Ly96-mediated activation of TGF-β1/Smad2/3 signaling in hepatocellular carcinoma and its potential for nanoparticle-based therapy

Abstract

Hepatocellular carcinoma (HCC) continues to pose a chief threat to the global healthcare landscape and is characterized by scarce therapeutic options and poor clinical outcomes, especially in advanced-stage disease. Although lymphocyte antigen 96 (LY96) is associated with immunogenic cell death, its specific role in HCC progression and therapeutic potential remains unclear. To identify prospective therapeutic targets in HCC, by combining the cancer-immunity cycle score with WGCNA and systems biology methods, we identify pivotal molecular interactions. By integrating the cancer-immunity cycle score with WGCNA and systems-level approaches, we systematically identify potential therapeutic targets in HCC. We evaluate LY96 expression at the transcriptomic and proteomic levels in HCC tissues and explore its prognostic relevance by drawing upon information from The Cancer Genome Atlas (TCGA) repository. The functional role of LY96 is delineated through a panel of cellular assays conducted in vitro, complemented by in vivo tumorigenesis models. To identify the downstream signaling cascades associated with LY96, gene set enrichment analysis (GSEA) is performed to elucidate the implicated pathways, which are then confirmed via experimental validation. Furthermore, we employ a lipid-polymer hybrid nanoparticle (NP) platform to facilitate the systemic delivery of an LY96 inhibitor and demonstrate its potential as a newly proposed intervention strategy for HCC. Clinically, marked LY96 overexpression occurs in HCC samples, where elevated LY96 expression is strongly associated with reduced overall survival (OS) among liver cancer patients. LY96 facilitates the progression of HCC via complementary in vitro and in vivo approaches. Mechanistically, LY96 induces the activation of the TGF-β1/Smad2/3 signaling axis in HCC. For therapeutic applications, we develop a liposome-based nanoparticle system that delivers the LY96 inhibitor L6H21 to tumor cells and effectively suppresses HCC progression through a combination of in vivo and in vitro studies. Taken together, the current observations identify LY96 as a promising diagnostic indicator and a viable intervention for therapeutic modulation to improve HCC treatment.

1. Introduction

Hepatocellular carcinoma (HCC), the predominant histological subtype of primary liver cancer, continues to constitute a formidable global medical concern, with projections indicating that the annual incidence of HCC will surpass one million cases worldwide by 2025 [1]. Despite notable advances in prevention and treatment, HCC continues to rank among the foremost causes of cancer-associated deaths, driven by frequent recurrence and limited therapeutic outcomes [2–4]. Following the guidelines set forth by the 2022 Barcelona Clinic Liver Cancer (BCLC) consortium, surgical strategies, including tumor resection and liver transplantation, remain central to the current therapeutic paradigm for HCC. This method is complemented by multimodal approaches, including transarterial chemoembolization (TACE), radiofrequency ablation (RFA), and systemic therapies [5–9]. This multimodal approach has demonstrably extended patient survival [10]. However, there remains a substantial gap between treatment efficacy and long-term prognosis, particularly in advanced HCC, where malignancy is more aggressive [11].

The progression of HCC is frequently accompanied by complex molecular alterations, including the dysregulation of multiple genes that not only drive tumor development but also offer opportunities for prognostic assessment and targeted therapeutic strategies [12]. Consequently, a critical unmet need persists in uncovering robust biomarkers and delineating the mechanistic basis of HCC progression to advance individualized and efficacious therapeutic strategies. Despite their clinical adoption, the therapeutic efficacy of immune checkpoint blockade modulators (ICIs) for the care of patients with advanced HCC across both frontline and subsequent lines of therapy continues to be critically examined. Despite extensive evaluation, phase III clinical trials have largely fallen short of confirming a meaningful survival advantage of ICIs in patients with unresectable HCC [13,14]. When administered as monotherapy, ICIs yield an objective response rate of only 15–20% [15], a limitation attributed to the intricate interplay of multiple immune checkpoint pathways within the immunological milieu of HCC, the paucity of intertumoral immune cell infiltrates, and the excessive exhaustion of immune effector cells [16,17]. Nonetheless, findings from several phase III trials indicate that compared with sorafenib monotherapy, combined administration of anti-PD-1/PD-L1 and anti-CTLA-4 agents or antiangiogenic therapies confers superior survival benefits for unresectable HCC patients [18,19]. Collectively, these insights highlight the critical importance of elucidating cancer