ABSTRACT: MAPK scaffolds, such as IQGAP1, assemble pathway kinases together to effect signal transmission and disrupting scaffold function therefore offers a potentially orthogonal approach to MAPK cascade inhibition. Consistent with this possibility, we observed an IQGAP1 requirement in Ras-driven tumorigenesis in mouse and human tissue. Delivery of the IQGAP1 WW peptide sequence that mediates Erk1/4 binding, moreover, disrupted IQGAP1-Erk1/2 interactions, abolished Ras/Raf-driven tumorigenesis, bypassed acquired resistance to the B-Raf inhibitor vemurafinib (PLX- 4032), and acts as a systemically deliverable therapeutic to significantly increase lifespan of tumor bearing mice. Scaffold-kinase interaction blockade (SKIB) acts by a mechanism distinct from direct kinase inhibition and represents a strategy to target over-active oncogenic kinase cascades in cancer. Gene expression profiling: Fragmented cRNA was hybridized to the Mouse Gene 1.0 ST Array (Affymetrix). Iqgap1 wild-type and Iqgap1 knockout mouse treated with topical 4OHT for 0 days and 6 days days are compared.
Project description:BRAF, one of three RAF serine/threonine kinases (ARAF, BRAF and CRAF), plays a major role in the RAS-RAF-MEK-ERK mitogen-activated protein kinase (MAPK) signaling pathway, which mediates cellular responses to growth signals. Recently a high frequency (~60%-70%) of activating BRAF mutations (predominantly V600E) has been reported in malignant melanoma. In order to identify the downstream effects of BRAF signaling on melanoma cell growth and gene expression, cDNA microarray analysis was carried out following BRAF siRNA or MEK1/2 inhibitor (U0126) treatment. Keywords: time series, siRNA time series, siRNA, drug treatment
Project description:Angiogenesis is a complex process orchestrated by both growth factors and cell adhesion to the extracellular matrix and is initiated by focal degradation of the vascular basement membrane with subsequent migration and proliferation of endothelial cells (EC). The Ras/Raf/MEK/ERK pathway is critical for EC function during angiogensis. Although in vitro studies implicate ERK1 and ERK2 in EC survival, their precise role in EC function in vivo remains poorly defined. Cre/loxP technology was used to inactivate Erk1 and Erk2 in EC during murine development, resulting in embryonic lethality due to a drastic reduction in angiogenesis. The angiogenic defect was linked to diminished EC proliferation and migration, but not to increased cell apoptosis. Expression of key cell cycle regulators was diminished in the double knockout cells. In addition, both Paxillin and Focal Adhesion Kinase were expressed at lower levels and failed to correctly localize to the cell membrane in EC lacking Erk1 and Erk2, leading to defects in the organization of the cytoskeleton and in cell motility. The results demonstrate that ERK1 and ERK2 coordinate cell proliferation and migration during angiogenesis. Overall design: Lentivirus infected cells to generate ERK1/2 WT and ERK1/2 DKO endothelial cells were cultured, RNA was extracted and Affymetrix gene expression arrays were performed.
Project description:The most common oncogenic mutations in multiple myeloma (MM) affect N- and K-RAS leading to constitutive activation of RAS-dependent signaling. Signal transduction via RAS, Raf and MAPK has been well described as a canonical pathway. In accordance with this assumption, we showed that the activity of the MEK/ERK module is strictly dependent on pan-Raf activity. However, inhibition of MEK/ERK has no or only minor effects on MM cell survival, whereas oncogenic Ras and pan-Raf critically contribute to survival of multiple myeloma cells. Therefore, we aimed to learn more about Raf-dependent but MEK-independent signaling effectors. We analyzed gene expression profiles in INA-6 cells after either pan-Raf inhibition with SB-590885 or MEK inhibition with PD-325901. Overall design: Four biological replicates from each experimental group were analyzed with Affymetrix HGU133 Plus 2.0 microarrays.
Project description:Angiogenesis is a complex process orchestrated by both growth factors and cell adhesion to the extracellular matrix and is initiated by focal degradation of the vascular basement membrane with subsequent migration and proliferation of endothelial cells (EC). The Ras/Raf/MEK/ERK pathway is critical for EC function during angiogensis. Although in vitro studies implicate ERK1 and ERK2 in EC survival, their precise role in EC function in vivo remains poorly defined. Cre/loxP technology was used to inactivate Erk1 and Erk2 in EC during murine development, resulting in embryonic lethality due to a drastic reduction in angiogenesis. The angiogenic defect was linked to diminished EC proliferation and migration, but not to increased cell apoptosis. Expression of key cell cycle regulators was diminished in the double knockout cells. In addition, both Paxillin and Focal Adhesion Kinase were expressed at lower levels and failed to correctly localize to the cell membrane in EC lacking Erk1 and Erk2, leading to defects in the organization of the cytoskeleton and in cell motility. The results demonstrate that ERK1 and ERK2 coordinate cell proliferation and migration during angiogenesis. Lentivirus infected cells to generate ERK1/2 WT and ERK1/2 DKO endothelial cells were cultured, RNA was extracted and Affymetrix gene expression arrays were performed.
Project description:ABSTRACT: Despite major advances in targeted melanoma therapies, drug resistance limits their efficacy. Long noncoding RNAs (lncRNAs) are transcriptome elements that do not encode proteins but are important regulatory molecules. LncRNAs have been implemented in cancer development and response to different therapeutics and are thus potential treatment targets; however, the majority of their functions and molecular interactions remain unexplored. In this study we identify a cytoplasmic intergenic lincRNA (MIRAT), differentially expressed in drug-resistant melanoma samples. MIRAT is upregulated in early drug tolerance to MAPK inhibition and modulates MAPK signaling by binding to the MEK scaffold protein IQGAP1. Collectively, our results present MIRAT’s direct modulatory effect on the MAPK pathway and highlight the relevance of cytoplasmic lncRNA’s as potential targets for drug resistant cancer. Overall design: RNA-seq data from parental cell lines and their cell clones that are resistant to the MEK inhibitor Trametinib or BRAF inhibitor PLX-4720 (suffix RM; Resistant to MAPK inhibitor)
Project description:About 50% of human malignancies exhibit unregulated signalling through the Ras-ERK1/2 (ERK) pathway, as a consequence of activating mutations in members of Ras and Raf families. However, the quest for alternative Ras-ERK pathway-directed therapies is desirable. Upon phosphorylation ERK dimerize. We had previously demonstrated that dimerization is essential for ERK extranuclear but not nuclear signaling. Furthermore, by molecular biology approaches, we showed that specifically inhibiting ERK extranuclear component, by impeding ERK dimerization, is sufficient for curtailing tumor progression. Here, we have identified a small molecule inhibitor for ERK dimerization in vitro and in vivo that, without affecting ERK phosphorylation, prevents tumorigenesis driven by Ras-ERK pathway oncogenes, both in cellular and animal models. Importantly, this compound is unaffected by resistance-acquisition processes that hamper “classical” Ras-ERK pathway inhibitors. Thus, ERK dimerization inhibitors provide the proof of principle for two novel concepts in cancer therapy: 1) The blockade of sublocalization-specific sub-signals, rather than total signals, as a means of effectively counteracting oncogenic Ras-ERK signaling. 2) Targeting regulatory protein-protein interactions such as dimerization, rather than catalytic activities, within a signaling route, as an approach for producing effective anti-tumoral agents. Strategies aimed at preventing aberrant flux through this route remain an attractive option for therapeutic intervention in cancer. In this respect, drugs inhibiting the kinase activities of BRaf and MEK have yielded promising results. A375p cells treated with10 μM of either DEL22379, SCH772984 or DMSO as a control for two hours. mRNA from A375p cells was extrated using RNeasy mini kit (Qiagen, Germany) according to the manufacturer's instructions. Cells were previously treated with10 μM of either DEL22379, SCH772984 or DMSO as a control for two hours.
Project description:Unraveling the underlying mechanisms of cetuximab resistance in head and neck squamous cell carcinoma (HNSCC) is of major importance as many tumors remain non-responsive or become resistant. Out microarray results suggest that resistant cells still exhibit RAS-MAPK pathway signaling contributing to drug resistance, as witnessed by low expression of DUSP 5 and DUSP6, negative regulators of ERK1/2, and increased expression of AURKB, a key regulator of mitosis. Therefore, interrupting the RAS-MAPK pathway by an ERK1/2 inhibitor (apigenin) or an AURKB inhibitor (barasertib) might be a new strategy for overcoming cetuximab resistance in HNSCC 4 head and neck squamous cell carcinoma (HNSCC) cell lines were treated with either 15 nM cetuximab or PBS during 13 hours. For each cell line, differential gene expression was assessed between cetuximab and PBS treatments.
Project description:Selective RAF inhibitors including vemurafenib (PLX4032) have demonstrated clinical efficacy in mutant BRAF driven metastatic melanoma. The clinical effectiveness of RAF inhibitors depends on near complete abolition of the MAPK pathway output in tumors harboring BRAF mutations. However these compounds paradoxically activate the MAPK pathway in cells bearing oncogenic RAS or elevated upstream receptor signaling. This paradox can promote cellular proliferation and can manifest clinically with progression of secondary malignancies such as cutaneous squamous cell carcinomas (cuSCC). We have identified next generation RAF inhibitors (“paradox breakers”, e.g. PLX7904) that inhibit mutant BRAF cells without activating the MAPK pathway in cells bearing upstream activation. In murine cuSCC B9 cells that express the same HRAS mutation prevalent in squamous tumors from patients treated with RAF inhibitors, the first-generation RAF PLX4032 stimulated in vitro and in vivo growth; by contrast the paradox breaker PLX7904 had no effect. Here we compared the gene expression changes in B9 cells treated overnight with PLX4032 and PLX7904. Overall design: B9 cells were plated in 1 µM vemurafenib, 1 µM PLX7904 or 0.2% DMSO vehicle control and incubated for 17 hours. Cell lysates were harvested for RNA extraction and hybridization on Affymetrix
Project description:AIMS/HYPOTHESIS: Manoeuvres aimed at increasing beta cell mass have been proposed as regenerative medicine strategies for diabetes treatment. Raf-1 kinase inhibitor protein 1 (RKIP1) is a common regulatory node of the mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) pathways and therefore may be involved in regulation of beta cell homeostasis. The aim of this study was to investigate the involvement of RKIP1 in the control of beta cell mass and function. METHODS: Rkip1 (also known as Pebp1) knockout (Rkip1 (-/-)) mice were characterised in terms of pancreatic and glucose homeostasis, including morphological and functional analysis. Glucose tolerance and insulin sensitivity were examined, followed by assessment of glucose-induced insulin secretion in isolated islets and beta cell mass quantification through morphometry. Further characterisation included determination of endocrine and exocrine proliferation, apoptosis, MAPK activation and whole genome gene expression assays. Capacity to reverse a diabetic phenotype was assessed in adult Rkip1 (-/-) mice after streptozotocin treatment. RESULTS: Rkip1 (-/-) mice exhibit a moderately larger pancreas and increased beta cell mass and pancreatic insulin content, which correlate with an overall improvement in whole body glucose tolerance. This phenotype is established in young postnatal stages and involves enhanced cellular proliferation without significant alterations in cell death. Importantly, adult Rkip1 (-/-) mice exhibit rapid reversal of streptozotocin-induced diabetes compared with control mice. CONCLUSIONS/INTERPRETATION: These data implicate RKIP1 in the regulation of pancreatic growth and beta cell expansion, thus revealing RKIP1 as a potential pharmacological target to promote beta cell regeneration. Pancreatic gene expression of Rkip-1 (Raf kinase inhibitor 1) knockout (KO) and wild type (WT) mice, including three biological replicates in each group.