin treatment led to decreased levels of phosphorylated p70 S6 kinase (Thr(389)) in gallbladder tissue as assessed by both Western blot and immunofluorescence analyses. Finally, immunofluorescence staining revealed elevated phosphorylated Akt (Ser(473)) and phosphorylated mammalian target of rapamycin (mTOR; Ser(2448)) in human gallbladder cancer compared with normal gallbladder tissue. Based on our results using a novel genetically engineered mouse model and the fact that the Akt/mTOR pathway is activated in human gallbladder cancer, rapamycin and related drugs may be effective therapeutic agents for the treatment of human gallbladder cancer.

Evidence that arachidonate 15-lipoxygenase 2 is a negative cell cycle regulator in normal prostate epithelial cells.

15-Lipoxygenase 2 (15-LOX2) is a recently cloned human lipoxygenase that shows tissue-restricted expression in prostate, lung, skin, and cornea. The protein level and enzymatic activity of 15-LOX2 have been shown to be down-regulated in prostate cancers compared with normal and benign prostate tissues. The biological function of 15-LOX2 and the role of loss of 15-LOX2 expression in prostate tumorigenesis, however, remain unknown. We report the cloning and functional characterization of 15-LOX2 and its three splice variants (termed 15-LOX2sv-a, 15-LOX2sv-b, and 15-LOX2sv-c) from primary prostate epithelial cells. Western blotting with multiple primary prostate cell strains and prostate cancer cell lines reveals that the expression of 15-LOX2 is lost in all prostate cancer cell lines, accompanied by decreased enzymatic activity revealed by liquid chromatography/tandem mass spectrometry analyses. Further experiments show that the loss of 15-LOX2 expression results from transcriptional repression caused by mechanism(s) other than promoter hypermethylation or histone deacetylation. Subsequent functional studies indicate the following: 1) the 15-LOX2 product, 15(S)-hydroxyeicosatetraenoic acid, inhibits prostate cancer cell cycle progression; 2) 15-LOX2 expression in primary prostate epithelial cells is inversely correlated with cell cycle; and 3) restoration of 15-LOX2 expression in prostate cancer cells partially inhibits cell cycle progression. Taken together, these results suggest that 15-LOX2 could be a suppressor of prostate cancer development, which functions by restricting cell cycle progression.