A RasGAP SH3 Peptide Aptamer Inhibits RasGAP-Aurora Interaction and Induces Caspase-Independent Tumor Cell Death

The Ras GTPase-activating protein RasGAP catalyzes the conversion of active GTP-bound Ras into inactive GDP-bound Ras. However, RasGAP also acts as a positive effector of Ras and exerts an anti-apoptotic activity that is independent of its GAP function and that involves its SH3 (Src homology) domain. We used a combinatorial peptide aptamer approach to select a collection of RasGAP SH3 specific ligands. We mapped the peptide aptamer binding sites by performing yeast two-hybrid mating assays against a panel of RasGAP SH3 mutants. We examined the biological activity of a peptide aptamer targeting a pocket delineated by residues D295/7, L313 and W317. This aptamer shows a caspase-independent cytotoxic activity on tumor cell lines. It disrupts the interaction between RasGAP and Aurora B kinase. This work identifies the above-mentioned pocket as an interesting therapeutic target to pursue and points its cognate peptide aptamer as a promising guide to discover RasGAP small-molecule drug candidates

Exposure–response relationship of AMG 386 in combination with weekly paclitaxel in recurrent ovarian cancer and its implication for dose selection

To characterize exposure-response relationships of AMG 386 in a phase 2 study in advanced ovarian cancer for the facilitation of dose selection in future studies.A population pharmacokinetic model of AMG 386 (N = 141) was developed and applied in an exposure-response analysis using data from patients (N = 160) with recurrent ovarian cancer who received paclitaxel plus AMG 386 (3 or 10 mg/kg once weekly) or placebo. Reduction in the risk of progression or death with increasing exposure (steady-state area under the concentration-versus-time curve [AUC(ss)]) was assessed using Cox regression analyses. Confounding factors were tested in multivariate analysis. Alternative AMG 386 doses were explored with Monte Carlo simulations using population pharmacokinetic and parametric survival models.There was a trend toward increased PFS with increased AUC(ss) (hazard ratio [HR] for each one-unit increment in AUC(ss), 0.97; P = 0.097), suggesting that the maximum effect on prolonging PFS was not achieved at the highest dose tested (10 mg/kg). Among patients with AUC(ss) ≥ 9.6 mg h/mL, PFS was 8.1 months versus 5.7 months for AUC(ss) < 9.6 mg h/mL and 4.6 months for placebo. No relationship between AUC(ss) and grade ≥ 3 adverse events was observed. Simulations predicted that AMG 386 15 mg/kg once weekly would result in an AUC(ss) ≥ 9.6 mg h/mL in > 90% of patients with median PFS of 8.2 months versus 5.0 months for placebo (HR [15 mg/kg vs. placebo], 0.56).Increased exposure to AMG 386 was associated with improved clinical outcomes in recurrent ovarian cancer, supporting the evaluation of a higher dose in future studies

Alendronate Inhibits VEGF Expression in Growth Plate Chondrocytes by Acting on the Mevalonate Pathway

Bisphosphonates decrease chondrocyte turnover at the growth plate and impact bone growth. Likewise vascular endothelial growth factor (VEGF) plays an important role in endochondral bone elongation by influencing chondrocyte turnover at the growth plate. To investigate whether the action of bisphosphonate on the growth plate works through VEGF, VEGF protein expression and isoform transcription in endochondral chondrocytes isolated from growing mice and treated with a clinically used bisphosphonate, alendronate, were assessed. Alendronate at 10µM and 100µM concentrations decreased secreted VEGF protein expression but not cell associated protein. Bisphosphonates are known to inhibit the mevalonate intracellular signaling pathway used by VEGF. Addition of the mevalonate pathway intermediates farnesol (FOH) and geranylgeraniol (GGOH) interacted with the low concentration of alendronate to further decrease secreted VEGF protein whereas FOH partially restored VEGF protein secretion when combined with the high alendronate. Similar to the protein data, the addition of alendronate decreased VEGF mRNA isoforms. VEGF mRNA levels were rescued by the GGOH mevalonate pathway intermediate at the low alendronate dose whereas neither intermediate consistently restored the VEGF mRNA levels at the high alendronate dose. Thus, the bisphophonate alendronate impairs growth plate chondrocyte turnover by down-regulating the secreted forms of VEGF mRNA and protein by inhibiting the mevalonate pathway

Effect of a farnesyl transferase inhibitor (R115777) on ductal carcinoma in situ of the breast in a human xenograft model and on breast and ovarian cancer cell growth in vitro and in vivo

INTRODUCTION: The ras pathway is essential for cell growth and proliferation. The effects of R115777, a farnesyl transferase inhibitor, were investigated in cancer cell lines expressing varying levels of growth factor receptors and with differing ras status. Effects on tumour xenografts and human ductal carcinoma in situ (DCIS) of the breast in a xenograft mouse model were also tested. METHOD: In vitro, the concentrations required to reduce cell numbers by 50% (50% inhibitory concentration) were established (MDA-MB231, MCF-7, MCF-7/HER2-18, BT-474, SK-BR3 and SKOV3). Human DCIS was implanted in nude mice or, in separate experiments, cultured cells were injected (MDA-MB231, MCF-7/HER2-18, SKOV3) and allowed to form tumours. Proliferation and apoptosis were determined by immunohistochemistry in xenografts and cell tumours. RESULTS: The 50% inhibitory concentrations varied a hundred-fold, from 39 nmol/l (± 26 nmol/l) for SKBR3 to 5.9 μmol/l(± 0.8 μmol/l) for MDA-MB231. In MCF-7/HER2-18 and SKOV3 cells the levels of tumour growth inhibition were approximately 85% and 40%, respectively. There was a significant decrease in the cell turnover index (CTI; proliferation/apoptosis). In MDA-MB 231 with activated k-ras no inhibition was observed. In treated DCIS xenografts proliferation decreased and apoptosis increased. The CTI ratio between the start and 1 and 2 weeks of treatment were 1.99 and 1.50, respectively, for controls and 0.85 (P = 0.005) and 0.75 (P = 0.08) for treated xenografts. CONCLUSION: Treatment with the farnesyl transferase inhibitor reduced cell growth in vitro and cell tumour growth in vivo. In DCIS treatment resulted in a reduced CTI. R115777 is a promising treatment for breast cancer but the relation between effect and growth factor receptor and ras status has to be established

Prenylation Inhibition-Induced Cell Death in Melanoma: Reduced Sensitivity in BRAF Mutant/PTEN Wild-Type Melanoma Cells.

While targeted therapy brought a new era in the treatment of BRAF mutant melanoma, therapeutic options for non-BRAF mutant cases are still limited. In order to explore the antitumor activity of prenylation inhibition we investigated the response to zoledronic acid treatment in thirteen human melanoma cell lines with known BRAF, NRAS and PTEN mutational status. Effect of zoledronic acid on proliferation, clonogenic potential, apoptosis and migration of melanoma cells as well as the activation of downstream elements of the RAS/RAF pathway were investigated in vitro with SRB, TUNEL and PARP cleavage assays and videomicroscopy and immunoblot measurements, respectively. Subcutaneous and spleen-to-liver colonization xenograft mouse models were used to evaluate the influence of zoledronic acid treatment on primary and disseminated tumor growth of melanoma cells in vivo. Zoledronic acid more efficiently decreased short-term in vitro viability in NRAS mutant cells when compared to BRAF mutant and BRAF/NRAS wild-type cells. In line with this finding, following treatment decreased activation of ribosomal protein S6 was found in NRAS mutant cells. Zoledronic acid demonstrated no significant synergism in cell viability inhibition or apoptosis induction with cisplatin or DTIC treatment in vitro. Importantly, zoledronic acid could inhibit clonogenic growth in the majority of melanoma cell lines except in the three BRAF mutant but PTEN wild-type melanoma lines. A similar pattern was observed in apoptosis induction experiments. In vivo zoledronic acid did not inhibit the subcutaneous growth or spleen-to-liver colonization of melanoma cells. Altogether our data demonstrates that prenylation inhibition may be a novel therapeutic approach in NRAS mutant melanoma. Nevertheless, we also demonstrated that therapeutic sensitivity might be influenced by the PTEN status of BRAF mutant melanoma cells. However, further investigations are needed to identify drugs that have appropriate pharmacological properties to efficiently target prenylation in melanoma cells

Clinical trials update: endocrine and biological therapy combinations in the treatment of breast cancer

A greater understanding of the biological mechanisms responsible for de novo and acquired endocrine resistance has led to the rational design of clinical trials exploring the benefit of combining hormonal therapies with novel biological agents in an effort to enhance the efficacy of ER+ breast cancer treatment. These studies are increasingly including parallel biological analyses to elucidate the molecular characteristics of those tumors that are most likely to respond to specific targeted/endocrine combinations in an effort to develop a tailored approach to the management of individual patients. Unfortunately despite encouraging preclinical data, some of these combinations have yielded disappointing results in the clinical setting. This article will review the results of clinical trials of endocrine/biological combinations conducted in early and advanced breast cancer as well as provide an update on ongoing studies

Evidence-based Kernels: Fundamental Units of Behavioral Influence

This paper describes evidence-based kernels, fundamental units of behavioral influence that appear to underlie effective prevention and treatment for children, adults, and families. A kernel is a behavior–influence procedure shown through experimental analysis to affect a specific behavior and that is indivisible in the sense that removing any of its components would render it inert. Existing evidence shows that a variety of kernels can influence behavior in context, and some evidence suggests that frequent use or sufficient use of some kernels may produce longer lasting behavioral shifts. The analysis of kernels could contribute to an empirically based theory of behavioral influence, augment existing prevention or treatment efforts, facilitate the dissemination of effective prevention and treatment practices, clarify the active ingredients in existing interventions, and contribute to efficiently developing interventions that are more effective. Kernels involve one or more of the following mechanisms of behavior influence: reinforcement, altering antecedents, changing verbal relational responding, or changing physiological states directly. The paper describes 52 of these kernels, and details practical, theoretical, and research implications, including calling for a national database of kernels that influence human behavior