Combined Menin and EGFR Inhibitors Synergize to Suppress Colorectal Cancer via EGFR-Independent and Calcium-Mediated Repression of SKP2 Transcription

Abstract

Menin is a nuclear epigenetic regulator that can both promote and suppress tumor growth in a highly tissue-specific manner. The role of menin in colorectal cancer, however, remains unclear. Here, we demonstrate that menin was overexpressed in colorectal cancer and that inhibition of menin synergized with small-molecule inhibitors of EGFR (iEGFR) to suppress colorectal cancer cells and tumor xenografts in vivo in an EGFR-independent manner. Mechanistically, menin bound the promoter of SKP2, a pro-oncogenic gene crucial for colorectal cancer growth, and promoted its expression. Moreover, the iEGFR gefitinib activated endoplasmic reticulum calcium channel inositol trisphosphate receptor 3 (IP3R3)-mediated release of calcium, which directly bound menin. Combined inhibition of menin and iEGFR-induced calcium release synergistically suppressed menin-mediated expression of SKP2 and growth of colorectal cancer. Together, these findings uncover a molecular convergence of menin and the iEGFR-induced, IP3R3-mediated calcium release on SKP2 transcription and reveal opportunities to enhance iEGFR efficacy to improve treatments for colorectal cancer. SIGNIFICANCE: Menin acts as a calcium-responsive regulator of SKP2 expression, and small molecule EGFR inhibitors, which induce calcium release, synergize with Menin inhibition to reduce SKP2 expression and suppress colorectal cancer.

Figure 1:. Menin is upregulated in colon cancer and provides resistance to gefitinib.

A) TCGA database analysis comparing menin expression in non-cancerous colonic epithelial samples compared to colon cancer samples. Log2-transformed, normalized counts are shown for menin, dividing the plot to separate normal and tumor samples. *** p = 3.31E-14. B) Menin IHC in colon cancer and adjacent normal colonic mucosa, with two representative examples shown, 100X magnification. C-D) HT-29 cells transduced with either scrambled or menin shRNAs (C), then cell growth was assessed after 96 hours by the MTS assay (D). E-F) HT-29 cells transduced with either vector or menin (E), then cell growth was assessed after 96 hours by the MTS assay (F). G) HT-29 cells transduced with either scrambled or menin shRNAs, then treated with varying concentrations of gefitinib. Cell growth was assessed after 96 hours by the MTS assay. H) HT-29 cells transduced with either lentiCRISPRv2 vector or menin sgRNA, then treated with varying concentrations of gefitinib. Cell growth was assessed after 96 hours by the MTS assay. I) HT-29 cells transduced with scrambled or menin shRNAs, then protein levels were assessed by western blot after 96 hours. 10 μM gefitinib. J) HT-29 cells transduced with either lentiCRISPRv2 vector or menin sgRNA, then protein levels were assessed by western blot after 96 hours. 10 μM gefitinib. * p < 0.05.

Figure 2:. Menin inhibition sensitizes colon cancer cells to iEGFRs in an EGFR-independent manner.

A) Treatment of HT-29 cells with various concentrations of gefitinib, with and without 1 μM MI-2–2, with cell growth assessed after 96 hours by the MTS assay. B) HCT-15 cells treated with varying concentrations of gefitinib with and without 1 μM MI-2–2. MTS assay performed after 96 hours. C) HT-29 cells treated with varying concentrations of lapatinib with and without 1 μM MI-2–2. MTS assay performed after 96 hours. D) HT-29 cells treated with varying concentrations of gefitinib with and without 1 μM MI-503. MTS assay performed after 96 hours. E) HT-29 cells treated for 96 hours followed by analysis of protein levels by western blotting, 10 μM gefitinib, 1 μM MI-2–2. F) HT-29 cells treated for 96 hours and then analyzed by western blotting. 10 μM gefitinib, 1 μg/mL C225. G) HT-29 cells treated with varying concentrations of C225 with or without 1 μM MI-2–2 for 96 hours, and then cell growth was assessed by the MTS assay. H) HT-29 cells transduced with scrambled or EGFR shRNAs, with EGFR protein expression analyzed by western blot. I) Scrambled and EGFR shRNA transduced HT-29 cells were treated with vehicle or 1 μM MI-2–2 for 96 hours, and then cell growth was assessed by the MTS assay. J) EGFR expression was assessed by western blotting in HT-29 and SW620 cells. K) SW620 cells treated with varying concentrations of gefitinib with and without 1 μM MI-2–2. MTS assay was performed after 96 hours to assess cell growth. L) SW620 cells treated with varying concentrations of gefitinib with and without 1 μM MI-2–2 for 96 hours, then protein levels were analyzed by western blotting. * p < 0.05.

Figure 3:. Increased cytosolic calcium is important for gefitinib mediated suppression of CRC cells.

A-B) HT-29 cells treated for 48 hours and then CHOP mRNA (A) and spliced XBP1 mRNA (B) were assessed by RT-PCR and plotted relative to actin. 10 μM gefitinib. C-E) HT-29 cells treated with varying concentrations of thapsigargin [TG] (C), tunicamycin (D), and brefeldin A (E), with and without 1 μM MI-2–2. Cell growth was assessed after 96 hours by the MTS assay. F-K) HT-29-GCaMP6f cells treated for 24 hours. Images were obtained at 200X. 1 μM MI-2–2, 10 μM gefitinib, 2 nM TG. L-M) HT-29-GCaMP6f cells treated with either gefitinib (L) or 2nM TG for 72 hours and then cytosolic calcium levels were analyzed by flow cytometry, with the median FLH-1 values reported (M). N) HT-29 cells treated for 96 hours with cell growth assessed by the MTS assay. 10 μM ionomycin, 1 μM MI-2–2. O) HT-29 cells treated for 96 hours, with protein analyzed by western blot. 5 μM ionomycin, 1 μM MI-2–2. P-Q) HT-29 cells treated for 96 hours with cell growth assessed by the MTS assay (P), and treated for 48 hours with protein analysis by western blot (Q). 10 μM gefitinib, 1 μM MI-2–2, 5 μM BAPTA-AM. * p < 0.05.

Figure 4:. Gefitinib induces CRC repression through activation of IP3R3.

A-B) HT-29 cells transduced with either scrambled or IP3R3 shRNAs, then treated with vehicle or 10 μM gefitinib/1 μM MI-2–2. Cell growth was assessed after 96 hours by the MTS assay (A) and protein levels were assessed after 48 hours by western blot (B). C) Quantitation and normalization of cleaved and uncleaved PARP levels on western blot from Figure 4B. D) HT-29 cells treated with vehicle or 10 μM gefitinib/1 μM MI-2–2 with different concentrations of caffeine for 96 hours with cell growth assessed by the MTS assay. E-F) HT-29-GCaMP6f cells treated for 24 hours with cytosolic calcium levels analyzed by flow cytometry (E), with the median FLH-1 values reported (F). 10 μM gefitinib, 2 mM caffeine. *p < 0.05.

Figure 5:. Gefitinib and menin inhibition synergistically decrease SKP2 expression in a calcium-dependent manner.

A) HT-29 cells treated for 48 hours followed by analysis of protein levels by western blotting. 10 μM gefitinib, 1 μM MI-2–2. B) After 48 hours of treatment in HT-29 cells SKP2 mRNA was assessed by RT-PCR and plotted relative to actin. 1 μM MI-2–2,10 μM gefitinib, 5 nM TG. C-D) A time course was performed in HT-29 cells treated with either vehicle or 10 μM gefitinib/1 μM MI-2–2, with analysis of protein levels by western blotting (C) and SKP2 mRNA assessment by RT-PCR, plotted relative to actin, and normalized to DMSO for each time point (D). E) HT-29 cells treated for 48 hours with protein analysis by western blot. 10 μM gefitinib, 1 μM MI-2–2, 5 μM BAPTA-AM. F) Quantitation and normalization of cleaved and uncleaved PARP levels on western blot from Figure 5E. G-H) HT-29 cells transduced with either scrambled or SKP2 shRNAs, then treated with 10 μM gefitinib. After 96 hours, cell growth was assessed by the MTS assay (G) and after 48 hours protein levels were assessed by western blot (H). * p < 0.05.

Figure 6:. Menin interacts directly with calcium and regulates the levels of active histone marks and SKP2 transcription.

A-B) HT-29 cells were treated for 30 hours, then ChIP assay was performed to look for menin (A), RNA polymerase II, H3K4me3, and total H3 (B) at amplicon 1 of the SKP2 promoter. 10 μM gefitinib, 1 μM MI-2–2. *p < 0.05 compared to Control. C-D) HT-29 cells were treated for 30 hours, then ChIP assay was performed to look for menin (C) and RNA polymerase II (D) at amplicon 1 of the SKP2 promoter. 10 μM gefitinib, 1 μM MI-2–2, 2 μM BAPTA-AM. *p < 0.05 compared to Control and BAPTA-AM. E) Chelex resin eluent after incubation with HT-29 cell lysate. Uncharged resin or resin prepared with NaOH or CaCl₂. Equal volumes of eluent were utilized to analyze protein by western blot. Ponceau S demonstrated no significant non-specific protein binding to resin. F) Chelex resin eluent after incubation with purified recombinant menin protein. Uncharged resin or resin prepared with NaOH or CaCl₂. Equal volumes of eluent were utilized to analyze protein by western blot. G) After incubation with recombinant menin protein, uncharged and CaCl₂ Chelex resin was washed with different calcium concentrations with resulting protein eluent examined by western blot. H-I) Differential scanning fluorimetry was performed on purified recombinant menin protein with different concentrations of CaCl₂, with example fitted curves (H) and average melting temperatures (I).

Figure 7:. Combined menin inhibition and gefitinib reduces colon cancer xenograft growth and suppresses SKP2.

Nude mice were transplanted with HT-29 cells, and once tumor size reached approximately 100mm³, treatment was started with either gefitinib (100 mg/kg daily by oral gavage), MI-463 (35 mg/kg daily by IP/SQ injection), the combination of gefitinib plus MI-463, or vehicle control. A) Tumor size was measured with a Vernier caliber every 3 days. Error bars indicate +/− SEM. B) Mice were weighed every 3 days. Error bars indicate +/− SD. C-D) SKP2 immunofluorescence was performed in the xenografts with quantitation of SKP2 immunofluorescence (C) and representative images from each of the 4 treatment groups (D). E) A representative model of menin and iEGFR involvement in SKP2 regulation. * p < 0.05.