Testing the pattern of AKT activation by variational parameter estimation

Dynamic modeling has become one of the pillars of understanding complex biological systems from a mechanistic point of view. In particular, ordinary differential equations are frequently used to model the dynamics of the interacting states, e.g., molecular species in cell signaling pathways. The equations typicallycontain many unknown parameters, such as reaction rates and initial conditions, but also time-dependent parameters, i.e., input functions driving the system. Both are a priori unknown and need to be estimated from experimental, time-resolved data. Here, we discuss an application of indirect optimal control methods for input estimation and parameter estimation in the mammalian target of rapamycin(mTOR) signaling. Whereas the direct identification and quantification of different active mTOR complexes, e.g., mTOR complex 2 (mTORC2), is only possible by highly challenging experiments, the mathematical framework allows to reconstruct its dynamics by solving an appropriate Euler–Lagrange equation based on Pontryagin’s maximum principle. The inherently large search space underlying this approach allows to test specific biological hypotheses about the activation ofprotein kinase B (AKT) by mTORC2 and to reject an alternative model with high statistical power. Hereby, we identify a minimal model that has AKT threonine phosphorylation as a prerequisite for serine phosphorylation by mTORC2. Based on this model, the activation of mTORC2 is predicted to be inhibited by drugs, targeting the receptors of the ERBB receptor family

Membrane potential differences between adriamycin-sensitive and -resistant cells as measured by flow cytometry

Using the fluorescent membrane potential probe, 3,3'-dihexyl-oxacarbocyanine (DiOC6(3)), we found a 4-fold higher uptake in Adriamycin (ADM)-sensitive versus -resistant Friend leukemia cells (FLC). When sensitive cells were treated in the presence of high potassium (120 mM K +), there was a greater than 80% reduction of DiOC6(3) uptake. Using carbonylcyanide 4-trinuoromethoxyphenylhydrazone (FCCP), a specific inhibitor of mitochondrial membrane potential, DiOC6(3) accumulation was reduced by less than 30% in these cells. Both results support the conclusion that a greater uptake of DiOC6(3) in ADM-sensitive than in -resistant cells indicates an increased plasma transmembrane potential. Since electronegative plasma membrane potentials are a driving force for the transport of lipophilic positively-charged compounds, differences in membrane potentials between sensitive and multiple drug resistant (MDR) tumor cells could have an important influence on drug accumulation and cytotoxicity. The drugs which our ADM-resistant FLC display multiple drug resistance to are positively charged. In MDR FLC, the calcium channel antagonist, verapamil, has been shown to block the efflux of Rhodamine 123 (Rho 123) and other positively-charged compounds. Since DiOC6(3) is also positivelycharged, we used verapamil to investigate its effects on drug uptake. In MDR FLC, verapamil increased DiOC6(3) accumulation by 1.9-fold, whereas in sensitive cells it was increased 1.5-fold. In contrast, verapamil increased the levels of Rho 123 in resistant cells 7.8-fold but lowered them in sensitive cells 1.5-fold. The minimal loss of DiOC6(3) from both sensitive and MDR cells and the above results can best be interpreted as indicating that DiOC6(3) is not transported by the efflux “pump” system but that verapamil induces a plasma membrane potential increase in sensitive and resistant cells that DiOC6(3) is sensitive to. On the other hand, since Rho 123 did appear to be actively effluxed from these resistant cells, the enhancement of this compound by verapamil was more likely due to inhibition of the MDR “pump”. How, or whether, plasma membrane potentials and the MDR efflux “pump” are related remains to be investigated. In the resistant cells, verapamil also induced an increase (13-fold) in the accumulation of the electrically neutral fluorescent probe for calcium, INDO-1/AM. However, verapamil had no effect on the efflux of this compound, which was equivalent in both resistant and sensitive cells. Thus, a new effect of verapamil on drug accumulation in MDR cells is identified here

Abstract 537: Is interrupting IGF1R signaling enough to overcome resistance to trastuzumab in HER2+ breast cancer models?.

Abstract Background: Trastuzumab resistance hampers its well-known efficacy in controlling HER2+ breast cancer progression. IGF1R is associated with worse outcomes in the HER2+ subtype of breast cancer (Br Cancer Res Treat 2012 132:131). Indeed, there is in vitro evidence showing that increased signaling through IGF1R confers resistance to HER2-targeting agents. However, the involvement of IGF1R and its downstream signaling pathways in this mechanism have yet to be confirmed. Purpose: Since, IGF1R is involved in the progression of breast cancers and resistance to trastuzumab therapy, co-suppression of this pathway with IGF1R mAB plus trastuzumab (T) or T-DM1 may, therefore, be efficacious in T-resistant breast cancer models. Methods: Here, we investigated the preclinical efficacy of IGF1R mAB (R7072) alone or in combination with T or T-DM1 in BT474/HerR (T-resistant) and MCF7/HER2-18 (HER2 stably overexpressed) cell lines. The inhibition of IGF1R pathway effectors was evaluated by Western blotting. Tumor growth inhibition after treatment of R7072, T/T-DM1 or the combination was examined in cell-based xenograft models. Results: 1) Doses as low as 10 μg/ml, R7072 abrogated p- AKT (S473 and T308) within 1hr of treatment in both MC7/HER2-18 and MCF7 cells (parental), 2) R7072 transiently blocked phosphorylation of downstream effector of mTOR, P70S6K (in 1hr treatment) in MCF7/HER2-18 cells but completely blocked of phosphorylation of P70S6K in MCF7 cells in both time points, 3) IGF1-induced AKT activation was inhibited in both HER2 overexpressed MCF7 and parental cells, however, both T and T-DM1 failed to block AKT activation following IGF1 stimulation in both cell lines, 4) in both cell lines, treatment of R7072 resulted in upregulation of p-ERK at higher time points, 5) R7072 was unable to block PI3K and its downstream effectors in HER2+/T-resistant cells, 6) in vivo xenograft data show that the combination of R7072 and T/T-DM1 has strongly enhanced antitumor effects in both MCF7/HER2-18 and BT474/HerR cells, but as a single agent, R7072 failed to block tumor growth in HER2+/T-resistant cells, and 5) similarly this combination significantly blocked the expression Ki67 and CD31 in tumor tissues. Conclusion: Our data suggest that the combination of R7072 and anti-HER2 antibody (T or T-DM1) is highly effective in the HER2+/T-resistant model and that similar level of inhibition cannot be achieved by either monotherapy. Citation Format: Yuliang Sun, Nandini Dey, Max Hasmann, Pradip De, Brian Leyland-Jones. Is interrupting IGF1R signaling enough to overcome resistance to trastuzumab in HER2+ breast cancer models?. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 537. doi:10.1158/1538-7445.AM2013-537 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.</jats:p

Strongly Enhanced Antitumor Activity of Trastuzumab and Pertuzumab Combination Treatment on HER2-Positive Human Xenograft Tumor Models

Abstract The human epidermal growth factor receptor (HER) family plays an important role in cell survival and proliferation, and is implicated in oncogenesis. Overexpression of HER2 is associated with aggressive disease and poor prognosis. Trastuzumab is a humanized monoclonal antibody targeting HER2 and has proven survival benefit for women with HER2-positive early and metastatic breast cancer. Pertuzumab, another monoclonal antibody, is a HER2 dimerization inhibitor that binds to a different epitope on HER2 than trastuzumab and inhibits HER2 dimer formation with other HER family members such as HER3 and HER1. We investigated the antitumor activity of these agents alone and in combination in HER2-positive breast and non–small cell lung cancer xenografts. Our data show that the combination of trastuzumab and pertuzumab has a strongly enhanced antitumor effect and induces tumor regression in both xenograft models, something that cannot be achieved by either monotherapy. The enhanced efficacy of the combination was also observed after tumor progression during trastuzumab monotherapy. Near-IR fluorescence imaging experiments confirm that pertuzumab binding to tumors is not impaired by trastuzumab pretreatment. Furthermore, we show by in vitro assay that both trastuzumab and pertuzumab potently activate antibody-dependent cellular cytotoxicity. However, our data suggest that the strongly enhanced antitumor activity is mainly due to the differing but complementary mechanisms of action of trastuzumab and pertuzumab, namely inhibition of HER2 dimerization and prevention of p95HER2 formation. [Cancer Res 2009;69(24):9330–6]</jats:p

Modulation of HER3 is a marker of dynamic cell signaling in ovarian cancer Implications for Pertuzumab Sensitivity:implications for Pertuzumab sensitivity

This study was designed to evaluate the expression of HER receptors as a marker of sensitivity to the humanized anti-HER2 monoclonal antibody pertuzumab in ovarian cancer cells. In a recent clinical trial, low levels of HER3 mRNA have been shown to associate with pertuzumab response when combined with gemcitabine. We sought to define how pertuzumab modulated HER expression levels in ovarian cancer using cell line models to better understand differential and dynamic receptor expression in therapeutic response. Changes in HER3 mRNA expression were also assessed in pertuzumab-treated xenografts. HER3 mRNA and, to a lesser extent, HER2, were down-regulated after stimulation both with heregulin-beta 1 and epidermal growth factor in a range of ovarian cancer cell lines either growth sensitive or growth resistant to pertuzumab. Pertuzumab reversed this down-regulation and the magnitude of the reversal correlated with pertuzumab sensitivity. The change in HER3 mRNA expression correlated inversely to how much the extracellular signal-regulated kinase and phosphoinositide 3-kinase pathways were dynamically activated with stimulation. Finally, up-regulation of HER3 mRNA was found in cancer xenografts treated with pertuzumab. We conclude that HER3 mRNA is down-regulated by both heregulin-beta 1 and epidermal growth factor activation. This suggests that in some tumors, low HER3 mRNA expression is driven by, or dependent on, growth factor. HER3 mRNA expression is effectively reversed in pertuzumab-sensitive tumors. These data are consistent with low HER3 mRNA identifying a pertuzumab-sensitive phenotype. (Mol Cancer Res 2009;7(9):1563-71)</p

Sensitivity to pertuzumab (2C4) in ovarian cancer models:cross-talk with estrogen receptor signaling

Pertuzumab (Omnitarg, rhuMab 2C4) is a humanized monoclonal antibody, which inhibits HER2 dimerization. Because it has shown some clinical activity in ovarian cancer, this study sought to identify predictors of response to this agent in a model of ovarian cancer. A panel of 13 ovarian cancer cell lines was treated with heregulin beta1 (HRGbeta1) or transforming growth factor-alpha, and cell proliferation was assessed. Both agents increased cell number in the majority of cell lines studied, the response to both being similar (r = 0.83; P = 0.0004, Pearson test). HRGbeta1 stimulation could be partially reversed by pertuzumab in 6 of 13 cell lines, with complete reversal in PE04 and PE06 cells. Addition of pertuzumab to transforming growth factor-alpha-stimulated cells produced growth inhibition in 3 of 13 cell lines (PE01, PE04, and PE06). The magnitude of HRGbeta1-driven growth stimulation correlated significantly with an increase in extracellular signal-regulated kinase 2 (P = 0.037) but not Akt (P = 0.99) phosphorylation. Such HRGbeta1-driven phosphorylation of extracellular signal-regulated kinase 1/2 and Akt could be reduced with pertuzumab, accompanied by changes in cell cycle distribution. In cell lines responsive to pertuzumab, HRGbeta1-enhanced phosphorylation of HER2 (Tyr(877)) was reduced. Estrogen-stimulated changes in growth, cell cycle distribution, and signaling were reversed by pertuzumab, indicating cross-talk between HER2 and estrogen signaling. These data indicate that there is a subset of ovarian cancer cell lines sensitive to pertuzumab and suggest possible predictors of response to identify patients who could benefit from this therapy. Furthermore, we have identified an interaction between HER2 and estrogen signaling in this disease.</p