Using DNA microarrays together with quantitative proteomic techniques (ICAT reagents, two-dimensional DIGE, and MS), we evaluated the correlation of mRNA and protein levels in two hematopoietic cell lines representing distinct stages of myeloid differentiation, as well as in the livers of mice treated for different periods of time with three different peroxisome proliferative activated receptor agonists. We observe that the differential expression of mRNA (up or down) can capture at most 40% of the variation of protein expression. Although the overall pattern of protein expression is similar to that of mRNA expression, the incongruent expression between mRNAs and proteins emphasize the importance of posttranscriptional regulatory mechanisms in cellular development or perturbation that can be unveiled only through integrated analyses of both proteins and mRNAs. Molecular & Cellular Proteomics 3:960 -969, 2004.Genome-wide mRNA expression profiling by means of DNA microarrays has proven to be a powerful approach in characterizing the changes in biological processes such as disease states, developmental stages, and responses to drugs or genetic perturbations (1). However, DNA arrays measure only the changes at the mRNA level. Most biological functions are executed by the proteins rather than mRNAs. While the expression of many genes is controlled at the transcriptional level, other genes also employ posttranscriptional regulation processes involving mRNA stability, translation initiation, and protein stability. An important issue is the extent to which the changing expression patterns of mRNAs reflect corresponding changes in their cognate proteins. Recent advances in quantitative proteomics, especially the application of ICAT reagents in conjunction with MS, have made possible simultaneous quantitative comparison of hundreds of proteins between two complex mixtures (2). Integrated analyses of mRNA and protein expression data by concurrent measurement of both have revealed moderate to poor correlation in yeast and Halobacteria (3-5). Discordant expression of protein and mRNA was also observed in lung adenocarcinomas (6). However, these analyses examined only one aspect of a biological system, i.e. the steady-state levels of mRNAs and proteins. Another important aspect that concerns the kinetic process of perturbation and how the correlation of mRNA and protein evolves during this process was not addressed. Here, we evaluated the correlation of mRNA and protein expression in mammalian systems under two experimental conditions. In the first, we compared steady-state levels of mRNAs and proteins between two related cell lines representing distinct hematopoietic stages, i.e. multipotent myeloid precursors versus lineage-committed promyelocytic cells. In the second condition, we used a mouse model to demonstrate the kinetic changes in liver mRNA and protein levels in response to treatment with three different drugs. In both cases, we observed a moderate correlation between mRNA and protein levels with the expression of m...

High-throughput screening (HTS) plays a central role in modern drug discovery, allowing the rapid screening of large compound collections against a variety of putative drug targets. HTS is an industrial-scale process, relying on sophisticated automation, control, and state-of-the art detection technologies to organize, test, and measure hundreds of thousands to millions of compounds in nano- to microliter volumes. Despite this high technology, hit selection for HTS is still typically done using simple data analysis and basic statistical methods. The authors discuss in this article some shortcomings of these methods and present alternatives based on modern methods of statistical data analysis. Most important, they describe and show numerous real examples from the biologist-friendly Stat Server HTS application (SHS), a custom-developed software tool built on the commercially available S-PLUS and StatServer statistical analysis and server software. This system remotely processes HTS data using powerful and sophisticated statistical methodology but insulates users from the technical details by outputting results in a variety of readily interpretable graphs and tables.

Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate consisting of the anti-HER2 antibody trastuzumab linked via a nonreducible thioether linker to the maytansinoid antitubulin agent DM1. T-DM1 has shown favorable safety and efficacy in patients with HER2-positive metastatic breast cancer. In previous animal studies, T-DM1 exhibited better pharmacokinetics (PK) and slightly more efficacy than several disulfide-linked versions. The efficacy findings are unique, as other disulfide-linked antibody-drug conjugates (ADC) have shown greater efficacy than thioether-linked designs. To explore this further, the in vitro and in vivo activity, PK, and target cell activation of T-DM1 and the disulfide-linked T-SPP-DM1 were examined. Both ADCs showed high in vitro potency, with T-DM1 displaying greater potency in two of four breast cancer cell lines. In vitro target cell processing of T-DM1 and T-SPP-DM1 produced lysine-N e -MCC-DM1, and lysine-N e -SPP-DM1 and DM1, respectively; in vivo studies confirmed these results. The in vitro processing rates for the two conjugate to their respective catabolites were similar. In vivo, the potencies of the conjugates were similar, and T-SPP-DM1 had a faster plasma clearance than T-DM1. Slower T-DM1 clearance translated to higher overall tumor concentrations (conjugate plus catabolites), but unexpectedly, similar levels of tumor catabolite. These results indicate that, although the ADC linker can have clear impact on the PK and the chemical nature of the catabolites formed, both linkers seem to offer the same payload delivery to the tumor.

Purpose: Inhibition of the vascular endothelial growth factor (VEGF) axis is the basis of all currently approved antiangiogenic therapies. In preclinical models, anti-VEGF blocking antibodies have shown broad efficacy that is dependent on both tumor context and treatment duration. We aimed to characterize this activity and to evaluate the effects of discontinuation of treatment on the dynamics of tumor regrowth. Experimental Design: We evaluated the effects of anti-VEGF treatment on tumor growth and survival in 30 xenograft models and in genetic mouse models of cancer. Histologic analysis was used to evaluate the effects of treatment on tumor vasculature. We used a variety of treatment regimens to allow analysis of the effects of treatment duration and cessation on growth rate, survival, and vascular density. Results: Preclinical tumor models were characterized for their varied dependence on VEGF, thereby defining models for testing other agents that may complement or augment anti-VEGF therapy. We also found that longer exposure to anti-VEGF monoclonal antibodies delayed tumor growth and extended survival in established tumors from both cell transplants and genetic tumor models and prevented regrowth of a subset of residual tumors following cytoablative therapy. Discontinuation of anti-VEGF in established tumors resulted in regrowth at a rate slower than that in control-treated animals, with no evidence of accelerated tumor growth or rebound. However, more rapid regrowth was observed following discontinuation of certain chemotherapies. Concurrent administration of anti-VEGF seemed to normalize these accelerated growth rates. Conclusions: In diverse preclinical models, continuous VEGF suppression provides maximal benefit as a single agent, combined with chemotherapy, or as maintenance therapy once chemotherapy has been stopped. Clin Cancer Res; 16(15); 3887–900. ©2010 AACR.

Purpose: Oncogenic activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway is prevalent in breast cancer and has been associated with resistance to HER2 inhibitors in the clinic.We therefore investigated the combinatorial activity of GDC-0941, a novel class IPI3K inhibitor, with standard-of-care therapies for HER2-amplified breast cancer. Experimental Design: Three-dimensional laminin-rich extracellular matrix cultures of human breast cancer cells were utilized to provide a physiologically relevant approach to analyze the efficacy and molecular mechanism of combination therapies ex vivo. Combination studies were done using GDC-0941 with trastuzumab (Herceptin), pertuzumab, lapatinib (Tykerb), and docetaxel, the principal therapeutic agents that are either approved or being evaluated for treatment of early HER2-positive breast cancer.Results: Significant GDC-0941 activity (EC 50 <1 Amol/L) was observed for >70% of breast cancer cell lines that were examined in three-dimensional laminin-rich extracellular matrix culture. Differential responsiveness to GDC-0941as a single agent was observed for luminal breast cancer cells upon stimulation with the HER3 ligand, heregulin. Combined treatment of GDC-0941, trastuzumab, and pertuzumab resulted in growth inhibition, altered acinar morphology, and suppression of AKT mitogen-activated protein kinase (MAPK) / extracellular signed-regulated kinase (ERK) kinase and MEK effector signaling pathways for HER2-amplified cells in both normal and heregulin-supplemented media. The GDC-0941 and lapatinib combination further showed that inhibition of HER2 activity was essential for maximum combinatorial efficacy. PI3K inhibition also rendered HER2-amplified BT-474M1cells and tumor xenografts more sensitive to docetaxel. Conclusions: GDC-0941 is efficacious in preclinical models of breast cancer. The addition of GDC-0941to HER2-directed treatment could augment clinical benefit in breast cancer patients.