Research Article: Master Regulators of Oncogenic KRAS Response in Pancreatic Cancer: An Integrative Network Biology Analysis

Date Published: January 31, 2017

Publisher: Public Library of Science

Author(s): Shivan Sivakumar, Ines de Santiago, Leon Chlon, Florian Markowetz, Marc Ladanyi

Abstract: BackgroundKRAS is the most frequently mutated gene in pancreatic ductal adenocarcinoma (PDAC), but the mechanisms underlying the transcriptional response to oncogenic KRAS are still not fully understood. We aimed to uncover transcription factors that regulate the transcriptional response of oncogenic KRAS in pancreatic cancer and to understand their clinical relevance.Methods and FindingsWe applied a well-established network biology approach (master regulator analysis) to combine a transcriptional signature for oncogenic KRAS derived from a murine isogenic cell line with a coexpression network derived by integrating 560 human pancreatic cancer cases across seven studies. The datasets included the ICGC cohort (n = 242), the TCGA cohort (n = 178), and five smaller studies (n = 17, 25, 26, 36, and 36). 55 transcription factors were coexpressed with a significant number of genes in the transcriptional signature (gene set enrichment analysis [GSEA] p < 0.01). Community detection in the coexpression network identified 27 of the 55 transcription factors contributing to three major biological processes: Notch pathway, down-regulated Hedgehog/Wnt pathway, and cell cycle. The activities of these processes define three distinct subtypes of PDAC, which demonstrate differences in survival and mutational load as well as stromal and immune cell composition. The Hedgehog subgroup showed worst survival (hazard ratio 1.73, 95% CI 1.1 to 2.72, coxPH test p = 0.018) and the Notch subgroup the best (hazard ratio 0.62, 95% CI 0.42 to 0.93, coxPH test p = 0.019). The cell cycle subtype showed highest mutational burden (ANOVA p < 0.01) and the smallest amount of stromal admixture (ANOVA p < 2.2e–16). This study is limited by the information provided in published datasets, not all of which provide mutational profiles, survival data, or the specifics of treatment history.ConclusionsOur results characterize the regulatory mechanisms underlying the transcriptional response to oncogenic KRAS and provide a framework to develop strategies for specific subtypes of this disease using current therapeutics and by identifying targets for new groups.

Partial Text: Pancreatic ductal adenocarcinoma (PDAC) is the most lethal human malignancy, with a 5-y survival of 4% [1]. There are very few treatment options, with the only chance of a cure being surgical resection if the tumour is detected at an early, confined stage. Chemotherapeutic options are used in the palliative setting but have toxic side effects and do not extend survival for more than a few months. Pancreatic cancers display vast intratumoural heterogeneity with respect to their mutational profiles [2], but more than 90% of cases have a mutation in the KRAS oncogene, which almost exclusively is located in codon 12 [3].

This study did not have a protocol or prospective analysis plan. To achieve the first aim, we used master regulator analysis, a well-established network biology strategy [13–16], which combines a transcriptional signature with a coexpression network to identify key transcription factors. For the second aim, we used clustering techniques to identify patient subtypes based on transcription factor activities and characterised these subtypes by survival analysis, integration of mutation data, and methods that infer immune activity from gene expression profiles. For clarity, the Methods description is split into three main sections: defining a transcriptional signature for oncogenic KRAS, identification of master regulators of KRAS response, and characterisation of PDAC subtypes. All code and scripts to reproduce our analysis are available as annotated documents as part of the supplementary information (S1 Computational Analysis, S2 Computational Analysis).

The first aim of our study was to identify transcription factors determining the transcriptional response to oncogenic KRAS. To address this aim, we used master regulator analysis to combine a murine gene expression signature of oncogenic KRAS with a coexpression network integrating data from 560 patients. We found that the KRAS-specific master regulators represented three main biological processes: Notch, repressed Hedgehog/Wnt, and the cell cycle. The second aim was to explore the extent to which activity of these biological processes had on the development of the disease and patient outcome. We demonstrated that the functional groupings of the transcription factors represented distinct clinical entities of PDAC, with differences in survival, mutational load, and immune activity. These three patient subtypes represent three routes of PDAC development, characterized by three different transcriptional programs.



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