A. El Khodiry (New Cairo, Egypt)
GUC - German University in CairoAuthor Of 1 Presentation
87P - Epigenetics of the epigenetics: impact of Let-7a expression restoration in CD8+ cells of HER2+ breast cancer patients
Abstract
Background
HER-2+ breast cancer (BC) remains challenging as the use of Trastuzumab is ineffective in some cases. Let-7a is considered as a well-known tumor suppressor microRNA. It is epigenetically regulated by hypermethylation and suppressed by lncRNA, lin28 in many cancer such as BC. Here, we aim at restoring the expression of Let-7a through epigenetic modulation to study the impact of Let-7a on PD-1, PD-L1 and CLDN1 expression levels in CD8+ cells of HER2+ BC patients.
Methods
65 biopsies/blood samples were withdrawn from BC patients, CD8+ cells isolated from PBMCS (MojoSort™). Cells were transfected with siRNAs of NEAT1/Lin28 (HiPerFect®) then with/without Decitabine (DAC) treatment. LDH and MTT assays were performed after co-culturing transfected/treated CD8+ cells with MDA-MB-231 and MCF-7 cells ratio (5:1). One-way ANOVA and Dunnett’s test were used in statistical analysis for multiple groups.
Results
Let-7a was found to be downregulated (p=0.0001) while NEAT1 and CLDN1 were overexpressed (p=0.0052, 0.0051, respectively) compared to controls. We reported before overexpression of PD-1 and PD-L1. Let-7a levels was restored upon silencing of lin28, NEAT1 and DAC treatment (p=0.0326,0.0326,0.0014 respectively) and upon different conditions of cotransfections and DAC treatment of CD8+ cells using siLin28/silNEAT1, siLin28/DAC, siNEAT1/DAC and silin28/NEAT1/DAC (p= 0.0447, 0.0245, 0.1628,0.0011 respectively). Let-7a potential downstream targets, PD-1, PD-L1 and CLDN1 were assessed upon restoration of let-7a using the conditions of transfections mentioned above. PD-1 was downregulated (p=<0.0001, in all conditions) as well as PD-L1 (p= 0.0017, 0.0010, 0.0013, 0.0009, 0.0052, 0.0020, 0.0015, respectively) and CLDN1 was upregulated (p=0.0077, 0.0650, 0.0037, 0.0486, 0.0011, 0.0316, 0.0427, respectively) compared to mock. Tumor cytotoxicity was found to be elevated in high-Let-7a-CD8+ cells where MDA-MB-231 cells (p=<0.0001 in all conditions, but p= 0.0018 in siNEAT1) and MCF-7 cells (p=0.0006, 0.0006, 0.0015, 0.0012, 0.0005, 0.0004 and 0.0080) compared to mock.
Conclusions
These data shed the light on the pivotal role of Let-7a in HER2+ BC in an attempt to develop combined therapy with immunotherapeutic agents.
Legal entity responsible for the study
German University in Cairo - GUC.
Funding
Has not received any funding.
Disclosure
All authors have declared no conflicts of interest.