Effects Of Pathway Activation Research Articles

Abstract 204: Quantitative evaluation of biomarkers for TGF-β-induced epithelial-mesenchymal transition in biochemical, cellular, and 3D spheroid model systems

Abstract Epithelial-mesenchymal transition (EMT) is a cellular differentiation process whereby epithelial cells lose epithelial features and acquire mesenchymal, fibroblast-like properties, leading to reduced cell-cell contacts and increased motility. EMT is a fundamental biological process required for normal embryonic development, but may be co-opted by malignant epithelial tumors to facilitate metastatic spread. A known driver of EMT is the TGF-β superfamily of growth factor ligands, which elicit receptor-mediated responses in cells, primarily via TGF-β/SMAD signaling pathways. In these pathways, receptor-mediated SMAD (R-SMAD) proteins serve as the primary downstream effector molecules, the activities of which are regulated through receptor-mediated phosphorylation. The magnitude and duration of ligand-induced receptor activation influences the level of SMAD phosphorylation, which in turn influences the magnitude of the downstream cellular response(s). In this study, we describe high-throughput methods to quantitatively evaluate the biochemical and cellular responses to TGF-β/SMAD pathway activation in a cellular model of TGF-β-induced EMT. Effects of pathway activation are examined at different levels of biological complexity (biochemical, cellular, and multicellular), using 2D and 3D (spheroid) models. Collectively, these approaches enable a comprehensive evaluation of TGF-β/SMAD pathway activation that is amenable to high-throughput analysis platforms. Citation Format: Antony W. Wood, Ernest Heimsath. Quantitative evaluation of biomarkers for TGF-β-induced epithelial-mesenchymal transition in biochemical, cellular, and 3D spheroid model systems [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 204.

Abstract 3436: Pan-cancer analysis of the HIF-transcriptional pathway and its association with genetic susceptibility to cancer

Abstract The purpose of this study was to determine whether specific components of the HIF-transcriptional pathway are under selective pressure during the development of cancer. Induction of HIF is a common feature of most cancers that frequently results from intra-tumour hypoxia but may also be activated as a result of oncogenic switching. Activation of such large transcriptional pathways commonly leads to heterogeneous effects on tumour biology that entrain multiple responses, which both promote and inhibit tumour formation. This generates a Darwinian pressure that selects for variants (either germline or somatic) that re-balance the numerous (often small) opposing effects of pathway activation to promote tumourigenesis. We have undertaken pangenomic analyses of the direct and indirect transcriptional targets of the HIF pathway in cell lines from six common cancer types (breast, prostate, lung, colon, kidney and liver) using combined ChIP-seq and RNA-seq assays. This identifies a small set of common HIF-target genes affecting discrete pathways as well as much larger sets of target genes that are cancer-type specific. Correlating these findings with RNA-seq analyses across 20 tumour types in the TCGA database confirms distinct patterns of expression and the importance of these genes to tumour biology. Furthermore, by relating our datasets to GWAS signals of cancer-susceptibility in each tumour type, we have identified a significant excess overlap between cancer-susceptibility loci and HIF-target genes. Notably, despite the importance of HIF in other tumour types, this overlap is specific to kidney cancer in which, by contrast, HIF activation, resulting from oncogenic switching, is inappropriate to the level of intra-tumour oxygenation. This analysis provides a comprehensive analysis of the HIF pathway across multiple cancer types and provides evidence that specific aspects of the HIF-pathway are under Darwinian selection in kidney cancer. Since these alter kidney cancer susceptibility, this analysis helps distinguish those components of the HIF transcriptional response that drive tumour formation from those that are simply co-activated as a consequence of large-pathway activation. Finally, this work supports a ‘pathway tuning' model of cancer and suggests that subtle re-balancing of inappropriately activated, cancer-specific pathways is important in oncogenesis. Citation Format: David R. Mole, Virginia Schmid, Olivia Lombardi, Silvia Halim, Veronique N. Lafleur, Ran Li, Rafik Salama, Leandro Colli, Stephen Chanock, Peter J. Ratcliffe. Pan-cancer analysis of the HIF-transcriptional pathway and its association with genetic susceptibility to cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3436.

A correlation study of the expression of HA-CD44st and HER-2 in breast cancer.

BackgroundThis study investigated the effect of hyaluronic acid (HA)-CD44st on the invasive ability of human breast cancer MCF-7 cells and the correlation between the expression of CD44st and human epidermal growth factor receptor-2 (HER-2) in postoperative breast cancer patients.Materials and methodsMCF-7 cells transfected with the eukaryotic expression vector pcDNA3.1-CD44st (MCF/CD44st) were used to examine the effect of the activation of the HA-CD44st-transforming growth factor β (TGFβ)-phosphatidylinositol-3-kinase (PI3K) signaling pathway on the invasive ability of MCF-7 cells. The expression of proteins related to this signaling pathway was assessed by flow cytometry, reverse transcription-polymerase chain reaction, and Western blotting, and the role of AP-1 in the pathway was investigated by electrophoretic mobility shift assay. The effect of pathway activation on the invasion of MCF-7 cells was assessed by Transwell assay, and CD44 expression in breast cancer tissue was detected by immunohistochemistry. Quantitative reverse transcription-polymerase chain reaction was used to detect the expression of CD44st and HER-2 in breast cancer tissue and their correlation was investigated.ResultsHA significantly upregulated HER-2 and TGFβ in MCF-7/CD44st cells, increased p-AKT expression and AP-1 activity, and promoted the invasive ability of tumor cells. CD44st mRNA expression had significant difference between breast cancer tissues and adjacent normal tissues (P < 0.05), and high expression of CD44st mRNA was closely correlated with HER-2 expression in breast cancer tissues.ConclusionBinding of HA to the CD44st receptor may regulate the invasiveness of MCF-7 cells through the CD44st/TGFβ/PI3K/AP-1 signaling pathway with increased expression of TGFβ and HER-2. The expression of CD44st mRNA is correlated with HER-2 expression in postoperative breast cancer patients.

Deletion of Tsc2 in Nociceptors Reduces Target Innervation, Ion Channel Expression, and Sensitivity to Heat.

The mechanistic target of rapamycin complex 1 (mTORC1) is known to regulate cellular growth pathways, and its genetic activation is sufficient to enhance regenerative axon growth following injury to the central or peripheral nervous systems. However, excess mTORC1 activation may promote innervation defects, and mTORC1 activity mediates injury-induced hypersensitivity, reducing enthusiasm for the pathway as a therapeutic target. While mTORC1 activity is required for full expression of some pain modalities, the effects of pathway activation on nociceptor phenotypes and sensory behaviors are currently unknown. To address this, we genetically activated mTORC1 in mouse peripheral sensory neurons by conditional deletion of its negative regulator Tuberous Sclerosis Complex 2 (Tsc2). Consistent with the well-known role of mTORC1 in regulating cell size, soma size and axon diameter of C-nociceptors were increased in Tsc2-deleted mice. Glabrous skin and spinal cord innervation by C-fiber neurons were also disrupted. Transcriptional profiling of nociceptors enriched by fluorescence-associated cell sorting (FACS) revealed downregulation of multiple classes of ion channels as well as reduced expression of markers for peptidergic nociceptors in Tsc2-deleted mice. In addition to these changes in innervation and gene expression, Tsc2-deleted mice exhibited reduced noxious heat sensitivity and decreased injury-induced cold hypersensitivity, but normal baseline sensitivity to cold and mechanical stimuli. Together, these data show that excess mTORC1 activity in sensory neurons produces changes in gene expression, neuron morphology and sensory behavior.

PI3K/Akt Signaling Interacts With Wnt/β-Catenin Signaling But Does Not Induce An Accumulation Of β-Catenin In The Nucleus Of Acute Lymphoblastic Leukemia Cell Lines

Signaling pathways play essential roles in biological processes as development, cell proliferation and homeostasis. The accurate modulation of signaling pathways, their adapted interaction and their time- and tissue-specific adjusted regulation are required for normal cell development. PI3K/Akt and Wnt/β-Catenin signaling pathways act as key regulators in cell proliferation, differentiation and growth. Both signaling pathways include GSK3β as a common protein, which may mediate an interaction and cross-talk between the pathways. Aberrant activation of PI3K/Akt signaling has been linked to different types of leukemia while Wnt/β-Catenin signaling is known to be deregulated in some solid tumors. However, a potential role of Wnt/β-Catenin signaling for pathogenesis of acute lymphoblastic leukemia (ALL) has not yet been analyzed.In our study we analyzed both signaling pathways in different B- and T-ALL cell lines (RS4;11, SEM, REH, CEM, Jurkat, MOLT-4), thereby focusing mainly on their potential interaction via the protein GSK3β. Western Blot experiments were performed to evaluate the expression of specific PI3K/Akt and Wnt/β-Catenin key proteins. To evaluate the activation status of Wnt signaling immunofluorescence and protein fractionation experiments were performed, analyzing the activation linked nucleic localization of β-Catenin. The effect of pathway activation and inhibition on cell proliferation via chemical compounds was analyzed by WST-1 test.High pAkt levels were detected in B-ALL cell line SEM and T-ALL cell line CEM, indicating a hyperactive PI3K/Akt signaling, whereas other analyzed cell lines diplayed lower pAkt status. Among all cell lines analyzed SEM and CEM also showed the highest cytoplasmic β-Catenin levels, indicating a direct interaction of both signaling pathways. However, immunofluorescence and fractionation experiments revealed that a translocation of β-Catenin into the nucleus did not occur. To further investigate the role and interaction of PI3K/Akt and Wnt/β-Catenin signaling, pathway inhibiting and stimulating experiments were performed. Treatment of cells with Wnt3a led to activation of the Wnt/β-Catenin signaling cascade, characterized by nuclear β-Catenin accumulation. Inhibition of cell proliferation was detected after treatment with high concentrations Wnt3a (≥ 500 ng/ml). PI3K inhibition by LY294002 led to decreased phosphorylation of GSK3β at Ser9 and an increased decay of β-Catenin. Stimulation of PI3K/Akt signaling using activating ligand FLT3L induced GSK3β phosphorylation at Ser9 and accumulation of cytoplasmic β-Catenin. However a translocation of β-Catenin into the nucleus seems not to occur.In summary our results indicate that PI3K/Akt and Wnt/β-Catenin signaling can interact through their common protein GSK3β, but stimulation of the PI3K/Akt signaling pathway by addition of PI3K/Akt specific activators does not fully activate Wnt/β-Catenin signaling in ALL cells. Complete activation of the Wnt cascade characterized by translocation of β-Catenin into the nucleus can only be induced by use of specific Wnt effectors. Disclosures:No relevant conflicts of interest to declare.