Regulation of oncogenic KRAS signaling via a novel KRAS-integrin-linked kinase-hnRNPA1 regulatory loop in human pancreatic cancer cells

Abstract

Integrin-linked kinase (ILK) is a mediator of aggressive phenotype in pancreatic cancer. On the basis of our finding that knockdown of either KRAS or ILK has a reciprocal effect on the other's expression, we hypothesized the presence of an ILK-KRAS regulatory loop that enables pancreatic cancer cells to regulate KRAS expression. This study aimed to elucidate the mechanism by which this regulatory circuitry is regulated and to investigate the translational potential of targeting ILK to suppress oncogenic KRAS signaling in pancreatic cancer. Interplay between KRAS and ILK and the roles of E2F1, c-Myc and heterogeneous nuclear ribonucleoprotein as intermediary effectors in this feedback loop was interrogated by genetic manipulations through small interfering RNA/short hairpin RNA knockdown and ectopic expression, western blotting, PCR, promoter-luciferase reporter assays, chromatin immunoprecipitation and pull-down analyses. In vivo efficacy of ILK inhibition was evaluated in two murine xenograft models. Our data show that KRAS regulated the expression of ILK through E2F1-mediated transcriptional activation, which, in turn, controlled KRAS gene expression via hnRNPA1-mediated destabilization of the G-quadruplex on the KRAS promoter. Moreover, ILK inhibition blocked KRAS-driven epithelial-mesenchymal transition and growth factor-stimulated KRAS expression. The knockdown or pharmacological inhibition of ILK suppressed pancreatic tumor growth, in part, by suppressing KRAS signaling. These studies suggest that this KRAS-E2F1-ILK-hnRNPA1 regulatory loop enables pancreatic cancer cells to promote oncogenic KRAS signaling and to interact with the tumor microenvironment to promote aggressive phenotypes. This regulatory loop provides a mechanistic rationale for targeting ILK to suppress oncogenic KRAS signaling, which might foster new therapeutic strategies for pancreatic cancer.

The cellular protein hnRNP A2/B1 enhances HIV-1 transcription by unfolding LTR promoter G-quadruplexes

“…The LTR G4 system displays regulating features similar to those described for the c-myc4344, HRAS26 and KRAS oncogene-promoters30454647. As in these eukaryotic G4-modulated promoters, the HIV-1 LTR promoter is processed by G4 stabilizing (nucleolin)22 and destabilizing proteins (hnRNP A2/B1).…”

Insight into the Complexity of the i-Motif and G-Quadruplex DNA Structures Formed in the KRAS Promoter and Subsequent Drug-Induced Gene Repression

“…33 In accord with our proposed model shown in Figure 9, the action of hnRNP A1 in dissolving the Near-region G-quadruplex structure causes transcription activation by allowing access to the Mid-region and the associated activating i-motif–hnRNP K complex. 72 During active transcription, the Mid-region element is engaged and acts as an “active switch” (Figure 9B). The more stable G-quadruplex predominates until hnRNP K is present and can actively compete with the protein that stabilizes the G-quadruplex (ON/OFF).…”

Substratum stiffness tunes proliferation downstream of Wnt3a in part by regulating integrin-linked kinase and frizzled-1

“…Fzd1 is the latest example of a protein whose expression is modulated by ILK. In pancreatic cancer cells, ILK enhances the expression of the KRAS oncogene by altering the levels of hnRNP1, which binds to the KRAS promoter (Chu et al, 2016). ILK also stabilizes Mucin-1 protein by decreasing its phosphorylation via protein kinase-C, thus altering Mucin-1 levels post-translationally (Huang et al, 2017).…”