Menin Maintains Cholesterol Content in Colorectal Cancer via Repression of LXR-Mediated Transcription

Abstract

Background and aims: Menin is a nuclear scaffold protein that regulates gene transcription in an oftentimes tissue-specific manner. Our previous work showed that menin is over-expressed in colorectal cancer (CRC); however, the full spectrum of menin function in colonic neoplasia remains unclear. Herein, we aimed to uncover novel menin-regulated pathways important for colorectal carcinogenesis.

Methods: RNA-Seq analysis identified that menin regulates LXR-target gene expressions in CRC cell lines. Isolated colonic epithelium from Men1^f/f;Vil1-Cre and Men1^f/f mice was used to validate the results in vivo. Cholesterol content was quantified via an enzymatic assay.

Results: RNA-Seq analysis in the HT-29 CRC cell line identified that menin inhibition upregulated LXR-target genes, specifically ABCG1 and ABCA1, with protein products that promote cellular cholesterol efflux. Similar results were noted across other CRC cell lines and with different methods of menin inhibition. Consistent with ABCG1 and ABCA1 upregulation, and similarly to LXR agonists, menin inhibition reduced the total cellular cholesterol in both HT-29 and HCT-15 cells. To confirm the effects of menin inhibition in vivo, we assessed Men1^f/f;Vil1-Cre mice lacking menin expression in the colonic epithelium. Men1^f/f;Vil1-Cre mice were found to have no distinct baseline phenotype compared to control Men1^f/f mice. However, similarly to CRC cell lines, Men1^f/f;Vil1-Cre mice showed an upregulation of Abcg1 and a reduction in total cellular cholesterol. Promoting cholesterol efflux, either via menin inhibition or LXR activation, was found to synergistically suppress CRC cell growth under cholesterol-depleted conditions and when administered concomitantly with small molecule EGFR inhibitors.

Conclusions: Menin represses the transcription of LXR-target genes, including ABCA1 and ABCG1 in the colonic epithelium and CRC. Menin inhibition conversely upregulates LXR-target genes and reduces total cellular cholesterol, demonstrating that menin inhibition may be an important mechanism for targeting cholesterol-dependent pathways in colorectal carcinogenesis.

Keywords: ABCA1; ABCG1; LXR; cholesterol; colorectal cancer; menin.

Figure 1

Menin inhibition in CRC cells leads to upregulation of LXR target genes. (A) RNA-Seq performed on HT-29 cells treated with either vehicle or 1 µM MI-2-2 for 30 h. Genes with a net log2-fold change ≥0.5 and p-value < 0.05 are displayed in a heatmap. Red coloring represents upregulation, and blue coloring represents downregulation. (B) Top regulated pathways after Ingenuity Pathway Analysis of RNA-Seq data from HT-29 cells. (C) Genes with the lowest p-values from HT-29 RNA-Seq data. Red coloring represent upregulation after MI-2-2 treatment, and blue coloring represents downregulation. (D) Relative expression differences between benign colonic epithelium and CRC from the TCGA. * p < 0.05.

Figure 2

Menin inhibition increases expression of LXR target genes in CRC cells. (A–F) HT-29 cells (A,B), HCT-15 cells (C,D), or HCT-116 cells (E,F) were treated with vehicle (DMSO) or 1 μM MI-2-2 for 96 h, and expression of ABCA1 and ABCG1 was analyzed using RT-qPCR relative to actin. (G,H) HT-29 cells were treated with empty vector (vehicle) or menin-directed sgRNA for 96 h, and expression of ABCA1 (G) and ABCG1 (H) was analyzed using RT-qPCR relative to actin. * p < 0.05. ** p < 0.01. *** p < 0.001. # p < 0.06.

Figure 3

Menin inhibition increases active gene transcription at LXR target gene promoters. (A) HT-29 cells were treated with either vector or an LXRα shRNA, and protein levels were assessed via western blot. (B,C) HT-29 cells were transduced with either vector or an LXRα shRNA and then treated with vehicle or 1 µM MI-2-2 for 72 h, followed by assessment of ABCA1 (B) and ABCG1 (C) expression relative to actin using RT-qPCR. (D,E) ChIP assay was performed at an LXRE at the promoter of ABCA1 (D) and ABCG1 (E) in HT-29 cells after treatment with vehicle or 1 μM MI-2-2 for 48 h. ** p < 0.01. *** p < 0.001.

Figure 4

Menin inhibition decreases cellular cholesterol. (A,B) HT-29 (A) and HCT-15 (B) cells were treated with 1 µM MI-2-2 and 5 µM GW3965 for 48 h; total cellular cholesterol was quantified and normalized to protein concentration. (C,D) HT-29 (C) and HCT-15 (D) cells were treated with 1 µM MI-2-2 and 5 µM GW3965 in media containing 10% FBS or serum-free media for 48 h; total cellular cholesterol was quantified and normalized to protein concentration. *** p < 0.001.

Figure 5

Targeted deletion of the Men1 gene in mouse colonic epithelium reduces colonic epithelial cholesterol content. (A) Representative picture of a Men1^fl/fl mouse and Men1^fl/fl;Vil1-Cre mouse. (B) Weight in grams of male and female Men1^fl/fl and Men1^fl/fl;Vil1-Cre mice at 8 weeks of age. (C) Representative H&E-stained sections of colonic epithelium from 8-week-old Men1^fl/fl and Men1^fl/fl;Vil1-Cre mice, scale bars = 50 µm. (D) Representative IHC staining for menin in the colonic epithelium of Men1^fl/fl mice and Men1^fl/fl;Vil1-Cre mice, scale bars = 25 µm. (E,F) Menin protein assessment using western blot from isolated colonic epithelium of female (E) and male (F) mice. (G,H) Relative total cellular cholesterol in isolated mouse colonic epithelium either with or without menin expression in female (G) and male (H) mice. (I,J) Abcg1 expression in isolated mouse colonic epithelium with and without menin in female (I) and male (J) mice assessed via RT-qPCR relative to GAPDH. * p < 0.05. ns = not significant (p > 0.05).

Figure 6

Menin inhibition enhances CRC cell death under lipid-poor conditions. (A,B) HT-29 cells were treated with 1 µM MI-2-2 for 96 h in media containing 10% FBS (serum) or serum-free media, with cell growth assessed via MTS assay (A) and protein assessed via western blot (B). (C) HT-29 cells were treated with 1 µM MI-2-2 for 72 h in media containing 10% FBS (serum) or serum-free media with apoptosis quantified with caspases-3/7 activity. (D–F) HT-29 cells were treated with 1 µM MI-2-2 for 96 h in media containing 10% FBS (serum) or 10% lipid-depleted FBS (LD-serum), with cell growth assessed via MTS assay (D), protein assessed via western blot (E), and apoptosis quantified with caspases-3/7 activity (F). ** p < 0.01. *** p < 0.001. **** p < 0.0001.

Figure 7

Similar to menin inhibitors, LXR agonists synergize with iEGFRs to suppress CRC. (A–C) HT-29 cells were treated with 10 µM GW3965 and/or 10 µM gefitinib for 96 h, with assessment of cell growth via MTS assay (A) and apoptosis via both PARP cleavage (B) and via caspases-3/7 activity (C). (D–F) HCT-15 cells were treated with 10 µM GW3965 and/or 7.5 µM gefitinib for 96 h with assessment of cell growth via MTS assay (D) and apoptosis via both PARP cleavage (E) and via caspases-3/7 activity (F). (G–I) HT-29 cells were treated with 10 µM T0901317 and/or 10 µM gefitinib for 96 h, with assessment of cell growth via MTS assay (G) and apoptosis via both PARP cleavage (H) and via caspases-3/7 activity (I). (J) Proposed model for menin repression of LXR-mediated transcription in CRC. * p < 0.05. ** p < 0.01. *** p < 0.001. **** p < 0.0001.