Pancreatic cancer is a devastating gastrointestinal cancer with a poor prognosis and high mortality rate. Exocrine pancreatic ductal adenocarcinoma (PDAC) accounts for approximately 95% of pathological types and can be regulated by both internal and external factors. (Park et al., 2021). Chemotherapy alone or in combination with radiation is the current standard treatment for advanced PDAC, but its clinical effectiveness is limited due to cancer cells’ ability to evade apoptosis and counteract the anti-cancer effects of chemotherapy drugs (De Dosso et al., 2021, Mohammad et al., 2015). Although small molecule drugs targeting KRAS-G12C, such as sotorasib (AMG 510) and adagrasib (MRTX849), are promising chemotherapeutic agents, their effectiveness in treating lung, colon, or pancreatic cancers is individualized and variable (Hong et al., 2020, Ou et al., 2022). Therefore, developing non-apoptotic cell death induced by chemotherapeutic agents could be an effective means of treating PDAC and overcoming multi-drug resistance (Chen et al., 2023a, Chen et al., 2024, Qi et al., 2023).
Alkaliptosis, a pH-dependent non-apoptotic cell death induced by JTC801 (an opioid analgesic drug), has shown promising anticancer activity, particularly for PDAC (Song et al., 2018). However, JTC801-induced alkaliptosis is not dependent on the OPRL1 receptor. Genetically, JTC801-induced cell death was not dependent on apoptosis, necroptosis, or ferroptosis, and the corresponding molecular markers were not significantly induced, such as PARP (cleavage of caspase 3 or poly [ADP ribose] polymerase) for apoptosis, MLKL (mixed lineage kinase domain like pseudokinase) and RIPK3 (receptor-interacting serine-threonine kinase 3) for necroptosis, or GPX4 (glutathione peroxidase 4) degradation for ferroptosis. More importantly, RNAi inhibition of these genes did not prevent JTC801-induced cell death. Pharmacologically, Z-VAD-FMK (for apoptosis), necrostatin-1 (for necroptosis), ferrostatin-1 (for ferroptosis) or hydroxychloroquine (for autophagy) also failed to inhibit the cytotoxicity of JTC801 (Chen et al., 2023b, Song et al., 2018). In contrast, acidic medium (pH 6.2), N-acetyl cysteine (NAC), or N-acetyl alanine acid (NAA) reversed JTC801-induced alkaliptosis (Song et al., 2018). Indeed, targeting pH is a potential treatment strategy, as cancer cells differ significantly from normal cells in genetics and various biological functions, including the reversal of pH gradients mediated by various ion channels and transporters (Huber et al., 2010, Koltai, 2017). Although the ATPase H+ transporting V0 subunit D1 (ATP6V0D1)-dependent lysosomal pathway and the nuclear factor-κB (NF-κB)-dependent carbonic anhydrase 9 (CA9) pathway have been identified as key signals in the regulation of alkaliptosis (Chen et al., 2023d), the regulatory network of alkaliptosis is still lacking.
Itchy E3 ubiquitin protein ligase (ITCH) is a highly conserved HECT-type E3 ubiquitin ligase and a member of the NEDD4 E3 ubiquitin protein ligase family. It shares a 91% homology with the mouse protein (Yin et al., 2020). As a key E3 ubiquitin ligase, ITCH has been extensively characterized for its function in the immune system and malignancy. ITCH labels different ubiquitinated substrates such as jun proto-oncogene, AP-1 transcription factor subunit (JUN; also known as c-JUN), CASP8 and FADD like apoptosis regulator (CFLAR; also known as c-FLIP), LATS1, TCR-ζ and B-raf proto-oncogene, serine/threonine kinase (BRAF) to control multiple signaling pathways (Piggott et al., 2018, Yin et al., 2019). The complexity of ITCH’s three-dimensional structure also means that it can be regulated by a variety of molecules, including Nedd4 family interacting protein (NDFIP), low density lipoprotein receptor class A domain containing 3 (LDLRAD3), NUMB endocytic adaptor protein (NUMB) and arrestin beta (ARRB2) (Abe et al., 2019, Mund and Pelham, 2009). In addition, the localization of ITCH to various intracellular organelles, mainly the cytoplasm, nucleus, plasma membrane and endosomes, suggests that it has many uncharacterized functions (Gahlot et al., 2024, Totland et al., 2024). In particular, alkaliptosis is a pH-dependent regulated cell death (RCD) involving several pH regulators and transporter proteins. While SLC16A1 (also known as MCT1) a member of the solute carrier family, is localized at cell membranes and cell junctions and facilitates the rapid passage of monocarboxylates (such as pyruvate or lactate) across cell membranes to maintain pH homeostasis (Singh et al., 2023). However, its role in regulating alkaliptosis in PDAC cells remains unidentified.
In this study, we have identified ITCH as a previously unrecognized repressor of alkaliptosis in PDAC cells. We have demonstrated that the downregulation of ITCH promotes alkaliptosis by inhibiting the YAP1-SLC16A1 signaling pathway through LATS1-dependent mechanisms. Our findings establish a new regulatory network for alkaliptosis and suggest that targeting ITCH could enhance susceptibility to alkaliptosis.