MICAL2 Promotes Pancreatic Cancer Growth and Metastasis

Abstract

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest solid cancers; thus, identifying more effective therapies is a major unmet need. In this study, we characterized the super-enhancer (SE) landscape of human PDAC to identify drivers of the disease that might be targetable. This analysis revealed MICAL2 as an SE-associated gene in human PDAC, which encodes the flavin monooxygenase enzyme that induces actin depolymerization and indirectly promotes serum response factor transcription by modulating the availability of serum response factor coactivators such as myocardin-related transcription factors (MRTF-A and MRTF-B). MICAL2 was overexpressed in PDAC, and high-MICAL2 expression correlated with poor patient prognosis. Transcriptional analysis revealed that MICAL2 upregulates KRAS and epithelial-mesenchymal transition signaling pathways, contributing to tumor growth and metastasis. In loss- and gain-of-function experiments in human and mouse PDAC cells, MICAL2 promoted both ERK1/2 and AKT activation. Consistent with its role in actin depolymerization and KRAS signaling, loss of MICAL2 also inhibited macropinocytosis. MICAL2, MRTF-A, and MRTF-B influenced PDAC cell proliferation and migration and promoted cell-cycle progression in vitro. Importantly, MICAL2 supported in vivo tumor growth and metastasis. Interestingly, MRTF-B, but not MRTF-A, phenocopied MICAL2-driven phenotypes in vivo. This study highlights the multiple ways in which MICAL2 affects PDAC biology and provides a foundation for future investigations into the potential of targeting MICAL2 for therapeutic intervention. Significance: Characterization of the epigenomic landscape of pancreatic cancer to identify early drivers of tumorigenesis uncovered MICAL2 as a super-enhancer-associated gene critical for tumor progression that represents a potential pharmacologic target.

Figure 1.

H3K27ac ChIP-seq in human PDAC identifies MICAL2 as an SE-associated gene. A, Differentially expressed SE-associated genes in tumor compared with normal tissue. The top 10 upregulated and downregulated genes as well as the MICAL2 gene are annotated. B, qPCR of MICAL2 using RNA extracted from the same patient samples used for ChIP-seq. Two-tailed t test P value is shown. C, H3K27ac ChIP-seq occupancy upstream and within the MICAL2 loci in normal and tumor samples. Norm, normal.

Figure 2.

MICAL2 is overexpressed in human and mouse PDAC. A, IHC for MICAL2 in normal pancreas and PDAC human tissues. B, IHC for MICAL2 in normal pancreas and KPC-derived PDAC mouse tissues. C and D, Survival analysis of patients with PDAC segregated by MICAL2 expression (high vs. low) in two datasets: PanCuRx (C) and COMPASS (D). panc, pancreas.

Figure 3.

MICAL2 promotes a KRAS and EMT phenotype in PDAC cells. A, GSEA enrichment plots in AsPC1 cells with MICAL2-KD compared with scramble control indicating normalized enrichment score (NES), adjusted P value, and FDR. B, Enrichment analysis of the Klomp and colleagues (35) KRAS upregulated (KRAS-UP) and KRAS downregulated (KRAS-DOWN) genes in ASPC1 MICAL2-KD compared with scramble control. P.adj <0.01. C, OncoGPS plot of Cancer Cell Line Encyclopedia cell lines showing MICAL2 expression. Red dots, cell lines with high MICAL2 expression; blue dots, low expression. D, Hematoxylin and eosin stain of mouse PDAC organoids derived from control KPC46 cells (SCR) and MICAL2-KD. E, Immunoblots of organoids shown in D. Quantification is shown with two-tailed t test P value. E-cad, E-cadherin; Vinc, vinculin. F and G, Immunoblot analysis of AsPC1 (F) and KPC46 (G) cells treated with SCR, MICAL2, MRTF-A, and MRTF-B siRNAs at 72 hours. Quantification is shown with ANOVA P values. H and I, Immunoblot analysis of BxPc3 cells expressing empty vector (EV) or MICAL2-OE vector at 72 hours for AKT and ERK signaling (H) and KRAS–GTP loading (I). Total KRAS and GAPDH were loaded at 2% input. Quantification is shown with two-tailed t test P value. J, Representative immunofluorescent images of AsPC1 cells transfected with siRNA control (SCR) or MICAL2-KD. DAPI, blue, cell nuclei. FITC-conjugated dextran (green) was used to label macropinosomes. Quantitation of the relative macropinosome index with two-tailed t test P value.

Figure 4.

MICAL2 drives cancer cell migration and proliferation in vitro. A and B, Quantification of wound healing assay of AsPC1 (A) and KPC46 (B) transfected with SCR, MICAL2, MRTF-A, and MRTF-B siRNAs at the time points indicated. C, Quantification of wound healing assay of BxPc3 cells expressing empty vector (EV) or MICAL2-OE vector at the time points indicated. D and E, Proliferation assay of AsPC1 (D) and KPC46 (E) transfected with SCR, MICAL2, MRTF-A, and MRTF-B siRNAs at the time points indicated. F, Proliferation assay of BxPc3 cells expressing empty vector or MICAL2-OE vector at the time points indicated. G–I, Cell-cycle analysis of AsPC1 (G) and KPC46 (H) transfected with SCR, MICAL2, MRTF-A, and MRTF-B siRNAs at 72 hours, and BxPc3 (I) cells expressing empty vector or MICAL2-OE vector. ANOVA was used for statistical analysis.

Figure 5.

MICAL2 and MRTF-B promote tumor growth. A, Representative images and weight quantification with ANOVA testing of subcutaneous AsPC1 tumors grown in immunocompromised mice. AsPC1 cells express shRNA vectors to silence MICAL2, MRTF-A, and MRTF-B. B, Representative images and weight quantification with ANOVA testing of subcutaneous KPC46 tumors grown in syngeneic mice. KPC46 cells express shRNA vectors to silence MICAL2, MRTF-A, and MRTF-B. C, Representative images and weight quantification with ANOVA testing of subcutaneous BxPc3 tumors grown in immunocompromised mice. BxPc3 parental (PT) cells express empty vectors (EV) or MICAL2-OE vectors. D, Tumor growth over time of subcutaneous KPC46 tumors grown in syngeneic mice. KPC46 cells express dox-inducible shRNA vectors to silence MICAL2. E, Quantification and t test of tumor sizes from D. Tumors grown with and without dox chow (regular diet control) are shown. F, Representative images and weight quantification with ANOVA testing of orthotopic AsPC1 tumors grown in immunocompromised mice. AsPC1 cells express shRNA vectors to silence MICAL2, MRTF-A, and MRTF-B. G, Representative images and weight quantification with ANOVA testing of orthotopic KPC46 tumors grown in syngeneic mice. KPC46 cells express shRNA vectors to silence MICAL2, MRTF-A, and MRTF-B. RegDiet, regular diet.

Figure 6.

MICAL2, MRTF-A, and MRTF-B promote metastatic spread in vivo. A, Representative images of liver metastatic burden after splenic injection of KPC46 cell into syngeneic mice. KPC46 cells express shRNA vectors to silence MICAL2, MRTF-A, and MRTF-B. B, Histologic quantification of liver metastasis area normalized to total liver area. ANOVA was used for statistical analysis. C, Representative images of liver metastatic burden after splenic injection of KPC46 cell into syngeneic mice. KPC46 cells express dox-inducible shRNA vectors to silence MICAL2. D, Histologic quantification of liver metastasis area normalized to total liver area. One-tailed t test was used for statistical analysis.