bms-536924 and Cell-Transformation--Neoplastic

Malignant transformation of normal hematopoietic progenitors is a multistep process that likely requires interaction between collaborating oncogenic signals at critical junctures. For instance, the MLL-AF9 fusion oncogene is thought to contribute to myeloid leukemogenesis by driving a hematopoietic stem cell-like "self-renewal" gene expression signature in committed myeloid progenitors. In addition, insulin-like growth factor (IGF) signaling has been implicated in self-renewal/pluripotency in hematopoietic and embryonic stem cell contexts and supports cell growth/survival by activation of downstream pathways, including phosphatidylinositol 3-kinase/Akt and Ras/Raf/extracellular signal-regulated kinase. We hypothesized that IGF signaling could be an important contributor in the process of cellular transformation and/or clonal propagation. Utilizing an MLL-AF9 mouse bone marrow transplantation model of acute myelogenous leukemia, we discovered that committed myeloid progenitor cells with genetically reduced levels of IGF1R were less susceptible to leukemogenic transformation due, at least in part, to a cell-autonomous defect in clonogenic activity. Rather unexpectedly, genetic deletion of IGF1R by inducible Cre recombinase had no effect on growth/survival of established leukemia cells. These findings suggest that IGF1R signaling contributes to transformation of normal myeloid progenitor cells, but is not required for propagation of the leukemic clone once it has become established. We also show that treatment of mouse MLL-AF9 acute myelogenous leukemia cells with BMS-536924, an IGF1R/insulin receptor-selective tyrosine kinase inhibitor, blocked cell growth, suggesting its efficacy in this model may be due to inhibition of insulin receptor and/or related tyrosine kinases, and raising the possibility that similar IGF1R inhibitors in clinical development may be acting through alternate/related pathways.

Topics: Animals; Benzimidazoles; Blotting, Western; Bone Marrow Transplantation; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; Female; Flow Cytometry; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid, Acute; Male; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Knockout; Oncogene Proteins, Fusion; Pyrazoles; Pyridones; Receptor, IGF Type 1; Signal Transduction; Survival Analysis; Triazines

The insulin-like growth factor (IGF) 1 receptor (IGF1R) is an important therapeutic target under study in many cancers. Here, we describe a breast cancer model based on expression of the ETV6-NTRK3 (EN) chimeric tyrosine kinase that suggests novel therapeutic applications of IGF1R inhibitors in secretory breast cancers. Originally discovered in congenital fibrosarcomas with t(12;15) translocations, EN was identified subsequently in secretory breast carcinoma (SBC) which represent a variant of invasive ductal carcinoma. Because fibroblast transformation by EN requires the IGF1R axis, we hypothesized a similar dependency may exist in mammary cells and, if so, that IGF1R inhibitors might be useful to block EN-driven breast oncogenesis. In this study, we analyzed EN expressing murine and human mammary epithelial cell lines for transformation properties. Various IGF1R signaling inhibitors, including the dual specificity IGF1R/insulin receptor (INSR) inhibitor BMS-536924, were then tested for effects on three-dimensional Matrigel cell growth, migration, and tumor formation. We found that EN expression increased acinar size and luminal filling in Matrigel cultures and promoted orthotopic tumor growth in mice. Tumors were well differentiated and nonmetastatic, similar to human SBC. The known EN effector pathway, PI3K-Akt, was activated in an IGF1- or insulin-dependent manner. BMS-536924 blocked EN transformation in vitro, whereas BMS-754807, another IGIFR/INSR kinase inhibitor currently in clinical trials, significantly reduced tumor growth in vivo. Importantly, EN model systems mimic the clinical phenotype observed in human SBC. Moreover, EN has a strict requirement for IGF1R or INSR in breast cell transformation. Thus, our findings strongly encourage the evaluation of IGF1R/INSR inhibitors to treat EN-driven breast cancers.

Topics: Animals; Benzimidazoles; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Transformation, Neoplastic; Epithelial-Mesenchymal Transition; Humans; Insulin Receptor Substrate Proteins; Mice; Mice, Transgenic; Molecular Targeted Therapy; Oncogene Protein v-akt; Oncogene Proteins, Fusion; Pyridones; Receptor, IGF Type 1; Signal Transduction; Transplantation, Heterologous

This study aimed to test the ability of a new insulin-like growth factor receptor (IGF-IR) tyrosine kinase inhibitor, BMS-536924, to reverse the ability of constitutively active IGF-IR (CD8-IGF-IR) to transform MCF10A cells, and to examine the effect of the inhibitor on a range of human breast cancer cell lines.. CD8-IGF-IR-MCF10A cells were grown in monolayer culture, three-dimensional (3D) culture, and as xenografts, and treated with BMS-536924. Proliferation, cell cycle, polarity, and apoptosis were measured. Twenty-three human breast cancer cell lines were treated in monolayer culture with BMS-536924, and cell viability was measured. MCF7, MDA-MB-231, and MDA-MB-435 were treated with BMS-536924 in monolayer and 3D culture, and proliferation, migration, polarity, and apoptosis were measured.. Treatment of CD8-IGF-IR-MCF10A cells grown in 3D culture with BMS-536924 caused a blockade of proliferation, restoration of apical-basal polarity, and enhanced apoptosis, resulting in a partial phenotypic reversion to normal acini. In monolayer culture, BMS-536924 induced a dose-dependent inhibition of proliferation, with an accumulation of cells in G(0)/G(1,), and completely blocked CD8-IGF-IR-induced migration, invasion, and anchorage-independent growth. CD8-IGF-IR-MCF10A xenografts treated with BMS-536924 (100 mg/kg/day) showed a 76% reduction in xenograft volume. In a series of 23 human breast cancer cell lines, BMS-536924 inhibited monolayer proliferation of 16 cell lines. Most strikingly, treatment of MCF7 cells grown in 3D culture with BMS-536924 caused blockade of proliferation, and resulted in the formation of hollow polarized lumen.. These results show that the new small molecule BMS-536924 is an effective inhibitor of IGF-IR, causing a reversion of an IGF-IR - mediated transformed phenotype.

Topics: Apoptosis; Benzimidazoles; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Polarity; Cell Proliferation; Cell Transformation, Neoplastic; Drug Screening Assays, Antitumor; Epithelial Cells; Female; Humans; Mammary Glands, Human; Pyridones; Receptors, Somatomedin

Type I insulin-like growth factor receptor (IGF-IR) can transform mouse fibroblasts; however, little is known about the transforming potential of IGF-IR in human fibroblasts or epithelial cells. We found that overexpression of a constitutively activated IGF-IR (CD8-IGF-IR) was sufficient to cause transformation of immortalized human mammary epithelial cells and growth in immunocompromised mice. Furthermore, CD8-IGF-IR caused cells to undergo an epithelial-to-mesenchymal transition (EMT) which was associated with dramatically increased migration and invasion. The EMT was mediated by the induction of the transcriptional repressor Snail and downregulation of E-cadherin. NF-kappaB was highly active in CD8-IGF-IR-MCF10A cells, and both increased levels of Snail and the EMT were partially reversed by blocking NF-kappaB or IGF-IR activity. This study places IGF-IR among a small group of oncogenes that, when overexpressed alone, can confer in vivo tumorigenic growth of MCF10A cells and indicates the hierarchy in the mechanism of IGF-IR-induced EMT.

Topics: Animals; Benzimidazoles; Cadherins; CD8 Antigens; Cell Transformation, Neoplastic; Collagen; Down-Regulation; Drug Combinations; Epithelial Cells; Genes, Regulator; Humans; Laminin; Mammary Glands, Human; Mesoderm; Mice; Models, Biological; Morphogenesis; NF-kappa B; Proteoglycans; Pyridones; Receptor, IGF Type 1; Recombinant Fusion Proteins; Snail Family Transcription Factors; Transcription Factors; Transplantation, Heterologous