Epigenetic silence of C1RL-AS1 by Adriamycin inhibits PD-L1 expression via suppression of miR-3150a-3p/IFNAR2 and JAK1/STAT1 signaling pathway in breast cancer

Technical Summary: Epigenetic Silencing of C1RL-AS1 by Adriamycin in Breast Cancer

Problem Statement
Adriamycin (Doxorubicin) resistance remains a primary obstacle to successful treatment and a cause of poor prognosis in breast cancer, particularly in luminal A subtypes. While Adriamycin is a first-line cytotoxic agent, surviving tumor cells often acquire resistance through the modulation of cellular signaling pathways, DNA repair mechanisms, and apoptotic deregulation. The specific molecular mechanisms driving this resistance, particularly involving long non-coding RNAs (lncRNAs) and epigenetic modifications, require further elucidation to improve therapeutic efficacy.

Methodology
The study employed a multi-faceted approach combining bioinformatics, molecular biology, and in vivo models:

• Bioinformatics Analysis: Researchers utilized the GSE155478 dataset to screen for differentially expressed lncRNAs between Adriamycin-resistant (MCF-7/ADR) and parental cells. They further analyzed TCGA datasets and GEPIA for expression correlations, survival analysis, and immune infiltration (using CIBERSORT).
• Cell Models: Adriamycin-resistant cell lines (MCF-7/ADR and ZR-75-1/ADR) were established. Standard cell lines (MCF-7, ZR-75-1, MDA-MB-231, HBL-100) were used for comparative analysis.
• Functional Assays:
  • Viability and Resistance: CCK-8 assays and colony formation assays were used to determine IC50 values and cell viability under Adriamycin treatment.
  • Apoptosis: Flow cytometry was used to assess apoptosis rates.
  • Epigenetic Regulation: Chromatin Immunoprecipitation (ChIP) was performed to analyze H3K27 acetylation levels at the C1RL-AS1 promoter. The histone acetyltransferase inhibitor C646 was used to validate the role of acetylation.
  • Mechanistic Interactions: RNA pull-down assays, luciferase reporter assays, and RIP (RNA Immunoprecipitation) were conducted to verify interactions between C1RL-AS1, miR-3150a-3p, and IFNAR2.
  • Protein Analysis: Western blotting was used to quantify PD-L1, JAK1, STAT1, and IFNAR2 protein levels.
• In Vivo Models: Xenograft tumors were established in nude mice using MCF-7/ADR cells transfected with lentiviral vectors (overexpression or control) to monitor tumor growth and response to Adriamycin administration.

Key Contributions and Results
The study identified C1RL-AS1 as a critical regulator of Adriamycin resistance in breast cancer through the following findings:

1. Epigenetic Silencing by Adriamycin:

   • C1RL-AS1 is significantly down-regulated in Adriamycin-resistant breast cancer cells and luminal A breast cancer tissues compared to normal adjacent tissues.
   • High expression of C1RL-AS1 correlates with better survival in HER2-negative patients.
   • Mechanistically, Adriamycin treatment reduces H3K27 acetylation at the C1RL-AS1 promoter region. This epigenetic modification leads to transcriptional silencing of C1RL-AS1. Treatment with the HAT inhibitor C646 mimicked this down-regulation, confirming the role of H3K27ac.

2. Restoration of Sensitivity via C1RL-AS1 Overexpression:

   • Overexpression of C1RL-AS1 in resistant cells (MCF-7/ADR, ZR-75-1/ADR) significantly reduced cell viability, decreased colony formation, and lowered IC50 values for Adriamycin.
   • In xenograft models, C1RL-AS1 overexpression slowed tumor growth and enhanced the inhibitory effect of Adriamycin compared to controls. Conversely, knockdown of C1RL-AS1 in sensitive cells increased viability and resistance.

3. The ceRNA Axis: C1RL-AS1 / miR-3150a-3p / IFNAR2:

   • C1RL-AS1 functions as a competing endogenous RNA (ceRNA) that directly sponges miR-3150a-3p.
   • miR-3150a-3p is up-regulated in Adriamycin-resistant cells and breast cancer tissues. Its knockdown sensitizes resistant cells to Adriamycin.
   • C1RL-AS1 overexpression inhibits miR-3150a-3p expression, while knockdown promotes miR-3150a-3p expression.
   • miR-3150a-3p directly targets and negatively regulates IFNAR2 (Interferon Alpha Receptor 2). Consequently, by sponging miR-3150a-3p, C1RL-AS1 releases IFNAR2 expression from miR-3150a-3p-mediated repression.

4. Activation of JAK1/STAT1 and PD-L1 Expression:

   • The restoration of IFNAR2 expression activates the JAK1/STAT1 signaling pathway.
   • This pathway activation promotes the transcription of PD-L1.
   • The study demonstrates that C1RL-AS1 knockdown reduces PD-L1 expression, an effect that is reversed by miR-3150a-3p inhibition but abolished if IFNAR2 is simultaneously silenced.
   • Lower IFNAR2 expression in patients correlates with reduced immune cell infiltration, suggesting C1RL-AS1 influences the tumor immune microenvironment.

Significance
The paper posits that the epigenetic silencing of C1RL-AS1 by Adriamycin is a key mechanism driving chemoresistance in breast cancer. By suppressing H3K27 acetylation, Adriamycin inadvertently down-regulates C1RL-AS1, which in turn fails to sponge miR-3150a-3p. This leads to the suppression of IFNAR2, inhibition of the JAK1/STAT1 pathway, and reduced PD-L1 expression.

The authors suggest that C1RL-AS1 may serve as a potential therapeutic target for overcoming chemotherapy resistance. Specifically, strategies to maintain or restore C1RL-AS1 expression could re-sensitize resistant tumors to Adriamycin. Furthermore, given the link between C1RL-AS1, PD-L1, and immune infiltration, the study implies that C1RL-AS1 status could be relevant for understanding the tumor immune microenvironment and potentially guiding combination therapies involving immunotherapy and chemotherapy. The study concludes that identifying these molecular mechanisms is crucial for improving clinical treatment outcomes and prognosis for breast cancer patients.