Gencefe Client Article |Immunosensitivity mediated by downregulated AKT1-SKP2 induces anti-PD-1-associated thyroid immune injury

The following article is transferred from GENCEFE BIOTECH （Disease Research Newsletter）

Immune therapy works by awakening the immune system to counteract immune evasion and immune silence in tumors. Immune checkpoint inhibitors (ICI) enhance the cytotoxic effects on tumor cells, including PD-1 and its ligand PD-L1, as well as CTLA-4[1][2]. PD-1/PD-L1 primarily function during the effector phase of the immune response, limiting the immune response of T cells under inflammatory conditions. CD8+ T cells are considered the most directly involved in cytotoxic effects and are closely associated with the response to anti-PD-1/PD-L1 treatment. Local responses to ICI occur in the tumor microenvironment (TME), with tumor-infiltrating lymphocytes (TIL) and PD-L1 expression considered as major predictive factors for TME responses to ICI[3]. Immune-related adverse events (irAEs) associated with ICI occur in all tissues[4]. The incidence of thyroid irAEs ranges from 6% to 20%. Uncontrolled immune damage may impede the progress of ICI, but its mechanisms are not yet clear.

In June 2023, Director Qiao Hong and her research team at Harbin Medical University published an article online in the journal "International Immunopharmacology" (scheduled for print in August), titled "Immunosensitivity mediated by downregulated AKT1-SKP2 induces anti-PD-1-associated thyroid immune injury." The article retrospectively analyzes the incidence and risk factors of thyroid irAEs, demonstrating that anti-PD-1 induces thyroid irAEs by enhancing thyroid immune sensitivity through downregulating AKT1-SKP2[5].

In this study, overexpression plasmids for AKT1, SKP2, and PD-L1 were designed and synthesized by GENCEFE BIOTECH, aiding the research team in identifying key proteins and pathways. These plasmids were utilized for in vitro and in vivo validation, contributing to a comprehensive understanding of the mechanisms involved in the research.

Research Results

Part One: Retrospective Analysis

Researchers collected data from cancer patients who underwent immune checkpoint inhibitor (ICI) treatment at Harbin Medical University Second Hospital between September 2019 and December 2022, with a total of 518 cases included. Through data analysis, the following findings were observed:

1. Approximately half of the patients experienced thyroid immune-related adverse events (irAEs) during anti-PD-1 treatment, and the risk differed with PD-L1 expression.

2. Thyroid irAEs were associated with gender, IgG subtype, FT4, TPOAb, TGAb, TSHI, TFQI, and TSH (see Figure 1A, B, C).

3. The highest risk of thyroid irAEs was observed in patients with head and neck cancer, with a potential association with tissue origin, especially squamous cell carcinoma (see Figure 1D, E).

4. Patients with enriched peripheral blood lymphocytes before treatment were more prone to irAE-related hypothyroidism. Those with a significant increase in lymphocytes before and after treatment were more likely to develop irAE-related hyperthyroidism and thyroiditis (see Figure 1F).

Part Two: In Vitro Mechanism Study

1. The human normal thyroid cell line Nthy-ori3-1 (NTHY) was divided into NIVO (Nivolumab, anti-PD-1) group and control group. It was found that NIVO induced G1 phase arrest in NTHY cells, reduced FT4 secretion, and increased NTHY cell immunosensitivity by downregulating PD-1 and upregulating IFN-γ. Differential protein expression (DEP) screening and analysis revealed that AKT1 is a key protein regulated by NIVO in modulating NTHY cell immunosensitivity.

2. NIVO downregulated AKT1 in NTHY cells, subsequently downregulated SKP2 and PD-L1, and upregulated IFN-γ.

The control group (NC group) and AKT1 overexpression plasmid-transfected NTHY cells (AKT1-OE group) were compared. AKT1 mRNA and protein were significantly upregulated in the AKT1-OE group (see Figure 2A, B). NTHY cells were divided into 6 groups to detect proliferation, apoptosis, and cell cycle. The experimental results showed that the overexpression of AKT1 could reverse the impact of NIVO on the viability of NTHY cells (see Figure 2 C-G); AKT1 overexpression antagonized the regulation of Fas and BAX by NIVO (see Figure 2 H, I); AKT1 overexpression reduced the sensitivity of NTHY cells induced by NIVO, protecting NTHY cells from cytotoxicity (see Figure 2 J); when NTHY was treated with NIVO, AKT1 overexpression inhibited the attraction of NTHY to CD8+ T cells by NIVO (see Figure 2 K).