Trp53 regulates Notch 4 signaling through Mdm2.

Notch receptors and their ligands have crucial roles in development and tumorigenesis. We present evidence demonstrating the existence of an antagonistic relationship between Notch 4 and Trp53, which is controlled by the Mdm2-dependent ubiquitylation and degradation of the Notch receptor. We show that this signal-controlling mechanism is mediated by physical interactions between Mdm2 and Notch 4 and suggest the existence of a trimeric complex between Trp53, Notch 4 and Mdm2, which ultimately regulates Notch activity. Functional studies indicate that Trp53 can suppress NICD4-induced anchorage-independent growth in mammary epithelial cells and present evidence showing that Trp53 has a pivotal role in the suppression of Notch-associated tumorigenesis in the mammary gland.

Insulin-mediated acceleration of breast cancer development and progression in a nonobese model of type 2 diabetes.

Epidemiologic studies suggest that type 2 diabetes (T2D) increases breast cancer risk and mortality, but there is limited experimental evidence supporting this association. Moreover, there has not been any definition of a pathophysiological pathway that diabetes may use to promote tumorigenesis. In the present study, we used the MKR mouse model of T2D to investigate molecular mechanisms that link T2D to breast cancer development and progression. MKR mice harbor a transgene encoding a dominant-negative, kinase-dead human insulin-like growth factor-I receptor (IGF-IR) that is expressed exclusively in skeletal muscle, where it acts to inactivate endogenous insulin receptor (IR) and IGF-IR. Although lean female MKR mice are insulin resistant and glucose intolerant, displaying accelerated mammary gland development and enhanced phosphorylation of IR/IGF-IR and Akt in mammary tissue, in the context of three different mouse models of breast cancer, these metabolic abnormalities were found to accelerate the development of hyperplastic precancerous lesions. Normal or malignant mammary tissue isolated from these mice exhibited increased phosphorylation of IR/IGF-IR and Akt, whereas extracellular signal-regulated kinase 1/2 phosphorylation was largely unaffected. Tumor-promoting effects of T2D in the models were reversed by pharmacological blockade of IR/IGF-IR signaling by the small-molecule tyrosine kinase inhibitor BMS-536924. Our findings offer compelling experimental evidence that T2D accelerates mammary gland development and carcinogenesis,and that the IR and/or the IGF-IR are major mediators of these effects.

Recombinant human insulin-like growth factor-I treatment inhibits gluconeogenesis in a transgenic mouse model of type 2 diabetes mellitus.

IGF-I and insulin are structurally related polypeptides that mediate a similar pattern of biological effects via receptors that display considerably homology. Administration of recombinant human IGF-I (rhIGF-I) has been proven to improve glucose control and liver and muscle insulin sensitivity in patients with type 2 diabetes mellitus (DM). The effect of rhIGF-I treatment was evaluated in a mouse model of type 2 DM (MKR mouse), which expresses a dominant-negative form of the human IGF-I receptor under the control of the muscle creatine kinase promoter specifically in skeletal muscle. MKR mice have impaired IGF-I and insulin signaling in skeletal muscle, leading to severe insulin resistance in muscle, liver, and fat, developing type 2 DM at 5 wk of age. Six-week-old MKR mice were treated with either saline or rhIGF-I for 3 wk. Blood glucose levels were decreased in response to rhIGF-I treatment in MKR mice. rhIGF-I treatment also increased body weight in MKR with concomitant changes in body composition such as a decrease in fat mass and an increase in lean body mass. Insulin, fatty acid, and triglyceride levels were not affected by rhIGF-I, nor were insulin or glucose tolerance in MKR mice. Hyperinsulinemic-euglycemic clamp analysis demonstrated no improvement in overall insulin sensitivity. Pyruvate and glutamine tolerance tests proved that there was a decrease in the rate of glucose appearance in MKR mice treated with rhIGF-I, suggesting a reduction in the gluconeogenic capacity of liver, kidney, and small intestine. Taken together these results demonstrate that the improvement of the hyperglycemia was achieved by inhibition of gluconeogenesis rather than an improvement in insulin sensitivity. Also, these results suggest that a functional IGF-I receptor in skeletal muscle is required for IGF-I to improve insulin sensitivity in this mouse model of type 2 DM.

Raised expression of the antiapoptotic protein ped/pea-15 increases susceptibility to chemically induced skin tumor development.

ped/pea-15 is a cytosolic protein performing a broad antiapoptotic function. We show that, upon DMBA/TPA-induced skin carcinogenesis, transgenic mice overexpressing ped/pea-15 (Tg(ped/pea-15)) display early development of papillomas and a four-fold increase in papilloma number compared to the nontransgenic littermates (P<0.001). The malignant conversion frequency was 24% for the Tg(ped/pea-15) mice and only 5% in controls (P<0.01). The isolated application of TPA, but not that of DMBA, was sufficient to reversibly upregulate ped/pea-15 in both untransformed skin and cultured keratinocytes. ped/pea-15 protein levels were also increased in DMBA/TPA-induced papillomas of both Tg(ped/pea-15) and control mice. Isolated TPA applications induced Caspase-3 activation and apoptosis in nontransformed mouse epidermal tissues. The induction of both Caspase-3 and apoptosis by TPA were four-fold inhibited in the skin of the Tg(ped/pea-15) compared to the nontransgenic mice, accompanied by a similarly sized reduction in TPA-induced JNK and p38 stimulation and by constitutive induction of cytoplasmic ERK activity in the transgenics. ped/pea-15 expression was stably increased in cell lines from DMBA/TPA-induced skin papillomas and carcinomas, paralleled by protection from TPA apoptosis. In the A5 spindle carcinoma cell line, antisense inhibition of ped/pea-15 expression simultaneously rescued sensitivity to TPA-induced Caspase-3 function and apoptosis. The antisense also reduced A5 cell ability to grow in semisolid media by 65% (P<0.001) and increased by three-fold tumor latency time (P<0.01). Thus, the expression levels of ped/pea-15 control Caspase-3 function and epidermal cell apoptosis in vivo and determine susceptibility to skin tumor development.

Protein kinase B/Akt binds and phosphorylates PED/PEA-15, stabilizing its antiapoptotic action.

The antiapoptotic protein PED/PEA-15 features an Akt phosphorylation motif upstream from Ser(116). In vitro, recombinant PED/PEA-15 was phosphorylated by Akt with a stoichiometry close to 1. Based on Western blotting with specific phospho-Ser(116) PED/PEA-15 antibodies, Akt phosphorylation of PED/PEA-15 occurred mainly at Ser(116). In addition, a mutant of PED/PEA-15 featuring the substitution of Ser(116)-->Gly (PED(S116-->G)) showed 10-fold-decreased phosphorylation by Akt. In intact 293 cells, Akt also induced phosphorylation of PED/PEA-15 at Ser(116). Based on pull-down and coprecipitation assays, PED/PEA-15 specifically bound Akt, independently of Akt activity. Serum activation of Akt as well as BAD phosphorylation by Akt showed no difference in 293 cells transfected with PED/PEA-15 and in untransfected cells (which express no endogenous PED/PEA-15). However, the antiapoptotic action of PED/PEA-15 was almost twofold reduced in PED(S116-->G) compared to that in PED/PEA-15(WT) cells. PED/PEA-15 stability closely paralleled Akt activation by serum in 293 cells. In these cells, the nonphosphorylatable PED(S116-->G) mutant exhibited a degradation rate threefold greater than that observed with wild-type PED/PEA-15. In the U373MG glioma cells, blocking Akt also reduced PED/PEA-15 levels and induced sensitivity to tumor necrosis factor-related apoptosis-inducing ligand apoptosis. Thus, phosphorylation by Akt regulates the antiapoptotic function of PED/PEA-15 at least in part by controlling the stability of PED/PEA-15. In part, Akt survival signaling may be mediated by PED/PEA-15.

Protein kinase Calpha activation by RET: evidence for a negative feedback mechanism controlling RET tyrosine kinase.

We have studied the role of protein kinase C (PKC) in signaling of the RET tyrosine kinase receptor. By using a chimeric receptor (E/R) in which RET kinase can be tightly controlled by the addition of epidermal growth factor (EGF), we have found that RET triggering induces a strong increase of PKCalpha, PKCdelta and PKCzeta activity and that PKCalpha, not PKCdelta and PKCzeta, forms a ligand-dependent protein complex with E/R. We have identified tyrosine 1062 in the RET carboxyl-terminal tail as the docking site for PKCalpha. Block of PKC activity by bisindolylmaleimide or chronic phorbol esters treatment decreased EGF-induced serine/threonine phosphorylation of E/R, while it caused a similarly sized increase of EGF-induced E/R tyrosine kinase activity and mitogenic signaling. Conversely, acute phorbol esters treatment, which promotes PKC activity, increased the levels of E/R serine/threonine phosphorylation and significantly decreased its phosphotyrosine content. A threefold reduction of tyrosine phosphorylation levels of the constitutively active RET/MEN2A oncoprotein was observed upon coexpression with PKCalpha. We conclude that RET binds to and activates PKCalpha. PKCalpha, in turn, causes RET phosphorylation and downregulates RET tyrosine kinase and downstream signaling, thus functioning as a negative feedback loop to modulate RET activity.