Cell-Autonomous versus Systemic Akt Isoform Deletions Uncovered New Roles for Akt1 and Akt2 in Breast Cancer.

Studies in three mouse models of breast cancer identified profound discrepancies between cell-autonomous and systemic Akt1- or Akt2-inducible deletion on breast cancer tumorigenesis and metastasis. Although systemic Akt1 deletion inhibits metastasis, cell-autonomous Akt1 deletion does not. Single-cell mRNA sequencing revealed that systemic Akt1 deletion maintains the pro-metastatic cluster within primary tumors but ablates pro-metastatic neutrophils. Systemic Akt1 deletion inhibits metastasis by impairing survival and mobilization of tumor-associated neutrophils. Importantly, either systemic or neutrophil-specific Akt1 deletion is sufficient to inhibit metastasis of Akt-proficient tumors. Thus, Akt1-specific inhibition could be therapeutic for breast cancer metastasis regardless of primary tumor origin. Systemic Akt2 deletion does not inhibit and exacerbates mammary tumorigenesis and metastasis, but cell-autonomous Akt2 deletion prevents breast cancer tumorigenesis by ErbB2. Elevated circulating insulin level induced by Akt2 systemic deletion hyperactivates tumor Akt, exacerbating ErbB2-mediated tumorigenesis, curbed by pharmacological reduction of the elevated insulin.

Sirt2 deacetylase is a novel AKT binding partner critical for AKT activation by insulin.

AKT/PKB kinases transmit insulin and growth factor signals downstream of phosphatidylinositol 3-kinase (PI3K). AKT activation involves phosphorylation at two residues, Thr(308) and Ser(473), mediated by PDK1 and the mammalian target of rapamycin complex 2 (mTORC2), respectively. Impaired AKT activation is a key factor in metabolic disorders involving insulin resistance, whereas hyperactivation of AKT is linked to cancer pathogenesis. Here, we identify the cytoplasmic NAD(+)-dependent deacetylase, Sirt2, as a novel AKT interactor, required for optimal AKT activation. Pharmacological inhibition or genetic down-regulation of Sirt2 diminished AKT activation in insulin and growth factor-responsive cells, whereas Sirt2 overexpression enhanced the activation of AKT and its downstream targets. AKT was prebound with Sirt2 in serum or glucose-deprived cells, and the complex dissociated following insulin treatment. The binding was mediated by the pleckstrin homology and the kinase domains of AKT and was dependent on AMP-activated kinase. This regulation involved a novel AMP-activated kinase-dependent Sirt2 phosphorylation at Thr(101). In cells with constitutive PI3K activation, we found that AKT also associated with a nuclear sirtuin, Sirt1; however, inhibition of PI3K resulted in dissociation from Sirt1 and increased association with Sirt2. Sirt1 and Sirt2 inhibitors additively inhibited the constitutive AKT activity in these cells. Our results suggest potential usefulness of Sirt1 and Sirt2 inhibitors in the treatment of cancer cells with up-regulated PI3K activity and of Sirt2 activators in the treatment of insulin-resistant metabolic disorders.

Non-cuffed central venous catheter for unplanned and urgent start haemodialysis in chronic kidney disease: A multi-centre experience from India.

BACKGROUND: Central Venous Catheter (CVC) is indispensable to unplanned and urgent start haemodialysis in chronic kidney disease (CKD). While cuffed CVC is preferred to non-cuffed CVC for urgent start haemodialysis, patient's clinical condition might warrant immediate insertion of non-cuffed CVC. In the resource poor setting, non-cuffed CVCs might have to be retained longer than guideline recommended limit of 2 weeks. In this multi-centre retrospective observational study, the real-world survival of non-cuffed CVC was assessed among CKD patients who initiated dialysis urgently. METHODS: CVC survival was assessed by Kaplan-Meier survival estimate. Predictors of premature CVC loss were assessed using multi-level multi-variate Cox frailty model wherein, each centre was provided with a random intercept to account for within-centre correlation of practice patterns. RESULTS: Among 433 non-cuffed CVCs, there were 393 removals out of which 80% were elective and 20% were premature. The median CVC survival was 37 days (95% CI: 35-41). The rate of premature CVC removal was 4.5/1000 CVC-days (95% CI: 3.6-5.6). Mechanical complications followed by central line associated blood stream infection (CLABSI) were the most common reasons for premature removal. Rate of CLABSI was 1.7/1000 CVC-days (95% CI: 1.2-2.5). Diabetic CKD significantly increased the hazard of premature CVC removal (HR 1.91, 95% CI: 1.01-3.63, p = 0.04) while right internal-jugular location decreased the hazard (HR 0.22, 95% CI: 0.13-0.38, p < 0.001). CONCLUSION: Prolonged retention of non-cuffed CVC (median 37 days) is common in resource-poor setting. It is worrisome and calls for pre-emptive access creation.

CD36 maintains lipid homeostasis via selective uptake of monounsaturated fatty acids during matrix detachment and tumor progression.

A high-fat diet (HFD) promotes metastasis through increased uptake of saturated fatty acids (SFAs). The fatty acid transporter CD36 has been implicated in this process, but a detailed understanding of CD36 function is lacking. During matrix detachment, endoplasmic reticulum (ER) stress reduces SCD1 protein, resulting in increased lipid saturation. Subsequently, CD36 is induced in a p38- and AMPK-dependent manner to promote preferential uptake of monounsaturated fatty acids (MUFAs), thereby maintaining a balance between SFAs and MUFAs. In attached cells, CD36 palmitoylation is required for MUFA uptake and protection from palmitate-induced lipotoxicity. In breast cancer mouse models, CD36-deficiency induced ER stress while diminishing the pro-metastatic effect of HFD, and only a palmitoylation-proficient CD36 rescued this effect. Finally, AMPK-deficient tumors have reduced CD36 expression and are metastatically impaired, but ectopic CD36 expression restores their metastatic potential. Our results suggest that, rather than facilitating HFD-driven tumorigenesis, CD36 plays a supportive role by preventing SFA-induced lipotoxicity.

FoxO transcription factors; Regulation by AKT and 14-3-3 proteins.

The forkhead box O (FoxO) transcription factor family is a key player in an evolutionary conserved pathway downstream of insulin and insulin-like growth factor receptors. The mammalian FoxO family consists of FoxO1, 3, 4 and 6, which share high similarity in their structure, function and regulation. FoxO proteins are involved in diverse cellular and physiological processes including cell proliferation, apoptosis, reactive oxygen species (ROS) response, longevity, cancer and regulation of cell cycle and metabolism. The regulation of FoxO protein function involves an intricate network of posttranslational modifications and protein-protein interactions that provide integrated cellular response to changing physiological conditions and cues. AKT was identified in early genetic and biochemical studies as a main regulator of FoxO function in diverse organisms. Though other FoxO regulatory pathways and mechanisms have been delineated since, AKT remains a key regulator of the pathway. The present review summarizes the current knowledge of FoxO regulation by AKT and 14-3-3 proteins, focusing on its mechanistic and structural aspects and discusses its crosstalk with the other FoxO regulatory mechanisms. This article is part of a Special Issue entitled: PI3K-AKT-FoxO axis in cancer and aging.

Bimodal regulation of FoxO3 by AKT and 14-3-3.

FoxO3 is a member of FoxO family transcription factors that mediate cellular functions downstream of AKT. FoxO3 phosphorylation by AKT generates binding sites for 14-3-3, which in-turn regulates FoxO3 transcriptional activity and localization. We examine here the functional significance of AKT-FoxO3 interaction and further detail the mechanistic aspects of FoxO3 regulation by AKT and 14-3-3. Our data show that AKT overexpression increases the steady-state levels of FoxO3 protein in a manner dependent on AKT activity and its ability to bind FoxO3. Characterization of the AKT-FoxO3 interaction shows that the three AKT phosphorylation-site-recognition motifs (RxRxxS/T) present on FoxO3, which are required for FoxO3 phosphorylation, are dispensable for AKT binding, suggesting that AKT has a docking point on FoxO3 distinct from the phosphorylation-recognition motifs. Development of a FoxO3 mutant deficient in 14-3-3 binding (P34A), which can be phosphorylated by AKT, established that 14-3-3 binding and not AKT phosphorylation per se controls FoxO3 transcriptional activity. Intriguingly, 14-3-3 binding was found to stabilize FoxO3 by inhibiting its dephosphorylation and degradation rates. Collectively, our data support a model where both AKT and 14-3-3 positively regulate FoxO3 in addition to their established negative roles and that 14-3-3 availability could dictate the fate of phosphorylated FoxO3 toward degradation or recycling.

NF1 loss of function as an alternative initiating event in pancreatic ductal adenocarcinoma.

A long-standing question in the pancreatic ductal adenocarcinoma (PDAC) field has been whether alternative genetic alterations could substitute for oncogenic KRAS mutations in initiating malignancy. Here, we report that Neurofibromin1 (NF1) inactivation can bypass the requirement of mutant KRAS for PDAC pathogenesis. An in-depth analysis of PDAC databases reveals various genetic alterations in the NF1 locus, including nonsense mutations, which occur predominantly in tumors with wild-type KRAS. Genetic experiments demonstrate that NF1 ablation culminates in acinar-to-ductal metaplasia, an early step in PDAC. Furthermore, NF1 haploinsufficiency results in a dramatic acceleration of KrasG12D-driven PDAC. Finally, we show an association between NF1 and p53 that is orchestrated by PML, and mosaic analysis with double markers demonstrates that concomitant inactivation of NF1 and Trp53 is sufficient to trigger full-blown PDAC. Together, these findings open up an exploratory framework for apprehending the mechanistic paradigms of PDAC with normal KRAS, for which no effective therapy is available.

A non-catalytic scaffolding activity of hexokinase 2 contributes to EMT and metastasis.

Hexokinase 2 (HK2), which catalyzes the first committed step in glucose metabolism, is induced in cancer cells. HK2's role in tumorigenesis has been attributed to its glucose kinase activity. Here, we describe a kinase independent HK2 activity, which contributes to metastasis. HK2 binds and sequesters glycogen synthase kinase 3 (GSK3) and acts as a scaffold forming a ternary complex with the regulatory subunit of protein kinase A (PRKAR1a) and GSK3ß to facilitate GSK3ß phosphorylation and inhibition by PKA. Thus, HK2 functions as an A-kinase anchoring protein (AKAP). Phosphorylation by GSK3ß targets proteins for degradation. Consistently, HK2 increases the level and stability of GSK3 targets, MCL1, NRF2, and particularly SNAIL. In addition to GSK3 inhibition, HK2 kinase activity mediates SNAIL glycosylation, which prohibits its phosphorylation by GSK3. Finally, in mouse models of breast cancer metastasis, HK2 deficiency decreases SNAIL protein levels and inhibits SNAIL-mediated epithelial mesenchymal transition and metastasis.

Antioxidant and anticancer efficacy of hesperidin in benzo(a)pyrene induced lung carcinogenesis in mice.

Chemoprevention is regarded as one of the most promising and realistic approaches in the prevention of cancer. Several bioactive compounds present in fruits and vegetables have revealed their cancer curative potential on lung cancer. Hesperidin is one such naturally occurring flavonoid widely found in citrus fruits. The aim of the present study is to divulge the chemopreventive nature of hesperidin during benzo(a)pyrene (B(a)P) induced lung cancer in Swiss albino mice. Administration of B(a)P (50 mg/kg body weight) to mice resulted in increased lipid peroxides (LPO), lung specific tumor marker carcinoembryonic antigen (CEA) and serum marker enzymes aryl hydrocarbon hydroxylase (AHH), gamma glutamyl transpeptidase (GGT), 5'nucleotidase (5'ND) and lactate dehydrogenase (LDH) with concomitant decrease in the levels of tissue antioxidants like superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH), vitamin E and vitamin C. Hesperidin supplementation (25 mg/kg body weight) significantly attenuated these alterations thereby showing potent anticancer effect in lung cancer. Further the antiproliferative effect of hesperidin was confirmed by histopathological analysis and proliferating cell nuclear antigen (PCNA) immunostaining. Overall, these findings substantiate the chemopreventive potential of hesperidin against chemically induced lung cancer in mice.

Silymarin attenuated mast cell recruitment thereby decreased the expressions of matrix metalloproteinases-2 and 9 in rat liver carcinogenesis.

Liver cancer is the sixth most common cancer worldwide but because of very poor prognosis, it is the third most common cause of death from cancer. There are currently limited therapeutic regimens available for effective treatment of this cancer. Silymarin is a naturally derived polyphenolic antioxidant, is the active constituent in a widely consumed dietary supplement milk thistle (Silybum marianum) extract. Mast cells play an important role in the inflammatory component of a developing neoplasm; they are also a major source for matrix metalloproteinases (MMPs), which are involved in invasion and angiogenesis. In the present study, we investigated whether dietary supplementation of silymarin has any role in mast cell density (MCD) and in the expressions of MMP-2 and MMP-9 in N-nitrosodiethylamine induced (NDEA) liver cancer in Wistar albino male rats. NDEA administered rats showed increased MCD as revealed by toluidine blue staining along with upregulated expressions of MMP-2 and MMP-9. Silymarin treatment inhibited this increase in MCD and downregulated the expressions of MMP-2 and MMP-9 as revealed by Western blotting and immunohistochemistry. In conclusion, silymarin exerted beneficial effects on liver carcinogenesis by attenuating the recruitment of mast cells and thereby decreased the expressions of MMP-2 and MMP-9.

Antiproliferative potential of gallic acid against diethylnitrosamine-induced rat hepatocellular carcinoma.

One of the focuses in current cancer chemoprevention studies is the search for nontoxic chemopreventive agents that inhibit the initiation of malignant transformation. Cancer biomarkers are quantifiable molecules involved in the physiologic or pathologic events occurring between exposure to carcinogens and the development, progression of cancer. Biomarkers may be the consequence of a continuous process, such as increased cell mass, or a discrete event, such as genetic mutation. Analysis of tumor markers can be used as an indicator of tumor response to therapy. Gallic acid is a naturally available polyphenol, possess strong antioxidant activity with a capacity to inhibit the formation of tumors in several cancer models. In the present study, we investigated the antiproliferative effect of gallic acid during diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) in male wistar albino rats. DEN treatment resulted in increased levels of aspartate transaminase, alanine transaminase, alkaline phosphatase, acid phosphatase, lactate dehydrogenase, gamma-glutamyltransferase, 5'-nucleotidase, bilirubin, alpha-fetoprotein, carcinoembryonic antigen, argyophillic nucleolar organizing regions, and proliferating cell nuclear antigen. Gallic acid treatment significantly attenuated these alterations and decreased the levels of AgNORs and PCNA. These finding suggests that gallic acid is a potent antiproliferative agent against DEN-induced HCC.

Depsipeptide a histone deacetlyase inhibitor down regulates levels of matrix metalloproteinases 2 and 9 mRNA and protein expressions in lung cancer cells (A549).

BACKGROUND: A novel histone deacetylase inhibitor, depsipeptide FR901228 (FK228), is a promising anticancer and antiproliferative agent and has been proposed to regulate gene transcription and reported to lower the risk of several cancers in different cell lines. Depsipeptide showed therapeutic efficacy in Phase I trial of patients with malignant lymphoma. Based on the recognition that basement membrane disruption occurs in acute lung injury and that matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) can degrade type IV collagen, one of the major components of the basement membrane and known to involve in tumor invasion and metastases. We hypothesized that depsipeptide would modulate MMP-2 and MMP-9 production in lung adenocarcinoma cells line (A549). METHODS: In this study, we observed the precise involvement of depsipeptide role on cancer metastasis. A549 cells were treated with depsipeptide at various concentrations (50 and 100nm), for 24h period and then subjected to mRNA levels with RT-PCR and protein levels with Western blot analysis to investigate the impact of depsipeptide on MMP-2 and MMP-9 expressions and further confirmed by using immunocytochemistry. RESULTS: The results showed that depsipeptide treatment decreased the expressions of MMP-2 and MMP-9 in dose-dependent manner. The level of mRNA and proteins expressions were significantly decreased in depsipeptide treated A549 cells in a dose-dependent manner and the level of pro-MMP-9 was found to be high in the 100nm depsipeptide-treated cell lysate of A549 cells, suggesting inhibitory role of depsipeptide on pro-MMP-9 activation. Further immunocytochemistry studies showed the weak expression of MMP-2 and MMP-9 in depsipeptide treated cells. CONCLUSION: We speculate that inhibition of metastasis-specific MMPs in cancer cells may be one of the targets for anticancer function of depsipeptide, and thus provides the molecular basis for the development of depsipeptide as a novel chemopreventive agent for metastatic lung cancer.

Study of low-density lipoprotein receptor regulation by oral (steroid) contraceptives: desogestrel, levonorgestrel and ethinyl estradiol in JEG-3 cell line and placental tissue.

BACKGROUND: The aim of this study was to compare in vitro the role of two oral contraceptives, desogestrel (a less androgenic derivative of levonorgestrel) and levonorgestrel--alone and in combination with ethinyl estradiol--on low-density lipoprotein (LDL) receptor regulation by assessing receptor protein expression and functional effectiveness. STUDY DESIGN: Placental tissue and cultured placental cells (JEG-3) were used to study the expression and endocytotic activity of LDL receptor protein. The expression of the receptor was assessed by immunocytochemistry and immunoblot assays with and without contraceptive challenge. Functioning activity of LDL receptor was studied by measuring the rate of uptake of LDL by placental cells. Quantification of LDL was based on the total cholesterol content of the lipoprotein. RESULTS: A combination of desogestrel (20 ng/mL of incubation medium) and ethinyl estradiol (10 ng/mL of incubation medium) maintained the LDL receptor at high level of expression and functioning mode. In contrast, the double-blind preparation of levonorgestrel (20 ng/mL) and ethinyl estradiol (10 ng/mL) had shown much lower expression as well as receptor-mediated LDL uptake. The concentration of contraceptives used in this study was similar to the prevailing concentration of oral contraceptives in clinical use. CONCLUSION: Higher expression of LDL receptor and enhanced rate of LDL uptake by the receptor protein projects the possibility that there might be less atherosclerosis-related disorders from the combination of desogestrol and ethinyl estradiol.

Suppression of N-nitrosodiethylamine induced hepatocarcinogenesis by silymarin in rats.

Antioxidants are one of the key players in tumorigenesis, several natural and synthetic antioxidants were shown to have anticancer effects. In the present investigation the efficacy of silymarin on the antioxidant status of N-nitrosodiethylamine (NDEA) induced hepatocarcinogenesis in Wistar albino male rats were assessed. The animals were divided into five groups. The animals in the groups 1 and 3 were normal control and silymarin control, respectively. Groups 2, 4 and 5 were administered with 0.01% NDEA in drinking water for 15 weeks to induce hepatocellular carcinoma (HCC). Starting 1 week prior to NDEA administration group 4 animals were treated with silymarin in diet for 16 weeks, 10 weeks after NDEA administration group 5 animals were treated with silymarin and continued till the end of the experiment period (16 weeks). After the experimental period the body weight, relative liver weight, number of nodules, size of nodules, the levels of lipid peroxidation, glutathione (GSH), and the activities of antioxidant enzymes were assessed in both haemolysate and liver tissue. In group 2 hepatocellular carcinoma induced animals there was an increase in the number of nodules, relative liver weight. The levels of lipid peroxides were elevated with subsequent decrease in the body weight, (glutathione) GSH, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6PD). In contrast, silymarin + NDEA treated groups 4 and 5 animals showed a significant decrease in the number of nodules with concomitant decrease in the lipid peroxidation status. The levels of GSH and the activities of antioxidant enzymes in both haemolysate and liver were improved when compared with hepatocellular carcinoma induced group 2 animals. The electron microscopy studies were also carried out which supports the chemopreventive action of the silymarin against NDEA administration during liver cancer progression. These findings suggest that silymarin suppresses NDEA induced hepatocarcinogenesis by modulating the antioxidant defense status of the animals.

Characterization of cancer-associated missense mutations in MDM2.

MDM2 is an E3 ubiquitin ligase that binds the N-terminus of p53 and promotes its ubiquitin-dependent degradation. Elevated levels of MDM2 due to overexpression or gene amplification can contribute to tumor development by suppressing p53 activity. Since MDM2 is an oncogene, we explored the possibility that other genetic lesions, namely missense mutations, might alter its activities. We selected mutations in MDM2 that reside in one of the 4 key regions of the protein: p53 binding domain, acidic domain, zinc finger domain, and the RING domain. Unexpectedly, we observed that individual mutations in several of these domains compromised the ability of MDM2 to degrade p53. Mutations in the N-terminal p53 binding domain prevented the formation of a p53-MDM2 complex, thereby protecting p53 from degradation. Additionally, as would be predicted, several cancer-associated mutations in the RING finger domain disrupted the ubiquitin ligase activity of MDM2 and prevented p53 degradation. Interestingly, we observed that amino acid substitutions at the same codon differentially affected MDM2 activity. Our data reveal that mutations in this oncogene can have the paradoxical effect of suppressing its activity. Further understanding of how these mutations perturb MDM2 function may yield novel approaches to inhibiting its activity.

Mutant p53 protects ETS2 from non-canonical COP1/DET1 dependent degradation.

Mutations in the tumor suppressor gene TP53 contribute to the development of approximately half of all human cancers. One mechanism by which mutant p53 (mtp53) acts is through interaction with other transcription factors, which can either enhance or repress the transcription of their target genes. Mtp53 preferentially interacts with the erythroblastosis virus E26 oncogene homologue 2 (ETS2), an ETS transcription factor, and increases its protein stability. To study the mechanism underlying ETS2 degradation, we knocked down ubiquitin ligases known to interact with ETS2. We observed that knockdown of the constitutive photomorphogenesis protein 1 (COP1) and its binding partner De-etiolated 1 (DET1) significantly increased ETS2 stability, and conversely, their ectopic expression led to increased ETS2 ubiquitination and degradation. Surprisingly, we observed that DET1 binds to ETS2 independently of COP1, and we demonstrated that mutation of multiple sites required for ETS2 degradation abrogated the interaction between DET1 and ETS2. Furthermore, we demonstrate that mtp53 prevents the COP1/DET1 complex from ubiquitinating ETS2 and thereby marking it for destruction. Mechanistically, we show that mtp53 destabilizes DET1 and also disrupts the DET1/ETS2 complex thereby preventing ETS2 degradation. Our study reveals a hitherto unknown function in which DET1 mediates the interaction with the substrates of its cognate ubiquitin ligase complex and provides an explanation for the ability of mtp53 to protect ETS2.

The Anticancer Role of Capsaicin in Experimentallyinduced Lung Carcinogenesis.

OBJECTIVES: Capsaicin (CAP) is the chief pungent principle found in the hot red peppers and the chili peppers that have long been used as spices, food additives and drugs. This study investigated the anticancer potential of CAP through its ability to modify extracellular matrix components and proteases during mice lung carcinogenesis. METHODS: Swiss albino mice were treated with benzo(a) pyrene (50 mg/kg body weight dissolved in olive oil) orally twice a week for four successive weeks to induce lung cancer at the end of 14(th) week. CAP was administrated (10 mg/kg body weight dissolved in olive oil) intraperitoneally. Extracellular matrix components were assayed; Masson's trichome staining of lung tissues was performed. Western blot analyses of matrix metalloproteases 2 and 9 were also carried out. RESULTS: In comparison with the control animals, animals in which benzo(a)pyrene had induced lung cancer showed significant increases in extracellular matrix components such as collagen (hydroxy proline), elastin, uronic acid and hexosamine and in glycosaminoglycans such as hyaluronate, chondroitin sulfate, keratan sulfate and dermatan sulfate. The above alterations in extracellular matrix components were effectively counteracted in benzo(a)pyrene along with CAP supplemented animals when compared to benzo(a) pyrene alone supplemented animals. The results of Masson's trichome staining for collagen and of, immunoblotting analyses of matrix metalloproteases 2 and 9 further supported the biochemical findings. CONCLUSION: The apparent potential of CAP in modulating extracellular matrix components and proteases suggests that CAP plays a chemomodulatory and anti- cancer role working against experimentally induced lung carcinogenesis.

AKT and 14-3-3 regulate Notch4 nuclear localization.

Members of the Notch family of transmembrane receptors, Notch1-4 in mammals, are involved in the regulation of cell fate decisions and cell proliferation in various organisms. The Notch4 isoform, which is specific to mammals, was originally identified as a viral oncogene in mice, Int3, able to initiate mammary tumors. In humans, Notch4 expression appears to be associated with breast cancer stem cells and endocrine resistance. Following ligand binding, the Notch4 receptor undergoes cleavage at the membrane and the Notch4-intracellular domain (ICD), translocates to the nucleus and regulates gene transcription. Little is known on the mechanisms regulating Notch4-ICD and its nuclear localization. Here, we describe the identification of four distinct AKT phosphorylation sites in human Notch4-ICD and demonstrate that AKT binds Notch4-ICD and phosphorylates all four sites in vitro and in vivo. The phosphorylation in cells is regulated by growth factors and is sensitive to phosphatidyl inositol-3 kinase (PI3K) inhibitors. This phosphorylation generates binding sites to the 14-3-3 regulatory proteins, which are involved in the regulation of nucleocytoplasmic shuttling of target proteins, restricting phosphorylated Notch4-ICD to the cytoplasm. Our findings provide a novel mechanism for Notch4-ICD regulation, suggesting a negative regulatory role for the PI3K-AKT pathway in Notch4 nuclear signaling.

Regulation of nucleotide metabolism by mutant p53 contributes to its gain-of-function activities.

Mutant p53 (mtp53) is an oncogene that drives cancer cell proliferation. Here we report that mtp53 associates with the promoters of numerous nucleotide metabolism genes (NMG). Mtp53 knockdown reduces NMG expression and substantially depletes nucleotide pools, which attenuates GTP-dependent protein activity and cell invasion. Addition of exogenous guanosine or GTP restores the invasiveness of mtp53 knockdown cells, suggesting that mtp53 promotes invasion by increasing GTP. In addition, mtp53 creates a dependency on the nucleoside salvage pathway enzyme deoxycytidine kinase for the maintenance of a proper balance in dNTP pools required for proliferation. These data indicate that mtp53-harbouring cells have acquired a synthetic sick or lethal phenotype relationship with the nucleoside salvage pathway. Finally, elevated expression of NMG correlates with mutant p53 status and poor prognosis in breast cancer patients. Thus, mtp53's control of nucleotide biosynthesis has both a driving and sustaining role in cancer development.

The association between insulin and low-density lipoprotein receptors.

The insulin receptor (IR) and low-density lipoprotein receptor (LDLR) maintain glucose and lipid metabolism, respectively. Diabetes is associated with increased blood glucose, dyslipidaemia and increased risk of atherosclerosis. We hypothesise that interactions between IR and LDLR play a role in the atherosclerotic process in subjects with diabetes. Therefore, in this work we studied potential interactions between these two receptors. Our data show an intracellular and surface membrane-bound co-association of IR and LDLR. The co-association makes LDLR functionally poor in clearing extra-cellular LDL particles. A short 10 min exposure of cells to insulin disrupts the association between the two receptors and generates LDLR with higher LDL clearing activity without any change in protein expression. This co-association of LDLR with IR and their dissociation by insulin may be an important part of the regulatory mechanism of the normal physiological receptor function in a biological system. Modulation of receptor co-association is potentially a therapeutic target to reduce cardiovascular risk, and further studies are needed to investigate this possibility.