Repression of the miR-143/145 cluster by oncogenic Ras initiates a tumor-promoting feed-forward pathway

Abstract

Although activating mutations in RAS oncogenes are known to result in aberrant signaling through multiple pathways, the role of microRNAs (miRNAs) in the Ras oncogenic program remains poorly characterized. Here we demonstrate that Ras activation leads to repression of the miR-143/145 cluster in cells of human, murine, and zebrafish origin. Loss of miR-143/145 expression is observed frequently in KRAS mutant pancreatic cancers, and restoration of these miRNAs abrogates tumorigenesis. miR-143/145 down-regulation requires the Ras-responsive element-binding protein (RREB1), which represses the miR-143/145 promoter. Additionally, KRAS and RREB1 are targets of miR-143/miR-145, revealing a feed-forward mechanism that potentiates Ras signaling.

Figure 1.

The miR-143/145 cluster is repressed by oncogenic Kras in multiple model systems. (A,B) Northern blot analysis of miRNA expression in HPNE cells (A) or PDAC cell lines (B). (C) Quantitative PCR (qPCR) analysis of relative miR-143/miR-145 expression in liver and pancreas of postnatal day 0 (P0) mice (n = 7 mice per genotype). Box plots show median (horizontal line), 25th and 75th percentiles (box), and full range of data (whiskers). (**) P < 0.01 (two-tailed t-test). (D) miR-143/miR-145 expression in normal zebrafish pancreas and pancreatic tumors induced by transgenic Kras^G12V expression (Park et al. 2008). (E) miR-143/145 expression in NIH3T3 and v-Kras-transformed NIH3T3 cells.

Figure 2.

Expression of miR-143/145 inhibits transformation and tumorigenesis in pancreatic cancer cells. (A) Anchorage-independent growth of retrovirally infected cell lines (average of three independent experiments shown). Error bars for this and subsequent panels represent standard deviations. (B) Tumorigenesis assays with retrovirally infected MiaPaCa2 cells. Images depict a representative animal injected in each flank with cells infected with empty virus or miR-143/145 virus. The graph shows the average tumor volume (n = 5 mice per cell line). (C) Anchorage-independent growth of retrovirally infected cell lines expressing either miR-143 or miR-145. (D) Tumorigenesis assays with MiaPaCa2 cells infected with empty virus or virus expressing miR-143 or miR-145 (n = 5 mice per cell line).

Figure 3.

Kras-mediated repression of miR-143/145 occurs at the level of transcription and requires RREB1. (A) Structures of the experimentally determined human and mouse miR-143/145 primary transcripts and the annotated human RefSeq transcript (LOC728264). The plot depicted below the transcripts shows evolutionary conservation (UCSC Genome Browser 28 species conservation track, NCBI36/hg18 assembly). (B) qPCR analysis of pri-miR-143/145 expression in HPNE and NIH3T3-derived cell lines. Error bars for this and subsequent panels represent standard deviations from three independent measurements. (C) Pri-miR-143/145 abundance in cells transfected with negative control or KRAS targeting siRNAs. (D) RREB1 expression in HPNE and NIH3T3-derived cell lines. (E) Mature miR-143 and miR-145 expression in HPNE-Kras^G12D cells following knockdown of RREB1.

Figure 4.

RREB1 negatively regulates the miR-143/145 promoter. (A) Activity of the miR-143/145 promoter reporter construct in NIH3T3 and HeLa cells. For this and subsequent panels, values represent firefly luciferase activity produced from each plasmid normalized to renilla luciferase activity produced from a cotransfected control vector. Error bars represent standard deviations from three independent transfections, each measured in triplicate. (B,C) Activity of the wild-type and mutant miR-143/145 promoter reporter constructs in NIH3T3 and NIH3T3-Kras cells (B) or PDAC cell lines (C). (D) RREB1 knockdown reverses repression of the miR-145/145 promoter reporter construct in NIH3T3-Kras cells. (E) qPCR analysis of Flag-RREB1 chromatin immunoprecipitates in NIH3T3 cells. Signal obtained from the miR-143/145 promoter amplicon containing the RREB1-binding site (RRE), an amplicon 300 base pairs (bp) downstream (Dn), and a negative control amplicon 5 kb upstream (5kb) are shown. Error bars represent standard deviations derived from three independent measurements.

Figure 5.

miR-143 and miR-145 target KRAS and RREB1, establishing a feed-forward circuit that potentiates Ras signaling. (A–D) qPCR analysis of KRAS (A,B) or RREB1 (C,D) transcript abundance in cells transfected with negative control, miR-143, or miR-145 mimics (A,C) or inhibitors (B,D). Error bars represent standard deviations from three independent measurements. (E) Western blot analysis of phospho-ERK1/2 (pERK1/2), total ERK1/2 (tERK1/2), phospho-AKT^S473 (pAKT), and total AKT (tAKT) in retrovirally infected cell lines. (F) Model of feed-forward regulatory circuit in which Kras signaling, via RREB1, represses miR-143/145 transcription, thereby relieving KRAS and RREB1 negative regulation and potentiating signaling through Ras effector pathways.