ß-catenin turnover is regulated by Nek10-mediated tyrosine phosphorylation in A549 lung adenocarcinoma cells.

ß-catenin has influential roles affecting embryonic development, tissue homeostasis, and human diseases including cancer. Cellular ß-catenin levels are exquisitely controlled by a variety of regulatory mechanisms. In the course of exploring the functions of the Nek10 tyrosine kinase, we observed that deletion of Nek10 in lung adenocarcinoma cells resulted in dramatic stabilization of ß-catenin, suggestive of a Nek10 role in the control of ß-catenin turnover. Nek10-deficient cells exhibited diminished ability to form tumorspheres in suspension, grow in soft agar, and colonize mouse lung tissue following tail vein injection. Mechanistically, Nek10 associates with the Axin complex, responsible for ß-catenin degradation, where it phosphorylates ß-catenin at Tyr30, located within the regulatory region governing ß-catenin turnover. In the absence of Nek10 phosphorylation, GSK3-mediated phosphorylation of ß-catenin, a prerequisite for its turnover, is impaired. This represents a divergent function within the Nek family, whose other members are serine-threonine kinases involved in different elements of the centrosomal cycle, primary cilia function, and DNA damage responses.

Effect of Metformin vs Placebo on Invasive Disease-Free Survival in Patients With Breast Cancer: The MA.32 Randomized Clinical Trial.

Importance: Metformin, a biguanide commonly used to treat type 2 diabetes, has been associated with potential beneficial effects across breast cancer subtypes in observational and preclinical studies. Objective: To determine whether the administration of adjuvant metformin (vs placebo) to patients with breast cancer without diabetes improves outcomes. Design, Setting, and Participants: MA.32, a phase 3 randomized, placebo-controlled, double-blind trial, conducted in Canada, Switzerland, US, and UK, enrolled 3649 patients with high-risk nonmetastatic breast cancer receiving standard therapy between August 2010 and March 2013, with follow-up to October 2020. Interventions: Patients were randomized (stratified for hormone receptor [estrogen receptor and/or progesterone receptor {ER/PgR}] status, positive vs negative; body mass index, ≤30 vs >30; human epidermal growth factor receptor 2 [ERBB2, formerly HER2 or HER2/neu], positive vs negative; and any vs no chemotherapy) to 850 mg of oral metformin twice a day (n = 1824) or oral placebo twice a day (n = 1825) for 5 years. Main Outcomes and Measures: The primary outcome was invasive disease-free survival in hormone receptor-positive breast cancer. Of the 8 secondary outcomes, overall survival, distant relapse-free survival, and breast cancer-free interval were analyzed. Results: Of the 3649 randomized patients (mean age, 52.4 years; 3643 women [99.8%]), all (100%) were included in analyses. After a second interim analysis, futility was declared for patients who were ER/PgR-, so the primary analysis was conducted for 2533 patients who were ER/PgR+. The median duration of follow-up in the ER/PgR+ group was 96.2 months (range, 0.2-121 months). Invasive disease-free survival events occurred in 465 patients who were ER/PgR+. The incidence rates for invasive disease-free survival events were 2.78 per 100 patient-years in the metformin group vs 2.74 per 100 patient-years in the placebo group (hazard ratio [HR], 1.01; 95% CI, 0.84-1.21; P = .93), and the incidence rates for death were 1.46 per 100 patient-years in the metformin group vs 1.32 per 100 patient-years in the placebo group (HR, 1.10; 95% CI, 0.86-1.41; P = .47). Among patients who were ER/PgR-, followed up for a median of 94.1 months, incidence of invasive disease-free survival events was 3.58 vs 3.60 per 100 patient-years, respectively (HR, 1.01; 95% CI, 0.79-1.30; P = .92). None of the 3 secondary outcomes analyzed in the ER/PgR+ group had statistically significant differences. Grade 3 nonhematological toxic events occurred more frequently in patients taking metformin than in patients taking placebo (21.5% vs 17.5%, respectively, P = .003). The most common grade 3 or higher adverse events in the metformin vs placebo groups were hypertension (2.4% vs 1.9%), irregular menses (1.5% vs 1.4%), and diarrhea (1.9% vs 7.0%). Conclusions and Relevance: Among patients with high-risk operable breast cancer without diabetes, the addition of metformin vs placebo to standard breast cancer treatment did not significantly improve invasive disease-free survival. Trial Registration: ClinicalTrials.gov Identifier: NCT01101438.

Impact of the obesity epidemic on cancer.

There is growing appreciation that the current obesity epidemic is associated with increases in cancer incidence at a population level and may lead to poor cancer outcomes; concurrent decreases in cancer mortality at a population level may represent a paradox, i.e., they may also reflect improvements in the diagnosis and treatment of cancer that mask obesity effects. An association of obesity with cancer is biologically plausible because adipose tissue is biologically active, secreting estrogens, adipokines, and cytokines. In obesity, adipose tissue reprogramming may lead to insulin resistance, with or without diabetes, and it may contribute to cancer growth and progression locally or through systemic effects. Obesity-associated changes impact cancer in a complex fashion, potentially acting directly on cells through pathways, such as the phosphoinositide 3-kinase (PI3K) and Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathways, or indirectly via changes in the tumor microenvironment. Approaches to obesity management are discussed, and the potential for pharmacologic interventions that target the obesity-cancer link is addressed.

Effects of metformin versus placebo on vitamin B12 metabolism in non-diabetic breast cancer patients in CCTG MA.32.

BACKGROUND: Metformin is associated with low levels of vitamin B12 (VitB12) in patients with diabetes. The CCTG/MA.32 trial investigates the effects of metformin vs placebo on breast cancer (BC) outcomes in non-diabetic high-risk BC patients. We analyzed VitB12 at baseline and after 6 months of metformin (versus placebo) in the first 492 patients with paired blood samples. METHODS: VitB12 was analyzed centrally in baseline and 6-month fasting plasma. Levels <181 pmol/L were considered deficient, 181-221 pmol/L borderline, and ≥222 pmol/L sufficient. Methylmalonic acid (MMA) and homocysteine (HC) were assayed in those with VitB12 levels <222 pmol/L. Statistical analyses used Spearman's rank correlation coefficients and Wilcoxon signed-rank test for continuous variables and Chi-square test for categorical variables. RESULTS: 237 patients received metformin and 255 received placebo; median (inter quartile range) baseline VitB12 levels were 390 (290, 552) and 370 (290, 552) pmol/L in the metformin and placebo arms, respectively (p = 0.97). At 6 months, the median levels were 320 (244, 419) in the metformin versus 380 (286, 546) pmol/L in the placebo arm (p = 0.0001). At baseline, 15 patients (11 metformin and 4 placebo) had VitB12 <181 pmol/L, and at 6 months, 18 patients (15 metformin and 3 placebo) (p = 0.004). Median hemoglobin was similar at baseline, metformin, 130 g/L (124-137), and placebo arms, 131 g/L (124-137) (p = 0.38), and at 6 months, metformin, 131 g/L (91-162), and 131 g/L (106-169) in placebo group (p = 0.11). Of the 74 subjects with vitamin B12 <222 pmol/L at either time point (45 metformin, 29 placebo), at baseline MMA was normal in all patients and two had elevated HC (>15µmol/L). At 6 months, one patient (metformin) had MMA >0.4µmol/L and 3 (2 metformin, 1 placebo) had HC > 15µmol/L. CONCLUSIONS: There was an increased rate of biochemical VitB12 deficiency after 6 months of metformin; this was not associated with anemia. Further research will investigate VitB12 levels in all subjects at baseline and at 6 and 60 months.

Estrogen controls the survival of BRCA1-deficient cells via a PI3K-NRF2-regulated pathway.

Mutations in the tumor suppressor BRCA1 predispose women to breast and ovarian cancers. The mechanism underlying the tissue-specific nature of BRCA1's tumor suppression is obscure. We previously showed that the antioxidant pathway regulated by the transcription factor NRF2 is defective in BRCA1-deficient cells. Reactivation of NRF2 through silencing of its negative regulator KEAP1 permitted the survival of BRCA1-null cells. Here we show that estrogen (E2) increases the expression of NRF2-dependent antioxidant genes in various E2-responsive cell types. Like NRF2 accumulation triggered by oxidative stress, E2-induced NRF2 accumulation depends on phosphatidylinositol 3-kinase-AKT activation. Pretreatment of mammary epithelial cells (MECs) with the phosphatidylinositol 3-kinase inhibitor BKM120 abolishes the capacity of E2 to increase NRF2 protein and transcriptional activity. In vivo the survival defect of BRCA1-deficient MECs is rescued by the rise in E2 levels associated with pregnancy. Furthermore, exogenous E2 administration stimulates the growth of BRCA1-deficient mammary tumors in the fat pads of male mice. Our work elucidates the basis of the tissue specificity of BRCA1-related tumor predisposition, and explains why oophorectomy significantly reduces breast cancer risk and recurrence in women carrying BRCA1 mutations.

Structure-guided mutation of the conserved G3-box glycine in Rheb generates a constitutively activated regulator of mammalian target of rapamycin (mTOR).

Constitutively activated variants of small GTPases, which provide valuable functional probes of their role in cellular signaling pathways, can often be generated by mutating the canonical catalytic residue (e.g. Ras Q61L) to impair GTP hydrolysis. However, this general approach is ineffective for a substantial fraction of the small GTPase family in which this residue is not conserved (e.g. Rap) or not catalytic (e.g. Rheb). Using a novel engineering approach, we have manipulated nucleotide binding through structure-guided substitutions of an ultraconserved glycine residue in the G3-box motif (DXXG). Substitution of Rheb Gly-63 with alanine impaired both intrinsic and TSC2 GTPase-activating protein (GAP)-mediated GTP hydrolysis by displacing the hydrolytic water molecule, whereas introduction of a bulkier valine side chain selectively blocked GTP binding by steric occlusion of the γ-phosphate. Rheb G63A stimulated phosphorylation of the mTORC1 substrate p70S6 kinase more strongly than wild-type, thus offering a new tool for mammalian target of rapamycin (mTOR) signaling.

Changes in insulin receptor signaling underlie neoadjuvant metformin administration in breast cancer: a prospective window of opportunity neoadjuvant study.

INTRODUCTION: The antidiabetic drug metformin exhibits potential anticancer properties that are believed to involve both direct (insulin-independent) and indirect (insulin-dependent) actions. Direct effects are linked to activation of AMP-activated protein kinase (AMPK) and an inhibition of mammalian target of rapamycin mTOR signaling, and indirect effects are mediated by reductions in circulating insulin, leading to reduced insulin receptor (IR)-mediated signaling. However, the in vivo impact of metformin on cancer cell signaling and the factors governing sensitivity in patients remain unknown. METHODS: We conducted a neoadjuvant, single-arm, "window of opportunity" trial to examine the clinical and biological effects of metformin on patients with breast cancer. Women with untreated breast cancer who did not have diabetes were given 500 mg of metformin three times daily for ≥2 weeks after diagnostic biopsy until surgery. Fasting blood and tumor samples were collected at diagnosis and surgery. Blood glucose and insulin were assayed to assess the physiologic effects of metformin, and immunohistochemical analysis of tumors was used to characterize cellular markers before and after treatment. RESULTS: Levels of IR expression decreased significantly in tumors (P = 0.04), as did the phosphorylation status of protein kinase B (PKB)/Akt (S473), extracellular signal-regulated kinase 1/2 (ERK1/2, T202/Y204), AMPK (T172) and acetyl coenzyme A carboxylase (S79) (P = 0.0001, P < 0.0001, P < 0.005 and P = 0.02, respectively). All tumors expressed organic cation transporter 1, with 90% (35 of 39) exhibiting an Allred score of 5 or higher. CONCLUSIONS: Reduced PKB/Akt and ERK1/2 phosphorylation, coupled with decreased insulin and IR levels, suggest insulin-dependent effects are important in the clinical setting. These results are consistent with beneficial anticancer effects of metformin and highlight key factors involved in sensitivity, which could be used to identify patients with breast cancer who may be responsive to metformin-based therapies. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT00897884. Registered 8 May 2009.

Evidence for biological effects of metformin in operable breast cancer: biomarker analysis in a pre-operative window of opportunity randomized trial.

Metformin has therapeutic potential against breast cancer, but the mechanisms of action in vivo remain uncertain. This study examined biomarker effects of metformin in primary breast cancer in a preoperative window of opportunity trial. Non-diabetic women with operable invasive breast cancer were randomized to receive open label pre-operative metformin (500 mg daily for 1 week then 1 g twice daily for a further week) or as controls, not receiving metformin. Patients in both arms had a core biopsy pre-randomisation and again at the time of surgery. Immunohistochemistry for phospho-AMPK (pAMPK), phospho-Akt (pAkt), insulin receptor, cleaved caspase-3, and Ki67 was performed on formalin-fixed paraffin-embedded cores, scored blinded to treatment and analysed by paired t test. In metformin-treated patients, significant up-regulation of pAMPK (paired t test, p = 0.04) and down-regulation of pAkt (paired t test, p = 0.043) were demonstrated compared to the control group. Insulin receptor and serum insulin remained similar following metformin treatment compared with a rise in insulin receptor and insulin in controls. Significant falls in Ki67 and cleaved caspase-3 (paired t test, p = 0.044) were seen in the metformin-treated patients but not in the control group. Changes were independent of body mass index. These biomarker data suggest mechanisms for metformin action in vivo in breast cancer patients via up-regulation of tumor pAMPK, down-regulation of pAkt, and suppression of insulin responses reflecting cytostatic rather than cytotoxic mechanisms.

Membrane-dependent modulation of the mTOR activator Rheb: NMR observations of a GTPase tethered to a lipid-bilayer nanodisc.

Like most Ras superfamily proteins, the GTPase domain of Ras homologue enriched in brain (Rheb) is tethered to cellular membranes through a prenylated cysteine in a flexible C-terminal region; however, little is known about how Rheb or other GTPases interact with the membrane or how this environment may affect their GTPase functions. We used NMR methods to characterize Rheb tethered to nanodiscs, monodisperse protein-encapsulated lipid bilayers with a diameter of 10 nm. Membrane conjugation markedly reduced the rate of intrinsic nucleotide exchange, while GTP hydrolysis was unchanged. NMR measurements revealed that the GTPase domain interacts transiently with the surface of the bilayer in two distinct preferred orientations, which are determined by the bound nucleotide. We propose models of membrane-dependent signal regulation by Rheb that shed light on previously unexplained in vivo properties of this GTPase. The study presented provides a general approach for direct experimental investigation of membrane-dependent properties of other Ras-superfamily GTPases.

Probing the GTPase cycle with real-time NMR: GAP and GEF activities in cell extracts.

The Ras superfamily of small GTPases is a large family of switch-like proteins that control diverse cellular functions, and their deregulation is associated with multiple disease processes. When bound to GTP they adopt a conformation that interacts with effector proteins, whereas the GDP-bound state is generally biologically inactive. GTPase activating proteins (GAPs) promote hydrolysis of GTP, thus impeding the biological activity of GTPases, whereas guanine nucleotide exchange factors (GEFs) promote exchange of GDP for GTP and activate GTPase proteins. A number of methods have been developed to assay GTPase nucleotide hydrolysis and exchange, as well as the activity of GAPs and GEFs. The kinetics of these reactions are often studied with purified proteins and fluorescent nucleotide analogs, which have been shown to non-specifically impact hydrolysis and exchange. Most GAPs and GEFs are large multidomain proteins subject to complex regulation that is challenging to reconstitute in vitro. In cells, the activities of full-length GAPs or GEFs are typically assayed indirectly on the basis of nucleotide loading of the cognate GTPase, or by exploiting their interaction with effector proteins. Here, we describe a recently developed real-time NMR method to assay kinetics of nucleotide exchange and hydrolysis reactions by direct monitoring of nucleotide-dependent structural changes in an isotopically labeled GTPase. The unambiguous readout of this method makes it possible to precisely measure GAP and GEF activities from extracts of mammalian cells, enabling studies of their catalytic and regulatory mechanisms. We present examples of NMR-based assays of full-length GAPs and GEFs overexpressed in mammalian cells.

Insulin receptor loss impairs mammary tumorigenesis in mice.

Breast cancer (BC) prognosis and outcome are adversely affected by obesity. Hyperinsulinemia, common in the obese state, is associated with higher risk of death and recurrence in BC. Up to 80% of BCs overexpress the insulin receptor (INSR), which correlates with worse prognosis. INSR's role in mammary tumorigenesis was tested by generating MMTV-driven polyoma middle T (PyMT) and ErbB2/Her2 BC mouse models, respectively, with coordinate mammary epithelium-restricted deletion of INSR. In both models, deletion of either one or both copies of INSR leads to a marked delay in tumor onset and burden. Longitudinal phenotypic characterization of mouse tumors and cells reveals that INSR deletion affects tumor initiation, not progression and metastasis. INSR upholds a bioenergetic phenotype in non-transformed mammary epithelial cells, independent of its kinase activity. Similarity of phenotypes elicited by deletion of one or both copies of INSR suggest a dose-dependent threshold for INSR impact on mammary tumorigenesis.

Effect of Metformin Versus Placebo on New Primary Cancers in Canadian Cancer Trials Group MA.32: A Secondary Analysis of a Phase III Randomized Double-Blind Trial in Early Breast Cancer.

Clinical trials frequently include multiple end points that mature at different times. The initial report, typically based on the primary end point, may be published when key planned coprimary or secondary analyses are not yet available. Clinical trial updates provide an opportunity to disseminate additional results from studies, published in JCO or elsewhere, for which the primary end point has already been reported.Metformin has been associated with lower cancer risk in epidemiologic and preclinical research. In the MA.32 randomized adjuvant breast cancer trial, metformin (v placebo) did not affect invasive disease-free or overall survival. Here, we report metformin effects on the risk of new cancer. Between 2010 and 2013, 3,649 patients with breast cancer younger than 75 years without diabetes with high-risk T1-3, N0-3 M0 breast cancer (any estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2) were randomly assigned to metformin 850 mg orally twice a day or placebo twice a day for 5 years. New primary invasive cancers (outside the ipsilateral breast) developing as a first event were identified. Time to events was described by the competing risks method; two-sided likelihood ratio tests adjusting for age, BMI, smoking, and alcohol intake were used to compare metformin versus placebo arms. A total of 184 patients developed new invasive cancers: 102 metformin and 82 placebo, hazard ratio (HR), 1.25; 95% CI, 0.94 to 1.68; P = .13. These included 48 contralateral invasive breast cancers (27 metformin v 21 placebo), HR, 1.29; 95% CI, 0.72 to 2.27; P = .40 and 136 new nonbreast primary cancers (75 metformin v 61 placebo), HR, 1.24; 95% CI, 0.88 to 1.74; P = .21. Metformin did not reduce the risk of new cancer development in these nondiabetic patients with breast cancer.

Metformin in early breast cancer: a prospective window of opportunity neoadjuvant study.

Metformin may exert anti-cancer effects through indirect (insulin-mediated) or direct (insulin-independent) mechanisms. We report results of a neoadjuvant "window of opportunity" study of metformin in women with operable breast cancer. Newly diagnosed, untreated, non-diabetic breast cancer patients received metformin 500 mg tid after diagnostic core biopsy until definitive surgery. Clinical (weight, symptoms, and quality of life) and blood [fasting serum insulin, glucose, homeostasis model assessment (HOMA), C-reactive protein (CRP), and leptin] attributes were compared pre- and post-metformin as were terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and Ki67 scores (our primary endpoint) in tumor tissue. Thirty-nine patients completed the study. Mean age was 51 years, and metformin was administered for a median of 18 days (range 13-40) up to the evening prior to surgery. 51 % had T1 cancers, 38 % had positive nodes, 85 % had ER and/or PgR positive tumors, and 13 % had HER2 overexpressing or amplified tumors. Mild, self-limiting nausea, diarrhea, anorexia, and abdominal bloating were present in 50, 50, 41, and 32 % of patients, respectively, but no significant decreases were seen on the EORTC30-QLQ function scales. Body mass index (BMI) (-0.5 kg/m(2), p < 0.0001), weight (-1.2 kg, p < 0.0001), and HOMA (-0.21, p = 0.047) decreased significantly while non-significant decreases were seen in insulin (-4.7 pmol/L, p = 0.07), leptin (-1.3 ng/mL, p = 0.15) and CRP (-0.2 mg/L, p = 0.35). Ki67 staining in invasive tumor tissue decreased (from 36.5 to 33.5 %, p = 0.016) and TUNEL staining increased (from 0.56 to 1.05, p = 0.004). Short-term preoperative metformin was well tolerated and resulted in clinical and cellular changes consistent with beneficial anti-cancer effects; evaluation of the clinical relevance of these findings in adequately powered clinical trials using clinical endpoints such as survival is needed.

Machine learning-enabled cancer diagnostics with widefield polarimetric second-harmonic generation microscopy.

The extracellular matrix (ECM) collagen undergoes major remodeling during tumorigenesis. However, alterations to the ECM are not widely considered in cancer diagnostics, due to mostly uniform appearance of collagen fibers in white light images of hematoxylin and eosin-stained (H&E) tissue sections. Polarimetric second-harmonic generation (P-SHG) microscopy enables label-free visualization and ultrastructural investigation of non-centrosymmetric molecules, which, when combined with texture analysis, provides multiparameter characterization of tissue collagen. This paper demonstrates whole slide imaging of breast tissue microarrays using high-throughput widefield P-SHG microscopy. The resulting P-SHG parameters are used in classification to differentiate tumor from normal tissue, resulting in 94.2% for both accuracy and F1-score, and 6.3% false discovery rate. Subsequently, the trained classifier is employed to predict tumor tissue with 91.3% accuracy, 90.7% F1-score, and 13.8% false omission rate. As such, we show that widefield P-SHG microscopy reveals collagen ultrastructure over large tissue regions and can be utilized as a sensitive biomarker for cancer diagnostics and prognostics studies.

Regulation of adipocyte differentiation by distinct subcellular pools of protein kinase B (PKB/Akt).

The phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB)/Akt-PTEN signal transduction pathway orchestrates a variety of fundamental cell processes and its deregulation is implicated in many human diseases. Although the importance of this pathway to many cellular functions is well established, the mechanisms by which it achieves context-specific physiological outcomes in response to a variety of stimuli, using a relatively limited pool of effectors, remain largely unknown. Spatial restriction of signaling events is one means by which cells coordinate specific responses using common molecules. To investigate the subcellular location-specific roles of the major PI3K effector PKB/Akt in various cell processes, we have developed a novel experimental system employing cellular compartment-directed PKB/Akt pseudosubstrate inhibitors. Subcellular location-restricted PKB/Akt inhibition in the 3T3L1 adipocyte differentiation model revealed that nuclear and plasma membrane, but not cytoplasmic, PKB/Akt activity is required for terminal adipocyte differentiation. Nuclear and plasma membrane pools of PKB/Akt were found to contribute to distinct stages of adipocyte differentiation, revealing that PKB/Akt activity impacts multiple points of this program. Our work establishes the use of localized pseudosubstrate PKB/Akt inhibitors as an effective method for the dissection of PKB/Akt signaling.

Real-time NMR study of three small GTPases reveals that fluorescent 2'(3')-O-(N-methylanthraniloyl)-tagged nucleotides alter hydrolysis and exchange kinetics.

The Ras family of small GTPases control diverse signaling pathways through a conserved "switch" mechanism, which is turned on by binding of GTP and turned off by GTP hydrolysis to GDP. Full understanding of GTPase switch functions requires reliable, quantitative assays for nucleotide binding and hydrolysis. Fluorescently labeled guanine nucleotides, such as 2'(3')-O-(N-methylanthraniloyl) (mant)-substituted GTP and GDP analogs, have been widely used to investigate the molecular properties of small GTPases, including Ras and Rho. Using a recently developed NMR method, we show that the kinetics of nucleotide hydrolysis and exchange by three small GTPases, alone and in the presence of their cognate GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors, are affected by the presence of the fluorescent mant moiety. Intrinsic hydrolysis of mantGTP by Ras homolog enriched in brain (Rheb) is approximately 10 times faster than that of GTP, whereas it is 3.4 times slower with RhoA. On the other hand, the mant tag inhibits TSC2GAP-catalyzed GTP hydrolysis by Rheb but promotes p120 RasGAP-catalyzed GTP hydrolysis by H-Ras. Guanine nucleotide exchange factor-catalyzed nucleotide exchange for both H-Ras and RhoA was inhibited by mant-substituted nucleotides, and the degree of inhibition depends highly on the GTPase and whether the assay measures association of mantGTP with, or dissociation of mantGDP from the GTPase. These results indicate that the mant moiety has significant and unpredictable effects on GTPase reaction kinetics and underscore the importance of validating its use in each assay.

Real-time NMR study of guanine nucleotide exchange and activation of RhoA by PDZ-RhoGEF.

Small guanosine triphosphatases (GTPases) become activated when GDP is replaced by GTP at the highly conserved nucleotide binding site. This process is intrinsically very slow in most GTPases but is significantly accelerated by guanine nucleotide exchange factors (GEFs). Nucleotide exchange in small GTPases has been widely studied using spectroscopy with fluorescently tagged nucleotides. However, this method suffers from effects of the bulky fluorescent moiety covalently attached to the nucleotide. Here, we have used a newly developed real-time NMR-based assay to monitor small GTPase RhoA nucleotide exchange by probing the RhoA conformation. We compared RhoA nucleotide exchange from GDP to GTP and GTP analogues in the absence and presence of the catalytic DH-PH domain of PDZ-RhoGEF (DH-PH(PRG)). Using the non-hydrolyzable analogue guanosine-5'-O-(3-thiotriphosphate), which we found to be a reliable mimic of GTP, we obtained an intrinsic nucleotide exchange rate of 5.5 x 10(-4) min(-1). This reaction is markedly accelerated to 1179 x 10(-4) min(-1) in the presence of DH-PH(PRG) at a ratio of 1:8,000 relative to RhoA. Mutagenesis studies confirmed the importance of Arg-868 near a conserved region (CR3) of the Dbl homology (DH) domain and revealed that Glu-741 in CR1 is critical for full activity of DH-PH(PRG), together suggesting that the catalytic mechanism of PDZ-RhoGEF is similar to Tiam1. Mutation of the single RhoA (E97A) residue that contacts the pleckstrin homology (PH) domain rendered the mutant 10-fold less sensitive to the activity of DH-PH(PRG). Interestingly, this mutation does not affect RhoA activation by leukemia-associated RhoGEF (LARG), indicating that the PH domains of these two homologous GEFs may play different roles.

Evaluation of metformin in early breast cancer: a modification of the traditional paradigm for clinical testing of anti-cancer agents.

Metformin, an inexpensive oral agent commonly used to treat type 2 diabetes, has been garnering increasing attention as a potential anti-cancer agent. Preclinical, epidemiologic, and clinical evidences suggest that metformin may reduce overall cancer risk and mortality, with specific effects in breast cancer. The extensive clinical experience with metformin, coupled with its known (and modest) toxicity, has allowed the traditional process of drug evaluation to be shortened. We review the rationale for a modified approach to evaluation and outline the key steps that will optimize development of this agent in breast cancer, including discussion of a Phase III adjuvant trial (NCIC MA.32) that has recently been initiated.