Let-7a/cMyc/CCAT1/miR-17-5p Circuit Re-sensitizes Atezolizumab Resistance in Triple Negative Breast Cancer through Modulating PD-L1.

BACKGROUND: Triple negative breast cancer (TNBC) is an immunogenically hot tumor. The immune checkpoint blockades (ICBs) have been recently emerged as promising therapeutic candidates for several malignancies including TNBC. Yet, the development of innate and/or adaptive resistance by TNBC patients towards ICBs such as programmed death-ligand 1 (PD-L1) inhibitors (e.g. Atezolizumab) shed the light on importance of identifying the underlying mechanisms regulating PD-L1 in TNBC. Recently, it was reported that non-coding RNAs (ncRNAs) perform a fundamental role in regulating PD-L1 expression in TNBC. Hence, this study aims to explore a novel ncRNA axis tuning PD-L1 in TNBC patients and investigate its possible involvement in fighting Atezolizumab resistance. METHODS: In-silico screening was executed to identify ncRNAs that could potentially target PD-L1. Screening of PD-L1 and the nominated ncRNAs (miR-17-5p, let-7a and CCAT1 lncRNA) was performed in BC patients and cell lines. Ectopic expression and/or knockdown of respective ncRNAs were performed in MDA-MB-231. Cellular viability, migration and clonogenic capacities were evaluated using MTT, scratch assay and colony-forming assay, respectively. RESULTS: PD-L1 was upregulated in BC patients, especially in TNBC patients. PD-L1 is positively associated with lymph node metastasis and high Ki-67 in recruited BC patients. Let-7a and miR-17-5p were nominated as potential regulators of PD-L1. Ectopic expression of let-7a and miR-17-5p caused a noticeable reduction in PD-L1 levels in TNBC cells. In order to investigate the whole ceRNA circuit regulating PD-L1 in TNBC, intensive bioinformatic studies were performed. The lncRNA, Colon Cancer-associated transcript 1 (CCAT1), was reported to target PD-L1 regulating miRNAs. Results showed that CCAT1 is an upregulated oncogenic lncRNA in TNBC patients and cell lines. CCAT1 siRNAs induced a noticeable reduction in PD-L1 levels and a marked increase in miR-17-5p level, building up a novel regulatory axis CCAT1/miR-17-5p/PD-L1 in TNBC cells that was tuned by the let-7a/c-Myc engine. On the functional level, co-treatment of CCAT-1 siRNAs and let-7a mimics efficiently relieved Atezolizumab resistance in MDA-MB-231 cells. CONCLUSION: The present study revealed a novel PD-L1 regulatory axis via targeting let-7a/c-Myc/CCAT/miR-17-5p. Additionally, it sheds the light on the potential combinational role of CCAT-1 siRNAs and Let-7a mimics in relieving Atezolizumab resistance in TNBC patients.

MALAT-1/p53/miR-155/miR-146a ceRNA circuit tuned by methoxylated quercitin glycoside alters immunogenic and oncogenic profiles of breast cancer.

Triple-Negative Breast Cancer (TNBC) is one of the most aggressive and hot BC subtypes. Our research group has recently shed the light on the utility of natural compounds as effective immunotherapeutic agents. The aim of this study is to investigate the role of a methoxylated quercetin glycoside (MQG) isolated from Cleome droserifolia in harnessing TNBC progression and tuning the tumor microenvironment and natural killer cells cytotoxicity. Results showed that MQG showed the highest potency (IC50 = 12 µM) in repressing cellular proliferation, colony-forming ability, migration, and invasion capacities. Mechanistically, MQG was found to modulate a circuit of competing endogenous RNAs where it was found to reduce the oncogenic MALAT-1 lncRNA and induce TP53 and its downstream miRNAs; miR-155 and miR-146a. Accordingly, this leads to alteration in several downstream signaling pathways such as nitric oxide synthesizing machinery, natural killer cells' cytotoxicity through inducing the expression of its activating ligands such as MICA/B, ULBP2, CD155, and ICAM-1 and trimming of the immune-suppressive cytokines such as TNF-α and IL-10. In conclusion, this study shows that MQG act as a compelling anti-cancer agent repressing TNBC hallmarks, activating immune cell recognition, and alleviating the immune-suppressive tumor microenvironment experienced by TNBC patients.