Exercise Activates p53 and Negatively Regulates IGF-1 Pathway in Epidermis within a Skin Cancer Model.
ABSTRACT: Exercise has been previously reported to lower cancer risk through reducing circulating IGF-1 and IGF-1-dependent signaling in a mouse skin cancer model. This study aims to investigate the underlying mechanisms by which exercise may down-regulate the IGF-1 pathway via p53 and p53-related regulators in the skin epidermis. Female SENCAR mice were pair-fed an AIN-93 diet with or without 10-week treadmill exercise at 20 m/min, 60 min/day and 5 days/week. Animals were topically treated with TPA 2 hours before sacrifice and the target proteins in the epidermis were analyzed by both immunohistochemistry and Western blot. Under TPA or vehicle treatment, MDM2 expression was significantly reduced in exercised mice when compared with sedentary control. Meanwhile, p53 was significantly elevated. In addition, p53-transcriptioned proteins, i.e., p21, IGFBP-3, and PTEN, increased in response to exercise. There was a synergy effect between exercise and TPA on the decreased MDM2 and increased p53, but not p53-transcripted proteins. Taken together, exercise appeared to activate p53, resulting in enhanced expression of p21, IGFBP-3, and PTEN that might induce a negative regulation of IGF-1 pathway and thus contribute to the observed cancer prevention by exercise in this skin cancer model.
Project description:Loss of PTEN, the major negative regulator of the PI3K/AKT pathway induces a cellular senescence as a failsafe mechanism to defend against tumorigenesis, which is called PTEN-loss-induced cellular senescence (PICS). Although many studies have indicated that the mTOR pathway plays a critical role in cellular senescence, the exact functions of mTORC1 and mTORC2 in PICS are not well understood. In this study, we show that mTOR acts as a critical relay molecule downstream of PI3K/AKT and upstream of p53 in PICS. We found that PTEN depletion induces cellular senescence via p53-p21 signaling without triggering DNA damage response. mTOR kinase, a major component of mTORC1 and mTORC2, directly binds p53 and phosphorylates it at serine 15. mTORC1 and mTORC2 compete with MDM2 and increase the stability of p53 to induce cellular senescence via accumulation of the cell cycle inhibitor, p21. In embryonic fibroblasts of PTEN-knockout mice, PTEN deficiency also induces mTORC1 and mTORC2 to bind to p53 instead of MDM2, leading to cellular senescence. These results collectively demonstrate for the first time that mTOR plays a critical role in switching cells from proliferation signaling to senescence signaling via a direct link between the growth-promoting activity of AKT and the growth-suppressing activity of p53.
Project description:The MDM2-p53 pathway plays a prominent role in well-differentiated liposarcoma (LPS) pathogenesis. Here, we explore the importance of MDM2 amplification and p53 mutation in LPS independently, to determine whether HDACi are therapeutically useful in LPS. We demonstrated that simultaneous knockdown of MDM2 and p53 in p53-mutant LPS lines resulted in increased apoptosis, anti-proliferative effects, and cell cycle arrest, as compared to either intervention alone. HDACi treatment resulted in the dephosphorylation and depletion of MDM2 and p53 without affecting CDK4 and JUN expression, irrespective of p53 mutational status in MDM2-amplified LPS. In control mesothelioma cell lines, HDACi treatment resulted in down-regulation of p53 in the p53 mutant cell line JMN1B, but resulted in no changes of MDM2 and p53 in two mesothelioma lines with normal MDM2 and wild-type p53. HDACi treatment substantially decreased LPS and mesothelioma proliferation and survival, and was associated with upregulation of PTEN and p21, and inactivation of AKT. Our findings indicate that wild-type p53 depletion by HDACi is MDM2 amplification-dependent. These findings underscore the importance of targeting both MDM2 and p53 in LPS and other cancers harboring p53 mutations. Moreover, the pro-apoptotic and anti-proliferative effect of HDACi warrants further evaluation as a therapeutic strategy in MDM2-amplified LPS.
Project description:Mdm2, the principal negative regulator of p53, is critical for survival, a fact clearly demonstrated by the p53-dependent death of germline or conditional mice following deletion of Mdm2. On the other hand, Mdm2 hypomorphic (Mdm2Puro/?7-12) or heterozygous (Mdm2+/-) mice that express either 30 or 50% of normal Mdm2 levels, respectively, are viable but present distinct phenotypes because of increased p53 activity. Mdm2 levels are also transcriptionally regulated by p53. We evaluated the significance of this reciprocal relationship in a new hypomorphic mouse model inheriting an aberrant Mdm2 allele with insertion of the neomycin cassette and deletion of 184-bp sequence in intron 3. These mice also carry mutations in the Mdm2 P2-promoter and thus express suboptimal levels of Mdm2 entirely encoded from the P1-promoter. Resulting mice exhibit abnormalities in skin pigmentation and reproductive tissue architecture, and are subfertile. Notably, all these phenotypes are rescued on a p53-null background. Furthermore, these phenotypes depend on distinct p53 downstream activities as genetic ablation of the pro-apoptotic gene Puma reverts the reproductive abnormalities but not skin hyperpigmentation, whereas deletion of cell cycle arrest gene p21 does not rescue either phenotype. Moreover, p53-mediated upregulation of Kitl influences skin pigmentation. Altogether, these data emphasize tissue-specific p53 activities that regulate cell fate.
Project description:High levels of insulin-like growth factor (IGF)-I may increase the risk of common cancers in humans. We hypothesized that weight loss induced by diet and/or exercise would reduce IGF-I in postmenopausal women. Four hundred and thirty nine overweight or obese [body mass index (BMI) ? 25 kg/m(2)] women (50-75 years) were randomly assigned to: (i) exercise (N = 117), (ii) dietary weight loss (N = 118), (iii) diet + exercise (N = 117), or (iv) control (N = 87). The diet intervention was a group-based program with a 10% weight loss goal. The exercise intervention was 45 minutes/day, 5 days/week of moderate-to-vigorous intensity activity. Fasting serum IGF-I and IGF-binding protein (IGFBP)-3 were measured at baseline and 12 months by radioimmunoassay. Higher baseline BMI was associated with lower IGF-I and IGF-I/IGFBP-3 molar ratio. Although no significant changes in either IGF-I or IGFBP-3 were detected in any intervention arm compared with control, the IGF-I/IGFBP-3 ratio increased significantly in the diet (+5.0%, P < 0.01) and diet + exercise (+5.4%, P < 0.01) groups compared with control. Greater weight loss was positively associated with change in both IGF-I (P(trend) = 0.017) and IGF-I/IGFBP-3 ratio (P(trend) < 0.001) in the diet group, but inversely with change in IGFBP-3 in the diet + exercise group (P(trend) = 0.01). No consistent interaction effects with baseline BMI were detected. Modified IGF-I bioavailability is unlikely to be a mechanism through which caloric restriction reduces cancer risk in postmenopausal women.
Project description:Insulin-like growth factor-1 (IGF-1) promotes the survival of cardiomyocytes by activating type 1 IGF receptor (IGF-1R). Within the myocardium, IGF-1 action is modulated by IGF binding protein-3 (IGFBP-3), which sequesters IGF-1 away from IGF-1R. Since cardiomyocyte apoptosis is implicated in anthracycline cardiotoxicity, we investigated the effects of the anthracycline, doxorubicin, on the IGF-1 system in H9c2 cardiomyocytes.Besides inducing apoptosis, concentrations of doxorubicin comparable to those observed in patients after bolus infusion (0.1-1 µM) caused a progressive decrease in IGF-1R and increase in IGFBP-3 expression. Exogenous IGF-1 was capable to rescue cardiomyocytes from apoptosis triggered by 0.1 and 0.5 µM, but not 1 µM doxorubicin. The loss of response to IGF-1 was paralleled by a significant reduction in IGF-1 availability and signaling, as assessed by free hormone levels in conditioned media and Akt phosphorylation in cell lysates, respectively. Doxorubicin also dose-dependently induced p53, which is known to repress the transcription of IGF1R and induce that of IGFBP3. Pre-treatment with the p53 inhibitor, pifithrin-?, prevented apoptosis and the changes in IGF-1R and IGFBP-3 elicited by doxorubicin. The decrease in IGF-1R and increase in IGFBP-3, as well as apoptosis, were also antagonized by pre-treatment with the antioxidant agents, N-acetylcysteine, dexrazoxane, and carvedilol.Doxorubicin down-regulates IGF-1R and up-regulates IGFBP-3 via p53 and oxidative stress in H9c2 cells. This leads to resistance to IGF-1 that may contribute to doxorubicin-initiated apoptosis. Further studies are needed to confirm these findings in human cardiomyocytes and explore the possibility of manipulating the IGF-1 axis to protect against anthracycline cardiotoxicity.
Project description:Unlike most other matrix metalloproteinases (MMPs) MMP-19 is expressed in undifferentiated basal keratinocytes of healthy human skin. The human keratinocyte cell line HaCaT, which like basal keratinocytes constitutively expresses MMP-19, down-regulated the expression of MMP-19 at high calcium concentrations. Calcium-regulation occurred through E-cadherin mediated cell-cell contacts because neutralizing anti-E-cadherin antibodies restored MMP-19 expression in high calcium. Overexpression of MMP-19 in HaCaT cells (HaCaT-WT) increased cellular proliferation, as well as migration and adhesion on type I collagen. This was due to proteolysis of the insulin-like growth factor (IGF) binding protein-3 by MMP-19, which augmented signaling through the IGF-I receptor, as evidenced by its increased autophosphorylation. Conversely, these effects were not observed in cells transfected with MMP-2 or a catalytically inactive MMP-19 mutant. As further proof that increased IGF-signaling promoted adhesion and migration in HaCaT-WT cells, we reproduced these effects by treating parental HaCaT with IGF-I. We observed dephosphorylation of the focal adhesion kinase in HaCaT-WT as well as IGF-I-treated HaCaT cells, suggesting that inactivating focal adhesion kinase is a mechanism by which IGF-I enhances adhesion. Furthermore, IGF-I-triggered motility on type I collagen was mediated by MMP activity, which, however, was distinct from MMP-19. Considering the coexpression of IGFBP-3 and MMP-19 in the skin, we conclude that MMP-19 is a likely candidate to be the major IGFBP-3 degrading MMP in the quiescent epidermis. This activity might have widespread consequences for the behavior of epidermal keratinocytes.
Project description:Insulin-like growth factor 1 receptor (IGF-1R) is important in cancer cell growth and survival and has been implicated in cancer pathophysiology and treatment. Here we report a novel function for IGF-1R in p53-dependent apoptotic response. We show that inhibition or loss of IGF-1R activity reduces translational synthesis of p53 and Mdm2 protein. Notably, IGF-1R inhibition increases p53 protein stability by reducing p53 ubiquitination and maintains p53 at low levels by decreasing p53 synthesis, thus rendering p53 insensitive to stabilization after DNA damage. The accumulation and apoptosis of DNA-damage-induced p53 is therefore reduced in Igf-1r(-/-) mouse embryonic fibroblasts or tumor cells treated with the IGF-1R inhibitor. Furthermore, we find that inhibition of IGF-1R reduces p53 and Mdm2 translation through a gene-specific mechanism mediated by the respective 5' untranslated region of p53 and mdm2 messenger RNA. The eukaryotic translation initiation factor 4F complex is also involved in this translational inhibition. These results demonstrate an unexpected role for translational control by IGF-1R in p53-mediated apoptosis.
Project description:Activation of p53 is an important mechanism in apoptosis. However, whether the presence of p53 in mitochondria plays an important role in p53-mediated apoptosis is unclear. Here, we demonstrate that overexpression of NPM (nucleophosmin) significantly suppresses 12-O-tetradecanoylphorbol 13-acetate (TPA)-mediated apoptosis, in part, by blocking the mitochondrial localization of p53. Within 1 h following TPA treatment of skin epithelial (JB6) cells, p53 accumulated in mitochondria. Expression of NPM enhances p53 levels in the nucleus but reduces p53 levels in mitochondria, as detected by immunocytochemistry and Western blot analysis. The suppressive effect of NPM on p53 mitochondrial localization is also observed in TPA-treated primary epithelial cells and in JB6 cells treated with doxorubicin. NPM enhances the expression of p53 target gene p21 and bax. However, the increase in Bax level in the absence of p53 in mitochondria did not lead to an increase in TPA-induced apoptosis, suggesting that the presence of p53 in mitochondria is important. Suppression of NPM by NPM small interfering RNA leads to an increase of p53 levels in mitochondria and apoptosis. Furthermore, suppression of NPM in tumor cells with a high constitutive level of NPM results in p53 translocation to mitochondria and enhances TPA-mediated apoptosis. The results demonstrate the effect of NPM on p53 localization in mitochondria and apoptosis. Together, the data indicate that the presence of p53 in mitochondria plays an important role in stress-induced apoptosis and suggest that NPM may protect cells from apoptosis by reducing the mitochondrial level of p53.
Project description:Insulin-like growth factor binding protein-3 (IGFBP-3) is a p53 tumor suppressor-regulated protein and a major carrier for IGFs in circulation. Among six high-affinity IGFBPs, which are IGFBP-1 through 6, IGFBP-3 is the most extensively investigated IGFBP species with respect to its IGF/IGF-I receptor (IGF-IR)-independent biological actions beyond its endocrine/paracrine/autocrine role in modulating IGF action in cancer. Disruption of IGFBP-3 at transcriptional and post-translational levels has been implicated in the pathophysiology of many different types of cancer including breast, prostate, and lung cancer. Over the past two decades, a wealth of evidence has revealed both tumor suppressing and tumor promoting effects of IGF/IGF-IR-independent actions of IGFBP-3 depending upon cell types, post-translational modifications, and assay methods. However, IGFBP-3's anti-tumor function has been well accepted due to identification of functional IGFBP-3-interacting proteins, putative receptors, or crosstalk with other signaling cascades. This review mainly focuses on transmembrane protein 219 (TMEM219), which represents a novel IGFBP-3 receptor mediating antitumor effect of IGFBP-3. Furthermore, this review delineates the potential underlying mechanisms involved and the subsequent biological significance, emphasizing the clinical significance of the IGFBP-3/TMEM219 axis in assessing both the diagnosis and the prognosis of cancer as well as the therapeutic potential of TMEM219 agonists for cancer treatment.
Project description:Insulin-like growth factor (IGF)-I binds to the ECM protein vitronectin (VN) through IGF binding proteins (IGFBPs) to enhance proliferation and migration of skin keratinocytes and fibroblasts. Although evidence exists for the role of individual components of the complex (IGF-I, IGFBP-3 and VN), the cellular functions stimulated by these proteins together as a complex remains un-investigated in melanoma cells. We report here that the IGF-I:IGFBP-3:VN trimeric complex stimulates a dose-dependent increase in the proliferation and migration of WM35 and Sk-MEL28 melanoma cells. In 3D Matrigel™ and hydrogel cultures, both cell lines formed primary tumor-like spheroids, which increased in size in a dose-dependent manner in response to the trimeric complex. Furthermore, we reveal IGFBP-3:VN protein complexes in malignant melanoma and squamous cell carcinoma patient tissues, where the IGFBP-3:VN complex was seen to be predominantly tumor cell-associated. Peptide antagonists designed to target the binding of IGF-I:IGFBP-3 to VN were demonstrated to inhibit IGF-I:IGFBP-3:VN-stimulated cell migration, invasion and 3D tumor cell growth of melanoma cells. Overall, this study provides new data on IGF:ECM interactions in skin malignancies and demonstrates the potential usefulness of a growth factor:ECM-disrupting strategy for abrogating tumor progression.