The deubiquitinating enzyme Ubiquitin specific peptidase 5 (USP5) has attracted substantial notice for its vital role in cancer progression. However, the USP5-mediated deubiquitination of corresponding protein substrates and its functional role in hepatocellular carcinoma (HCC) have not been fully investigated. Here, we demonstrated that USP5 expression was significantly elevated in HCC tissues. The overexpression of USP5 was closely associated with larger tumor sizes, more satellite nodules and tumor emboli, and predicted unfavorable clinical outcome in HCC patients as well. Functionally, USP5 facilitated cell proliferation, migration, and invasion, and induced lipid accumulation in vitro, along with enhanced tumor growth in vivo. Moreover, knockdown of USP5 expression showed a profound effect on lipidomic profiling, specially reduced the content of palmitic acid (PA). Treatment of PA could partially rescue the suppression of HCC mediated by USP5 knockdown. Further mechanistic investigation uncovered that Fatty acid synthase (FASN), the crucial enzyme catalyzing PA synthesis, was a downstream target of USP5. USP5 interacted with FASN, repressing the ubiquitination modification of FASN and preventing its degradation. Notably, the positive correlation between USP5 and FASN expression in HCC tissues was observed, and USP5 exerted oncogenic effects partly via FASN. Our findings revealed that USP5 promotes HCC progression through deubiquitinating FASN, and targeting the USP5-FASN-PA axis could potentially serve as a strategic approach for the therapy of HCC.
Hepatocellular carcinoma (HCC), which comprises approximately 90% of primary liver cancer cases, stands as one of the most formidable and widespread malignancies in humans [1]. The heterogeneity and complexity of HCC leads to late-stage diagnoses and unfavorable overall survival outcomes for patients [2]. Metabolic reprogramming serves as a well-established hallmark of cancer [3]. The liver, being the central hub of metabolism within the body, experiences profound alterations in metabolic processes in HCC. The dysregulation of lipids assumes pivotal roles in the development and progression of HCC, owing to the vast and intricate nature of lipids as complex biomolecules. Lipids exert a critical role in multiple biological processes, encompassing not only energy storage and metabolism, but also the transmission of signals, the regulation of immune responses, the modulation of inflammatory cascades, and the intricate interplay of cellular recognition [4,5,6]. For example, elevated palmitic acid (PA), a saturated fatty acid with a 16-carbon chain, is strongly linked to tumor progression through multiple oncogenic mechanisms. PA induces a pro-metastatic memory via Schwann cells and fosters an immunosuppressive tumor microenvironment [7, 8]. PA drives metastasis by reinforcing YAP signaling through the ZDHHC15-YAP positive feedback loop [9]. Moreover, PA promotes RAS palmitoylation, a critical step for its oncogenic activation [10]. Given these multifaceted roles, elucidating the regulatory mechanisms of PA synthesis is crucial for understanding and potentially targeting cancer progression.
Fatty acid synthase (FASN) is an essential enzyme responsible for the de novo synthesis of long-chain fatty acids, particularly PA, a process dependent on nicotinamide adenine dinucleotide phosphate (NADPH) as a reducing cofactor [11]. In normal cells, lipid demands are primarily met through exogenous uptake, keeping FASN expression at basal levels due to sufficient fatty acid supply from dietary sources and hepatic or adipose stores. In contrast, cancer cells exhibit marked FASN upregulation, as their accelerated proliferation and survival hinge on enhanced de novo lipogenesis to meet heightened metabolic demands [12,13,14]. This metabolic reprogramming underscores FASN's critical role in sustaining tumor growth, making it a compelling therapeutic target for HCC [15]. FASN is highly expressed in HCC and correlates with sorafenib treatment response. Its transcription is regulated by upstream transcription factors, including liver X receptor (LXR) and sterol regulatory element-binding protein 1 (SREBP1) [16,17,18]. More importantly, several types of post-translational modification are also involved in FASN upregulation in HCC. Moreover, post-translational modifications contribute significantly to FASN upregulation in HCC. For example, S-palmitoylation of FASN mediated by the DHHC-type palmitoyltransferase ZDHHC20 enhances its protein stability, a process critical for hepatocarcinogenesis [19]. Additionally, heat shock protein 90α (Hsp90α) interacts with FASN and stabilizes it by suppressing ubiquitination [20]. Consequently, elucidating the mechanisms driving FASN overexpression in HCC is imperative for developing targeted interventions.
Ubiquitination represents a crucial post-translational modification that regulates protein activity, function, localization, and stability through a well-characterized enzymatic cascade. In this process, E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 (ubiquitin ligase) sequentially catalyze the transfer of activated ubiquitin molecules to lysine residues on target proteins [21]. The addition of ubiquitin signals by E3 ubiquitin ligases is counteracted by around 100 deubiquitinating enzymes (DUBs) in humans. DUBs and E3 ligases work together to regulate ubiquitin signaling by conferring specificity for various substrates and/or ubiquitin signals. The balance between ubiquitination and deubiquitination is meticulously regulated to maintain proper proteostasis and enable appropriate responses to cellular stimuli and stressors. Consequently, DUBs have been implicated in all fundamental characteristics of cancer [22]. Based on sequence and domain conservation, DUBs are classified into nine superfamilies, including ubiquitin-specific proteases (USPs), ovarian tumor proteases (OTUs), ubiquitin C-terminal hydrolases (UCHs), and JAMM/MPN domain-associated metalloproteases (JAMMs) [22, 23]. Ubiquitin-specific protease 5 (USP5) is a member of the USP family, which governs a multitude of cellular processes, such as the repair of DNA double-strand breaks, modulation of inflammatory reactions, regulation of stress responses, glucose metabolism reprogramming, and epithelial-mesenchymal transition (EMT) [24,25,26,27,28]. USP5 is frequently overexpressed in HCC and correlates with aggressive tumor behavior. It promotes epithelial-mesenchymal transition (EMT) by deubiquitinating SLUG and inosine monophosphate dehydrogenase 2 (IMPDH2) [25, 29]. USP5 also interacts with the oncoprotein c-Myc, stabilizing it by inhibiting K48-linked polyubiquitination and thereby modulating glucose metabolism [28]. Moreover, USP5-mediated deubiquitination of lymphoid-specific helicase (LSH) enhances hepatocarcinogenesis through upregulation of solute carrier family 7 member 11 (SLC7A11), which suppresses ferroptosis in HCC cells [30]. Despite these established roles, the key protein substrates of USP5 in lipid metabolism during HCC progression remain poorly defined, highlighting an important area for investigation.
In this study, we demonstrated that USP5 silencing markedly suppresses HCC cell proliferation, migration, invasion, and PA accumulation. Moreover, we elucidated the molecular interaction between USP5 and fatty acid synthase (FASN), revealing that USP5 inhibits FASN ubiquitination, thereby stabilizing its expression in HCC. These findings highlight the oncogenic role of USP5 in driving HCC progression and propose USP5 as a promising therapeutic target for HCC treatment.