The long non-coding RNA LINC01133 as a novel determinant of immune evasion in triple-negative breast cancer

Va Boston Health Care System, Boston MA

Investigators

Abstract

Triple-negative breast cancers (TNBCs) afflict younger women of African and Latino descent, present with aggressive clinical behavior, and are in their majority unresponsive to systemic treatments. Patients with refractory disease exhibit dismal survival rates with reliable therapies still undefined. To identify novel, disease- relevant therapeutic targets, we conducted transcriptomic analyses of highly metastatic and tumor-initiating TNBC cells, focusing specifically on long non-coding RNAs (lncRNAs), an emerging RNA family with pleiotropic contributions to essential cellular processes. These efforts identified the lncRNA LINC01133, which we found induced malignant tumor development by promoting the expansion of cancer stem cells (CSCs): tumor-initiating cells at the origin of metastasis and therapy resistance. Importantly, LINC01133 associated tightly with clinical TNBC and prognosticated poor patient survival. Further, LINC01133 expression, on its own, promoted cancer growth in vitro and tumorigenesis in vivo. Importantly, LINC01133 inhibition by anti-sense oligonucleotides (ASO) inhibited cancer cell growth, altogether ascribing LINC01133 critical role as a credible therapeutic target in TNBC. Interestingly, LINC01133 exerted additional, paracrine activities that regulated how TNBC cells interfaced with the tumor immune microenvironment (TIME). Indeed, LINC01133-generated syngeneic murine mammary tumors exhibited evident immune-suppressed phenotypes with increased myeloid-derived suppressor cells, M2 macrophages, and anergized 2B4+CD3+ T-cells, concomitant with striking PD-L1 induction in LINC01133-cancer cells. These results allow us to hypothesize that LINC01133 regulated immune evasion, in addition to CSC genesis, representing an opportune and novel dual target in immuno-oncology, and our objective is to verify these notions using three concerted aims. In Aim1, we will determine how LINC01133 promotes the expression of the checkpoint protein PD-L1 by probing the molecular events that foster PD-L1 accumulation (1.1), and by delineating the direct proximal molecular networks through which LINC01133 regulates PD-L1 expression (1.2 and 1.3). In Aim2, we will comprehensively determine the TIME constituents in LINC01133-expressing syngeneic immune-competent murine TNBC models using FACS and immunohistochemistry (IHC) (2.1). Then, we will use ex vivo phenotypic and functional assays to determine the extent to which tumor-purified infiltrating lymphocytes are exhausted/anergized in LINC01133- expressing tumors (2.2) and determine LINC01133 impact on metastasis (2.3). To clinically validate these findings, we will use quantitative PCR on cancer cells laser-captured from TNBC specimens to determine if LINC0113 levels correlate with patient metastasis and survival history and/or IHC-determined T-cell-specific exhaustion phenotypes in their tumors (2.4). In Aim 3, we will determine if LINC01133 inhibition stunts tumor/metastasis growth by reawakening anti-tumor responses. For this purpose, we will use in vivo tumor- targeted gene knockdown that uses Epcam RNA aptamers – structured RNAs that bind with high affinity to the cell receptor Epcam, highly expressed in LINC01133-expressing CSCs – to deliver covalently linked small interfering RNAs (siRNAs) against LINC01133 into TNBC growths in vivo. Knockdown occurs selectively in Epcam-bearing cancer cells when aptamer-siRNA chimeras (AsiCs) are internalized/cleaved intracellularly to liberate active LINC01133-siRNAs. Here, we will determine if Epcam-AsiC-LINC01133 causes immune- mediated cancer regression in syngeneic and genetically engineered mouse models (GEMM) by conducting comparative tumor growth kinetics, assessing cancer cell death/survival in treated tumors and metastases, and by characterizing the phenotype and functionality of tumor-infiltrating lymphocytes ex vivo (3.1). Finally, we will determine if AsiCs cooperate with immune checkpoint inhibitors in enhancing TIL-mediated cancer cell clearance (3.2). Our studies will thus introduce novel inhibitors directed against two major determinants of TNBC malignancy, CSC formation and immune escape, revolutionizing TNBC clinical management.