Abstract 1884: A PKCα-RAF-ERK signaling axis for downregulation of Id1 and Id3 in colorectal cancer cells

Members of the Id family (Id1-Id4) are emerging as attractive therapeutic targets in multiple tumor types. Id proteins are dominant negative antagonists of basic helix-loop-helix transcription factors, with recognized functions in development, stem cell maintenance/self-renewal, and cell fate determination. Recent studies have identified Id proteins as master regulators of cancer-initiating cells and tumor aggressiveness, with critical roles in regulation of central hallmarks of cancer, including cell proliferation, survival, senescence, angiogenesis, migration, metastasis, and chemoresistance. Deregulated Id1 and/or Id3 expression has been observed in more than twenty human cancers, including colorectal cancer (CRC). Based on their important roles in tumors, Id proteins are being actively explored as therapeutic targets, with promising results in mouse models and human tumor cell lines. Id1 and Id3 appear to have redundant functions in CRC; thus Id-based therapy would ideally involve a strategy that targets Id1 and Id3 simultaneously. We have identified a novel pathway of Id1 and Id3 repression involving the signal transduction molecule PKCα. Activation of PKCα in non-transformed intestinal cells (IEC-18 cells) and human CRC cells that retain PKCα potently suppresses Id1/Id3 mRNA and protein. Restoration of the enzyme in PKCα-deficient CRC cells also results in Id1/Id3 downregulation. Suppression occurs at the transcriptional level and is mediated by the proximal 932 bp of the promoter for Id1. Notably, the effects of PKCα were observed in CRC cell lines with diverse genetic backgrounds, differing in the status of APC, β-catenin, K-RAS, the PI3K/AKT pathway, and/or TP53. PKCα activates a growth inhibitory MEK/ERK signaling pathway in intestinal cells that is required for Id1/Id3 downregulation. Our demonstration that PKCα can activate ERK and suppress Id1/Id3 in KRAS-mutant CRC cells suggested that the effects of PKCα occur downstream of RAS activation. Notably, PKCα was unable to downregulate Id1/Id3 in CRC cells harboring V600E mutations in BRAF, indicating that RAF activation is a key mediator of PKCα-induced Id downregulation. This conclusion was further supported by the ability of the RAF inhibitor, sorafenib, to block the effects of PKCα on Id1/Id3 in BRAF-wild type cells. KSR1 is an important regulator of RAF function; however, PKCα retained its ability to downregulate Id1 in KSR1 knockdown CRC cells, excluding a role for altered KSR1-RAF interactions in the effects of PKCα. Collectively, these data indicate that PKCα regulates a novel signaling pathway for downregulation of Id1/Id3 that intersects the MEK/ERK pathway at or upstream of RAF, but downstream of RAS. Manipulation of this pathway for coincident downregulation of Id1 and Id3 offers a promising therapeutic strategy for treatment of CRC and other cancer types. Supported by NIH grants CA036727, CA016056, CA191894 and DK60632. Citation Format: Michelle A. Lum, Robert E. Lewis, Adrian R. Black, Jennifer D. Black. A PKCα-RAF-ERK signaling axis for downregulation of Id1 and Id3 in colorectal cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1884.