Pancreatic ductal adenocarcinoma (PDAC) is one of the most intractable malignancies, accounting for nearly eight percent of cancer-related deaths in the United States [1]. The 5-year overall survival rate is approximately 12 %, mainly as a result of late diagnosis at advanced stages for the majority of PDAC patients. These patients are ineligible for surgical resection, leaving systematic chemotherapy as the most commonly-used standard treatment. Gemcitabine-based monotherapy and combined therapies, such as gemcitabine plus nab-paclitaxel, provide survival benefits for patients with PDAC [[2], [3], [4]]; however, drug resistance may arise as early as several weeks after chemotherapy initiation, thus limiting the therapeutic efficacy of chemotherapy reagents [5]. Increasing evidence has demonstrated that acquired tolerance to apoptotic cell death induced by cytotoxic drugs is a critical cause of chemotherapy failure. Therefore, exploiting other forms of cell death might hold great potential for overcoming therapeutic resistance and improving the survival of patients with PDAC.

Ferroptosis was originally identified as a unique form of programmed cell death, characterized by shrunken mitochondria, condensed membranes and decreased crista, in drug screens for small molecules that can selectively kill RAS-expressing cancer cells [6]. Unlike apoptosis, ferroptosis is driven by iron-catalyzed excessive lipid peroxidation, ultimately leading to the rupture of the plasma membrane and cell death. Recent studies have indicated that ferroptosis induction is a promising therapeutic strategy in cancer treatment because it not only kills tumor cells directly but also synergizes with chemotherapy [7,8], radiotherapy [9], targeted therapy [10] and immunotherapy [11,12]. However, transformed malignant cells can acquire resistance to ferroptosis through multiple mechanisms, such as decreased coenzyme Q10 production mediated by the upregulation of apoptosis-inducing factor mitochondria-associated 2 (AIFM2/FSP1) and elevated expression of cytoprotective genes resulting from the activation of the antioxidant transcription factor nuclear factor erythroid 2-like 2 (NFE2L2/NRF2) [13]. In pancreatic cancer, activating transcription factor 4 (ATF4)-mediated upregulation of the molecular chaperone heat shock protein family A member 5 (HSPA5) directly binds to and stabilizes glutathione peroxidase 4 (GPX4) protein to suppress erastin-induced ferroptosis [14]. Therefore, unraveling the underlying mechanisms responsible for acquired resistance to ferroptosis inducers, such as erastin and RSL3, may facilitate the design of effective therapeutic regimens for patients with PDAC.

Ubiquitination is a multistep enzyme cascade in which E3 ubiquitin ligases attach ubiquitin moieties to specific lysine residues on protein substrates [15]. RanBP2-type and C3HC4-type zinc finger containing 1 (RBCK1), a RING-in-between-RING (RBR) E3 ubiquitin ligase, is well recognized for its roles as part of the linear ubiquitin chain assembly complex (LUBAC) in the context of inflammation and immunity [16]. Increasing evidence has shown that RBCK1 also plays multifaceted roles in other pathological processes, including cancer. For example, RBCK1 serves as an oncogene in renal cell carcinoma by promoting p53 polyubiquitination and degradation [17]. Additionally, RBCK1 facilitates the proliferation of breast cancer cells by upregulating the expression of estrogen receptor alpha and cyclin B1 at the transcriptional level [18]. In pancreatic cancer, a splice site mutation in RBCK1 transcripts was revealed by exome sequencing, the most likely consequence of which was increased nuclear retention of the RBCK1 protein owing to the absence of its entire ubiquitin domain and nuclear export signal [19]. However, the expression pattern and biological function of RBCK1 in PDAC remains unknown.

In this study, we concluded that ferroptosis is inhibited in PDAC because pancreatic cancer tissues are characterized by elevated expression of GSH metabolic enzymes, decreased labile iron pool (LIP), and universal upregulation of ferroptosis suppressors. Parallel RNA-seq and Ribo-seq data suggested that PDAC cells underwent dramatic transcriptional and translational changes following ferroptosis induction. The E3 ubiquitin ligase RBCK1 was upregulated in survived PDAC cells after IKE treatment and suppressed ferroptosis by polyubiquitinating MFN2 and promoting its proteasomal degradation to reduce mitochondrial ROS production. These findings provide a theoretical basis for RBCK1-mediated ferroptosis resistance as a potential target for designing combination therapies.