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.

Tumor-associated macrophages in cancers

“…Sites of [25][26][27] Gastric cancer TAM can promote angiogenesis reaction and the infiltration and metastasis of cancer cells. High expression of MIF and VEGF in tumor tissue hint that activation of TAM induces tumor angiogenesis [32][33][34] Breast cancer High expression of legumain by TAM is associated with significantly shorter survival time [44][45][46] Colorectal cancer The synergistic effect of CXCL16 and M1 can effectively inhibit colorectal liver metastasis [48,49] Lung cancer Bigger the ratio of the nest TAM and stromal TAM, more conducive to the survival of patients. M2 macrophages secrete IL-10 that induces CCR2 and CX3CR1 expression which can promote proliferation and/or migration [53][54][55] Esophageal carcinoma…”

“…High expression of legumain in patients with breast cancer is associated with significant shorter survival time. The high expression of legumain is an independent risk factor for clinical prognosis of breast cancer [44,45]. A legumain-based DNA vaccine induced a strong CD8 ?…”

Glycosylation is important for legumain localization and processing to active forms but not for cystatin E/M inhibitory functions

“…In breast cancer, cystatin E/M has been described as a candidate tumor suppressor [53][54][55] as epigenetic silencing of cystatin E/M occurs during breast cancer progression [56]. In tumor cells, legumain and cathepsin L appear to be regulated by cystatin E/M.…”

Haplotype-resolved and integrated genome analysis of the cancer cell line HepG2

“…It regulates cell cycle progression (Bilican and Goding 2006), and its overexpression has been demonstrated in promoting or maintaining the proliferation of many cancers including melanomas (Vance et al 2005), nasopharyngeal cancer (Lv et al 2017), breast cancer D'Costa et al 2014), prostate cancer (Du et al 2017), and gastric cancer (Yu et al 2015). Here, we show that three copies of the TBX2 gene exist in HepG2 cancer cells as a result of duplication in Haplotype 2.…”