Nan Li

miR-486 Promotes the Invasion and Cell Cycle Progression of Ovarian Cancer Cells by Targeting CADM1

Objective. To explore the role and possible underlying mechanism of miR-486 in ovarian cancer (OC) cells. Methods. The expression of miR-486 and CADM1 was detected by qRT-PCR in OC tissues and adjacent nontumor tissues and OC cell lines. The dual-luciferase reporter gene system was used to determine the targeting relationship between miR-486 and CADM1. CCK-8, colony formation assay, Transwell, and flow cytometry were performed to detect cell proliferation, cell invasion, cell cycle progression, and the apoptotic cell death, respectively. Western blot was carried out to detect the expression of CADM1 protein and the proteins associated with cell cycle progression. Results. miR-486 was significantly upregulated in OC tissues and cells, while CADM1 expression was significantly downregulated. Dual-luciferase reporter assays further confirmed that CADM1 was a target gene of miR-486. Interference with miR-486 could inhibit the proliferation and invasion and promoted the apoptosis of SKOV3 cells. Knocking down both miR-486 and CADM1 significantly increased the SKOV3 cell proliferation, invasion, and the number of cells transitioning from the G0/G1 phase into the S phase of cell cycle and reduced the cellular apoptosis. Western blot analysis revealed that the expression of cell cycle progression-related proteins (CyclinD1, CyclinE, and CDK6) was significantly reduced, and the p21 expression was increased when interfering with both miR-486 and CADM1 expression. Conclusion. Our results suggested that miR-486 could act as a tumor promoter by targeting CADM1 and be a potential therapeutic target for the treatment of OC.

Development of Highly Effective Anti-Mesothelin hYP218 Chimeric Antigen Receptor T Cells With Increased Tumor Infiltration and Persistence for Treating Solid Tumors

Abstract Mesothelin targeting CAR T cells have limited activity in patients. In this study, we sought to determine if efficacy of anti-mesothelin CAR T cells is dependent on the mesothelin epitopes that are recognized by them. To do so, we developed hYP218 (against membrane-proximal epitope) and SS1 (against membrane-distal epitope) CAR T cells. Their efficacy was assessed in vitro using mesothelin-positive tumor cell lines and in vivo in NSG mice with mesothelin-expressing ovarian cancer (OVCAR-8), pancreatic cancer (KLM-1), and mesothelioma patient-derived (NCI-Meso63) tumor xenografts. Persistence and tumor infiltration of CAR T cells was determined using flow cytometry. hYP218 CAR T cells killed cancer cells more efficiently than SS1 CAR T cells, with a two- to fourfold lower ET50 value (effector-to-target ratio for 50% killing of tumor cells). In mice with established tumors, single intravenous administration of hYP218 CAR T cells lead to improved tumor response and survival compared with SS1 CAR T cells, with complete regression of OVCAR-8 and NCI-Meso63 tumors. Compared with SS1 CAR T cells, there was increased peripheral blood expansion, persistence, and tumor infiltration of hYP218 CAR T cells in the KLM-1 tumor model. Persistence of hYP218 CAR T cells in treated mice led to antitumor immunity when rechallenged with KLM-1 tumor cells. Our results show that hYP218 CAR T cells, targeting mesothelin epitope close to cell membrane, are very effective against mesothelin-positive tumors and are associated with increased persistence and tumor infiltration. These results support its clinical development to treat patients with mesothelin-expressing cancers.