Assessment of Functional Phosphatidylinositol 3-Kinase Pathway Activity in Cancer Tissue Using Forkhead Box-O Target Gene Expression in a Knowledge-Based Computational Model

Abstract

The phosphatidylinositol 3-kinase (PI3K) pathway is commonly activated in cancer. Tumors are potentially sensitive to PI3K pathway inhibitors, but reliable diagnostic tests that assess functional PI3K activity are lacking. Because PI3K pathway activity negatively regulates forkhead box-O (FOXO) transcription factor activity, FOXO target gene expression is inversely correlated with PI3K activity. A knowledge-based Bayesian computational model was developed to infer PI3K activity in cancer tissue samples from FOXO target gene mRNA levels and validated in cancer cell lines treated with PI3K inhibitors. However, applied to patient tissue samples, FOXO was often active in cancer types with expected active PI3K. SOD2 was differentially expressed between FOXO-active healthy and cancer tissue samples, indicating that cancer-associated cellular oxidative stress alternatively activated FOXO. To enable correct interpretation of active FOXO in cancer tissue, threshold levels for normal SOD2 expression in healthy tissue were defined above which FOXO activity is oxidative stress induced and below which PI3K regulated. In slow-growing luminal A breast cancer and low Gleason score prostate cancer, FOXO was active in a PI3K-regulated manner, indicating inactive PI3K. In aggressive luminal B, HER2, and basal breast cancer, FOXO was increasingly inactive or actively induced by oxidative stress, indicating PI3K activity. We provide a decision tree that facilitates functional PI3K pathway activity assessment in tissue samples from patients with cancer for therapy response prediction and prognosis.

Quantitative measurement of activity of JAK-STAT signaling pathways in blood samples and immune cells to predict innate and adaptive cellular immune response to viral infection and accelerate vaccine development

“…To the best of our knowledge, sofar it has not been possible to measure the cellular adaptive immune response in a blood sample from a patient. To further complete our signal transduction pathway testing platform, we have now developed tests to quantitatively measure functional activity of the JAK-STAT1/2 and JAK-STAT3 signal transduction pathways in different immune cell types, based on the principle of measuring and interpreting target gene mRNA levels of the signaling pathway transcription factor (6), (7), (8). We provide evidence that measurement of activity of the JAK-STAT1/2 signaling pathway in whole blood, PBMCs, or T-cell subsets, from patients with an infectious disease allows distinction between viral and bacterial infection and is informative on the strength of the adaptive cellular immune response against a viral infection.…”

“…Development of tests to measure JAK-STAT1/2 and JAK-STAT3 pathway activity were developed as described before (6), (7), (8). In brief, target genes of the STAT transcription factors of the respective pathways were identified, and a Bayesian network computational model was created for interpretation of measured mRNA levels of the pathway target genes.…”

“…Following model calibration and model parameter freeze, mRNA probeset measurements of a new sample can be entered into the model, and Bayesian inference is used to calculate probability P that the transcription factor, and therefore the signalling pathway, is active. Pathway activity score can be presented as log2odds value log2 (P / (1 -P)), which can optionally be normalized on a scale from 0-100 (6), (7), (8). Quantitative log2odds pathway activity scores are used in the figures and results description.…”

“…We describe development, as well as evaluation in preclinical and clinical studies, of an mRNAbased test to quantify the immune response to a viral infection, based on measuring activity of the JAK-STAT1/2 and JAK-STAT3 signal transduction pathways in a whole blood or Peripheral Blood Mononuclear Cell (PBMC) sample, or in separate immune cell types. The technology used to develop tests to measure activity of signal transduction pathways is based on measuring the mRNA levels of target genes of the pathway-specific transcription factor and has been described before (6), (7),(8),(9),(10),(11), (12) , (13).…”

A novel dual antibody staining assay to measure estrogen receptor transcriptional activity

“…The calculated ER transcriptional activity score is taken as equivalent of the ER pathway activity score. Activity scores are presented on a log2 odds scale 5 , 6 .…”