Exploration of the ErbB/Ras/MAPK signaling pathway in cancer by gene expression profiling with isoform-specific assay development and microarray technology.

摘要

The ErbB/Ras/Mitogen Activated Protein Kinase (MAPK) signal transduction pathway is a highly conserved protein kinase cascade that plays a crucial role by altering gene expression and protein activity in many cell types within many species. The mechanisms and impacts of this signaling pathway are of great interest for various reasons. The ErbB/Ras/MAPK pathway is known to activate genes required for cell proliferation, differentiation, tissue development and cell survival. As such, a study of this particular pathway can reveal key proteins involved in many cellular outcomes. Additionally, while specific genes whose expression levels are altered through the activation of the ErbBRas/MAPK pathway have been identified, a comprehensive list of pathway targets still does not exist. At least two proteins within this pathway are affected by mutations in their encoding genes. Mutations in ras and raf are commonly found in cancer cells and can cause ErbB-independent, permanent activation of the MAPK signaling pathway. Understanding the effects of these mutations as well as the pathway as a whole holds promise for possible avenues for diagnosis, detection and treatment of cancer and other genetic diseases.; We present an investigation of the ErbB/Ras/MAPK signaling pathway to expose key characteristics of significant pathway participants as well as identify gene expression changes that occur through pathway activation. Specifically, we accomplish this objective by assessing gene expression at the transcript level. Because cells tightly regulate transcription in an effort to conserve cellular resources, trends observed at the RNA level provide insight into the function of translated proteins. Here, we use two of the latest technologies to measure gene expression at the transcript level. For highly quantitative measurements of transcripts on an individual gene basis, we employ immobilized PCR. Meanwhile, microarray technology allows for a full assessment of the entire transcriptome before and after altering the expression of a key protein within the pathway.; For gene-specific investigations of ras and raf isoforms, we have developed two assays employing immobilized PCR that allow us to distinguish between isoform transcripts. Through the use of these assays, we find that the ratio of k-ras4B:k-ras4A is nearly 10x higher in colon cancer cells and 50x higher in pancreatic cancer cells than in normal cells. Furthermore, this increase in k-ras4B:k-ras4A ratio is a result of both an increase in k-ras4B expression and decrease in k-ras4A expression. We observe that raf expression is observed to vary across all of the tissue types tested. Notably, c-Raf-1 expression is doubled and A-Raf expression is halved in pancreatic tumor cells as compared to normal pancreas cells. Also, B-raf is barely expressed in colon cells. This information could be used to (1) monitor disease progression, (2) diagnose disease, and/or (3) determine disease state.; In our evaluation of the entire transcriptome, we exploit naturally occurring RNA interference (RNAi) capabilities within the cell to silence the cancer-critical gene k-ras and prevent expression of the K-ras protein that activates the MAPK pathway. RNAi of k-ras within CAPAN-1 pancreatic cancer cells shows that various transcription regulators (ATF3, FOSB, EGR2, EFR4, HIST1H4H, HIST2H4A, H4, ZBTB10, LINCR, AFF4, EGR1) are induced upon k-ras silencing, resulting in apoptosis. Further, these results support the oncogene addiction theory that suggests the cancer cell's irreversible dependence upon K-ras for MAPK pathway activation. The k-ras-like genes, NRG2, GEM, and Rho, experienced increases in expression as well, indicating unsuccessful efforts by the cell to find alternate avenues for growth and differentiation. Additionally, the RNase H enzyme, RNASE7, was induced, perhaps playing a yet undiscovered role in the RNAi mechanism.