Gene Expression Profile Identifies Tyrosine Kinase c-Met as a Targetable Mediator of Antiangiogenic Therapy Resistance

PURPOSE: To identify mediators of glioblastoma anti-angiogenic therapy resistance and target these mediators in xenografts. EXPERIMENTAL DESIGN: We performed microarray analysis comparing bevacizumab-resistant glioblastomas (BRGs) to pre-treatment tumors from the same patients. We established novel xenograft models of anti-angiogenic therapy resistance to target candidate resistance mediator(s). RESULTS: BRG microarray analysis revealed upregulation versus pre-treatment of receptor tyrosine kinase c-Met, which underwent further investigation because of its prior biologic plausibility as a bevacizumab resistance mediator. BRGs exhibited increased hypoxia versus pre-treatment in a manner correlating with their c-Met upregulation, increased c-Met phosphorylation, and increased phosphorylation of c-Met-activated focal adhesion kinase (FAK) and STAT3. We developed two novel xenograft models of anti-angiogenic therapy resistance. In the first model, serial bevacizumab treatment of an initially responsive xenograft generated a xenograft with acquired bevacizumab resistance, which exhibited upregulated c-Met expression versus pre-treatment. In the second model, a BRG-derived xenograft maintained refractoriness to the MRI tumor vasculature alterations and survival-promoting effects of bevacizumab. Growth of this BRG-derived xenograft was inhibited by a c-Met inhibitor. Transducing these xenograft cells with c-Met shRNA inhibited their invasion and survival in hypoxia, disrupted their mesenchymal morphology, and converted them from bevacizumab-resistant to bevacizumab-responsive. Engineering bevacizumab-responsive cells to express constitutively active c-Met caused these cells to form bevacizumab-resistant xenografts. CONCLUSION: These findings support the role of c-Met in survival in hypoxia and invasion, features associated with anti-angiogenic therapy resistance; and growth and therapeutic resistance of xenografts resistant to anti-angiogenic therapy. Therapeutically targeting c-Met could prevent or overcome anti-angiogenic therapy resistance

Fully Automatic Quantification of Microarray Image Data

DNA microarrays are now widely used to measure expression levels and DNA copy number in biological samples. Ratios of relative abundance of nucleic acids are derived from images of regular arrays of spots containing target genetic material to which fluorescently labeled samples are hybridized. Whereas there are a number of methods in use for the quantification of images, many of the software systems in wide use either encourage or require extensive human interaction at the level of individual spots on arrays. We present a fully automatic system for microarray image quantification. The system automatically locates both subarray grids and individual spots, requiring no user identification of any image coordinates. Ratios are computed based on explicit segmentation of each spot. On a typical image of 6000 spots, the entire process takes less than 20 sec. We present a quantitative assessment of performance on multiple replicates of genome-wide array-based comparative genomic hybridization experiments. By explicitly identifying the pixels in each spot, the system yields more accurate estimates of ratios than systems assuming spot circularity. The software, called UCSF Spot, runs on Windows platforms and is available free of charge for academic use

General quantitative relations linking cell growth and the cell cycle in Escherichia coli

Growth laws emerging from studies of cell populations provide essential constraints on the global mechanisms that coordinate cell growth1-3. The foundation of bacterial cell cycle studies relies on two interconnected dogmas that were proposed more than 50 years ago-the Schaechter-Maaloe-Kjeldgaard growth law that relates cell mass to growth rate1 and Donachie's hypothesis of a growth-rate-independent initiation mass4. These dogmas spurred many efforts to understand their molecular bases and physiological consequences5-14. Although they are generally accepted in the fast-growth regime, that is, for doubling times below 1 h, extension of these dogmas to the slow-growth regime has not been consistently achieved. Here, through a quantitative physiological study of Escherichia coli cell cycles over an extensive range of growth rates, we report that neither dogma holds in either the slow- or fast-growth regime. In their stead, linear relations between the cell mass and the rate of chromosome replication-segregation were found across the range of growth rates. These relations led us to propose an integral-threshold model in which the cell cycle is controlled by a licensing process, the rate of which is related in a simple way to chromosomal dynamics. These results provide a quantitative basis for predictive understanding of cell growth-cell cycle relationships

Transcription and methylation analyses of preleukemic promyelocytes indicate a dual role for PML/RARA in leukemia initiation

International audienceAcute promyelocytic leukemia is an aggressive malignancy characterized by the accumulation of promyelocytes in the bone marrow. PML/RARA is the primary abnormality implicated in this pathology, but the mechanisms by which this chimeric fusion protein initiates disease are incompletely understood. Identifying PML/RARA targets in vivo is critical for comprehending the road to pathogenesis. Utilizing a novel sorting strategy, we isolated highly purified promyelocyte populations from normal and young preleukemic animals, carried out microarray and methylation profiling analyses, and compared the results from the two groups of animals. Surprisingly, in the absence of secondary lesions, PML/RARA had an overall limited impact on both the transcriptome and methylome. Of interest, we did identify down-regulation of secondary and tertiary granule genes as the first step engaging the myeloid maturation block. Although initially not sufficient to arrest terminal granulopoiesis in vivo, such alterations set the stage for the later, complete differentiation block seen in leukemia. Further, gene set enrichment analysis revealed that PML/RARA promyelocytes exhibit a subtle increase in expression of cell cycle genes, and we show that this leads to both increased proliferation of these cells and expansion of the promyelocyte compartment. Importantly, this proliferation signature was absent from the poorly leukemogenic p50/RARA fusion model, implying a critical role for PML in the altered cell-cycle kinetics and ability to initiate leukemia. Thus, our findings challenge the predominant model in the field and we propose that PML/RARA initiates leukemia by subtly shifting cell fate decisions within the promyelocyte compartment

Gene Expression Profile Identifies Tyrosine Kinase c-Met as a Targetable Mediator of Antiangiogenic Therapy Resistance

PurposeTo identify mediators of glioblastoma antiangiogenic therapy resistance and target these mediators in xenografts.Experimental designWe conducted microarray analysis comparing bevacizumab-resistant glioblastomas (BRG) with pretreatment tumors from the same patients. We established novel xenograft models of antiangiogenic therapy resistance to target candidate resistance mediator(s).ResultsBRG microarray analysis revealed upregulation versus pretreatment of receptor tyrosine kinase c-Met, which underwent further investigation because of its prior biologic plausibility as a bevacizumab resistance mediator. BRGs exhibited increased hypoxia versus pretreatment in a manner correlating with their c-Met upregulation, increased c-Met phosphorylation, and increased phosphorylation of c-Met-activated focal adhesion kinase and STAT3. We developed 2 novel xenograft models of antiangiogenic therapy resistance. In the first model, serial bevacizumab treatment of an initially responsive xenograft generated a xenograft with acquired bevacizumab resistance, which exhibited upregulated c-Met expression versus pretreatment. In the second model, a BRG-derived xenograft maintained refractoriness to the MRI tumor vasculature alterations and survival-promoting effects of bevacizumab. Growth of this BRG-derived xenograft was inhibited by a c-Met inhibitor. Transducing these xenograft cells with c-Met short hairpin RNA inhibited their invasion and survival in hypoxia, disrupted their mesenchymal morphology, and converted them from bevacizumab-resistant to bevacizumab-responsive. Engineering bevacizumab-responsive cells to express constitutively active c-Met caused these cells to form bevacizumab-resistant xenografts.ConclusionThese findings support the role of c-Met in survival in hypoxia and invasion, features associated with antiangiogenic therapy resistance, and growth and therapeutic resistance of xenografts resistant to antiangiogenic therapy. Therapeutically targeting c-Met could prevent or overcome antiangiogenic therapy resistance

Fractional Genomic Alteration Detected by Array-Based

Purpose: Although liver resection is the primary curative therapy for patients with colorectal hepatic metastases, most patients have a recurrence. Identification of molecular markers that predict patients at highest risk for recurrence may help to target further therapy