title: TBX2 represses CST6 resulting in uncontrolled legumain activity to sustain breast cancer proliferation: a novel cancer-selective target pathway with therapeutic opportunities.
abstract: TBX2 is an oncogenic transcription factor known to drive breast cancer proliferation. We have identified the cysteine protease inhibitor Cystatin 6 (CST6) as a consistently repressed TBX2 target gene, co-repressed through a mechanism involving Early Growth Response 1 (EGR1). Exogenous expression of CST6 in TBX2-expressing breast cancer cells resulted in significant apoptosis whilst non-tumorigenic breast cells remained unaffected. CST6 is an important tumor suppressor in multiple tissues, acting as a dual protease inhibitor of both papain-like cathepsins and asparaginyl endopeptidases (AEPs) such as Legumain (LGMN). Mutation of the CST6 LGMN-inhibitory domain completely abrogated its ability to induce apoptosis in TBX2-expressing breast cancer cells, whilst mutation of the cathepsin-inhibitory domain or treatment with a pan-cathepsin inhibitor had no effect, suggesting that LGMN is the key oncogenic driver enzyme. LGMN activity assays confirmed the observed growth inhibitory effects were consistent with CST6 inhibition of LGMN. Knockdown of LGMN and the only other known AEP enzyme (GPI8) by siRNA confirmed that LGMN was the enzyme responsible for maintaining breast cancer proliferation. CST6 did not require secretion or glycosylation to elicit its cell killing effects, suggesting an intracellular mode of action. Finally, we show that TBX2 and CST6 displayed reciprocal expression in a cohort of primary breast cancers with increased TBX2 expression associating with increased metastases. We have also noted that tumors with altered TBX2/CST6 expression show poor overall survival. This novel TBX2-CST6-LGMN signaling pathway, therefore, represents an exciting opportunity for the development of novel therapies to target TBX2 driven breast cancers.
authors.affiliation: Centre for Cancer Research and Cell Biology, Queen's University Belfast, Belfast, UK.
authors.forename: Zenobia C
authors.initials: ZC
authors.lastname: D'Costa
authors.forename: Catherine
authors.initials: C
authors.lastname: Higgins
authors.forename: Chee Wee
authors.initials: CW
authors.lastname: Ong
authors.forename: Gareth W
authors.initials: GW
authors.lastname: Irwin
authors.forename: David
authors.initials: D
authors.lastname: Boyle
authors.forename: Darragh G
authors.initials: DG
authors.lastname: McArt
authors.forename: Karen
authors.initials: K
authors.lastname: McCloskey
authors.forename: Niamh E
authors.initials: NE
authors.lastname: Buckley
authors.forename: Nyree T
authors.initials: NT
authors.lastname: Crawford
authors.forename: Lalitha
authors.initials: L
authors.lastname: Thiagarajan
authors.forename: James T
authors.initials: JT
authors.lastname: Murray
authors.forename: Richard D
authors.initials: RD
authors.lastname: Kennedy
authors.forename: Karl A
authors.initials: KA
authors.lastname: Mulligan
authors.forename: D Paul
authors.initials: DP
authors.lastname: Harkin
authors.forename: David J J
authors.initials: DJ
authors.lastname: Waugh
authors.forename: Chris J
authors.initials: CJ
authors.lastname: Scott
authors.forename: Manuel
authors.initials: M
authors.lastname: Salto-Tellez
authors.forename: Richard
authors.initials: R
authors.lastname: Williams
authors.forename: Paul B
authors.initials: PB
authors.lastname: Mullan
mesh_terms: D017209:Apoptosis; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D049109:Cell Proliferation; D047369:Chromatin Immunoprecipitation; D055332:Cystatin M / Q000235:genetics* / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D005260:Female; D005455:Fluorescent Antibody Technique; D015972:Gene Expression Regulation, Neoplastic; D006031:Glycosylation; D006801:Humans; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D020825:T-Box Domain Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D000099240:Asparaginyl Endopeptidase
chemical_list: D055332:Cystatin M; D003546:Cysteine Endopeptidases; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D020825:T-Box Domain Proteins; D000099240:Asparaginyl Endopeptidase; C525104:CST6 protein, human; C097348:T-Box Domain Protein 2
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