The supplementary information includes 9 supplementary Figures and 8 supplementary tables: Figure S1. Analysis of MORC2 mutational frequency in human cancer from the cBioPortal for Cancer Genomics database. Figure S2. Analysis of MORC2 mutational frequency in various types of human cancer in the COSMIC database. Figure S3. The mutational patterns of MORC2 in breast cancer. Figure S4. Clinical information of the TNBC patient harboring the M276I mutation. Figure S5. Detection of the MORC2 M276I mutation in TNBC cell lines by targeted exon sequencing. Figure S6. Schematic representation of the structural domains of human MORC2 protein. Figure S7. The M276I mutation does not affect cell proliferation and cell-cycle progression. Figure S8. The M276I mutation enhances migratory and invasive potential of TNBC cells. Figure S9. Both WT and M276I mutant MORC2 do not interact with hnRNPA1 and hnRNPL. Table S1. Information for the expression vectors used in this study. Table S2. Primers used for molecular cloning of expression vectors. Table S3. siRNA target sequences for hnRNPL. Table S4. Primary antibodies and antibody-conjugated magnetic beads used in this study. Table S5. Primers for qPCR analysis. Table S6. Primers used for targeted exon sequencing of MORC2-M276 mutation in TNBC cell lines. Table S7. The list of 48 proteins that interact with both WT and mutant M276I MORC2. Table S8. The GO molecular functions analyses of 48 proteins that interact with both WT and mutant M276I MORC2 using the PANTHER program.
Funding
National Natural Science Foundation of China
Shanghai Institutions of Higher Learning
Shanghai Municipal Science and Technology Commission
Fudan University
ARTICLE ABSTRACT
Triple-negative breast cancer (TNBC) is the most lethal subtype of breast cancer, with a high propensity for distant metastasis and limited treatment options, yet its molecular underpinnings remain largely unknown. Microrchidia family CW-type zinc finger 2 (MORC2) is a newly identified chromatin remodeling protein whose mutations have been causally implicated in several neurologic disorders. Here, we report that a cancer-associated substitution of methionine to isoleucine at residue 276 (M276I) of MORC2 confers gain-of-function properties in the metastatic progression of TNBC. Expression of mutant MORC2 in TNBC cells increased cell migration, invasion, and lung metastasis without affecting cell proliferation and primary tumor growth compared with its wild-type counterpart. The M276I mutation enhanced binding of MORC2 to heterogeneous nuclear ribonucleoprotein M (hnRNPM), a component of the spliceosome machinery. This interaction promoted an hnRNPM-mediated splicing switch of CD44 from the epithelial isoform (CD44v) to the mesenchymal isoform (CD44s), ultimately driving epithelial–mesenchymal transition (EMT). Knockdown of hnRNPM reduced the binding of mutant MORC2 to CD44 pre-mRNA and reversed the mutant MORC2-induced CD44 splicing switch and EMT, consequently impairing the migratory, invasive, and lung metastatic potential of mutant MORC2-expressing cells. Collectively, these findings provide the first functional evidence for the M276I mutation in promoting TNBC progression. They also establish the first mechanistic connection between MORC2 and RNA splicing and highlight the importance of deciphering unique patient-derived mutations for optimizing clinical outcomes of this highly heterogeneous disease.Significance:A gain-of-function effect of a single mutation on MORC2 promotes metastasis of triple-negative breast cancer by regulating CD44 splicing. Cancer Res; 78(20); 5780–92. ©2018 AACR.
