A Carbonic Anhydrase IX/SLC1A5 Axis Regulates Glutamine Metabolism Dependent Ferroptosis in Hypoxic Tumor Cells.

The ability of tumor cells to alter their metabolism to support survival and growth presents a challenge to effectively treat cancers. Carbonic anhydrase IX (CAIX) is a hypoxia-induced, metabolic enzyme that plays a crucial role in pH regulation in tumor cells. Recently, through a synthetic lethal screen, we identified CAIX to play an important role in redox homeostasis. In this study, we show that CAIX interacts with the glutamine (Gln) transporter, solute carrier family 1 member 5 (SLC1A5), and coordinately functions to maintain redox homeostasis through the glutathione/glutathione peroxidase 4 (GSH/GPX4) axis. Inhibition of CAIX increases Gln uptake by SLC1A5 and concomitantly increases GSH levels. The combined inhibition of CAIX activity and Gln metabolism or the GSH/GPX4 axis results in an increase in lipid peroxidation and induces ferroptosis, both in vitro and in vivo. Thus, this study demonstrates cotargeting of CAIX and Gln metabolism as a potential strategy to induce ferroptosis in tumor cells.

Genome-wide synthetic lethal screen unveils novel CAIX-NFS1/xCT axis as a targetable vulnerability in hypoxic solid tumors.

The metabolic mechanisms involved in the survival of tumor cells within the hypoxic niche remain unclear. We carried out a synthetic lethal CRISPR screen to identify survival mechanisms governed by the tumor hypoxia-induced pH regulator carbonic anhydrase IX (CAIX). We identified a redox homeostasis network containing the iron-sulfur cluster enzyme, NFS1. Depletion of NFS1 or blocking cyst(e)ine availability by inhibiting xCT, while targeting CAIX, enhanced ferroptosis and significantly inhibited tumor growth. Suppression of CAIX activity acidified intracellular pH, increased cellular reactive oxygen species accumulation, and induced susceptibility to alterations in iron homeostasis. Mechanistically, inhibiting bicarbonate production by CAIX or sodium-driven bicarbonate transport, while targeting xCT, decreased adenosine 5'-monophosphate-activated protein kinase activation and increased acetyl-coenzyme A carboxylase 1 activation. Thus, an alkaline intracellular pH plays a critical role in suppressing ferroptosis, a finding that may lead to the development of innovative therapeutic strategies for solid tumors to overcome hypoxia- and acidosis-mediated tumor progression and therapeutic resistance.

Interplay of Carbonic Anhydrase IX With Amino Acid and Acid/Base Transporters in the Hypoxic Tumor Microenvironment.

Solid tumors are challenged with a hypoxic and nutrient-deprived microenvironment. Hence, hypoxic tumor cells coordinatively increase the expression of nutrient transporters and pH regulators to adapt and meet their bioenergetic and biosynthetic demands. Carbonic Anhydrase IX (CAIX) is a membrane-bound enzyme that plays a vital role in pH regulation in the tumor microenvironment (TME). Numerous studies have established the importance of CAIX in mediating tumor progression and metastasis. To understand the mechanism of CAIX in mediating tumor progression, we performed an unbiased proteomic screen to identify the potential interactors of CAIX in the TME using the proximity-dependent biotin identification (BioID) technique. In this review, we focus on the interactors from this BioID screen that are crucial for nutrient and metabolite transport in the TME. We discuss the role of transport metabolon comprising CAIX and bicarbonate transporters in regulating intra- and extracellular pH of the tumor. We also discuss the role of amino acid transporters that are high confidence interactors of CAIX, in optimizing favorable metabolic state for tumor progression, and give our perspective on the coordinative interplay of CAIX with the amino acid transporters in the hypoxic TME.

Osteosarcoma progression is associated with increased nuclear levels and transcriptional activity of activated ß-Catenin.

Osteosarcoma (OS) is an aggressive primary bone malignancy that has peak incidence in children and young adults <25 years of age. Despite current multimodal treatments, no significant change in patient outcome has been observed in two decades. Presently, there is a lack of established, reliable baseline prognostic markers for aggressive OS, other than extent and site of disease involvement. The canonical Wnt/ß-catenin pathway controls multiple cellular processes, and is known to be a critical pathway in OS progression. This pathway regulates cellular levels of ß-catenin, which is a significant player in the oncogenesis and progression of many cancers. We investigated the relationship between ß-catenin, more specifically, the transcriptionally active form of ß-catenin, Activated ß-Catenin (ABC), and OS progression. Using an in vitro model, we observed that cellular/nuclear ABC levels, but not cellular/nuclear ß-catenin levels, increase with the degree of aggressiveness in OS. Our results demonstrate a strong association between nuclear-ABC levels and aggressive OS in vitro. Furthermore, we observed significant correlation between positive nuclear-ABC and patient age and tumor stage. Our results support the potential use of ABC as a predictive marker for risk stratification in OS.

Targeting Hypoxia-Induced Carbonic Anhydrase IX Enhances Immune-Checkpoint Blockade Locally and Systemically.

Treatment strategies involving immune-checkpoint blockade (ICB) have significantly improved survival for a subset of patients across a broad spectrum of advanced solid cancers. Despite this, considerable room for improving response rates remains. The tumor microenvironment (TME) is a hurdle to immune function, as the altered metabolism-related acidic microenvironment of solid tumors decreases immune activity. Here, we determined that expression of the hypoxia-induced, cell-surface pH regulatory enzyme carbonic anhydrase IX (CAIX) is associated with worse overall survival in a cohort of 449 patients with melanoma. We found that targeting CAIX with the small-molecule SLC-0111 reduced glycolytic metabolism of tumor cells and extracellular acidification, resulting in increased immune cell killing. SLC-0111 treatment in combination with immune-checkpoint inhibitors led to the sensitization of tumors to ICB, which led to an enhanced Th1 response, decreased tumor growth, and reduced metastasis. We identified that increased expression of CA9 is associated with a reduced Th1 response in metastatic melanoma and basal-like breast cancer TCGA cohorts. These data suggest that targeting CAIX in the TME in combination with ICB is a potential therapeutic strategy for enhancing response and survival in patients with hypoxic solid malignancies.

Regulation of pH by Carbonic Anhydrase 9 Mediates Survival of Pancreatic Cancer Cells With Activated KRAS in Response to Hypoxia.

BACKGROUND & AIMS: Most pancreatic ductal adenocarcinomas (PDACs) express an activated form of KRAS, become hypoxic and dysplastic, and are refractory to chemo and radiation therapies. To survive in the hypoxic environment, PDAC cells upregulate enzymes and transporters involved in pH regulation, including the extracellular facing carbonic anhydrase 9 (CA9). We evaluated the effect of blocking CA9, in combination with administration of gemcitabine, in mouse models of pancreatic cancer. METHODS: We knocked down expression of KRAS in human (PK-8 and PK-1) PDAC cells with small hairpin RNAs. Human and mouse (KrasG12D/Pdx1-Cre/Tp53/RosaYFP) PDAC cells were incubated with inhibitors of MEK (trametinib) or extracellular signal-regulated kinase (ERK), and some cells were cultured under hypoxic conditions. We measured levels and stability of the hypoxia-inducible factor 1 subunit alpha (HIF1A), endothelial PAS domain 1 protein (EPAS1, also called HIF2A), CA9, solute carrier family 16 member 4 (SLC16A4, also called MCT4), and SLC2A1 (also called GLUT1) by immunoblot analyses. We analyzed intracellular pH (pHi) and extracellular metabolic flux. We knocked down expression of CA9 in PDAC cells, or inhibited CA9 with SLC-0111, incubated them with gemcitabine, and assessed pHi, metabolic flux, and cytotoxicity under normoxic and hypoxic conditions. Cells were also injected into either immune-compromised or immune-competent mice and growth of xenograft tumors was assessed. Tumor fragments derived from patients with PDAC were surgically ligated to the pancreas of mice and the growth of tumors was assessed. We performed tissue microarray analyses of 205 human PDAC samples to measure levels of CA9 and associated expression of genes that regulate hypoxia with outcomes of patients using the Cancer Genome Atlas database. RESULTS: Under hypoxic conditions, PDAC cells had increased levels of HIF1A and HIF2A, upregulated expression of CA9, and activated glycolysis. Knockdown of KRAS in PDAC cells, or incubation with trametinib, reduced the posttranscriptional stabilization of HIF1A and HIF2A, upregulation of CA9, pHi, and glycolysis in response to hypoxia. CA9 was expressed by 66% of PDAC samples analyzed; high expression of genes associated with metabolic adaptation to hypoxia, including CA9, correlated with significantly reduced survival times of patients. Knockdown or pharmacologic inhibition of CA9 in PDAC cells significantly reduced pHi in cells under hypoxic conditions, decreased gemcitabine-induced glycolysis, and increased their sensitivity to gemcitabine. PDAC cells with knockdown of CA9 formed smaller xenograft tumors in mice, and injection of gemcitabine inhibited tumor growth and significantly increased survival times of mice. In mice with xenograft tumors grown from human PDAC cells, oral administration of SLC-0111 and injection of gemcitabine increased intratumor acidosis and increased cell death. These tumors, and tumors grown from PDAC patient-derived tumor fragments, grew more slowly than xenograft tumors in mice given control agents, resulting in longer survival times. In KrasG12D/Pdx1-Cre/Tp53/RosaYFP genetically modified mice, oral administration of SLC-0111 and injection of gemcitabine reduced numbers of B cells in tumors. CONCLUSIONS: In response to hypoxia, PDAC cells that express activated KRAS increase expression of CA9, via stabilization of HIF1A and HIF2A, to regulate pH and glycolysis. Disruption of this pathway slows growth of PDAC xenograft tumors in mice and might be developed for treatment of pancreatic cancer.

Active ß-catenin is regulated by the PTEN/PI3 kinase pathway: a role for protein phosphatase PP2A.

Dysregulation of Wnt/ß-catenin signaling has been associated with the development and progression of many cancers. The stability and subcellular localization of ß-catenin, a dual functional protein that plays a role in intracellular adhesion and in regulating gene expression, is tightly regulated. However, little is known about the transcriptionally active form of ß-catenin, Active Beta Catenin (ABC), that is unphosphorylated at serine 37 (Ser37) and threonine 41 (Thr41). Elucidating the mechanism by which ß-catenin is activated to generate ABC is vital to the development of therapeutic strategies to block ß-catenin signaling for cancer treatment. Using melanoma, breast and prostate cancer cell lines, we show that while cellular ß-catenin levels are regulated by the Wnt pathway, cellular ABC levels are mainly regulated by the PI3K pathway and are dependent on the phosphatase activity of the protein phosphatase PP2A. Furthermore, we demonstrate that although the PI3K/PTEN pathway does not regulate total ß-catenin protein levels within the cell, it plays a role in regulating the subcellular localization of ß-catenin. Our results support a novel functional interaction/cross-talk between the PTEN/PI3K and Wnt pathways in the regulation of the subcellular/nuclear levels of ABC, which is crucially important for the protein's activity as a transcription factor and its biological effects in health and disease.

ß-catenin is O-GlcNAc glycosylated at Serine 23: implications for ß-catenin's subcellular localization and transactivator function.

BACKGROUND: We have previously reported that ß-catenin is post-translationally modified with a single O-linked attachment of ß-N-acetyl-glucosamine (O-GlcNAc). We showed that O-GlcNAc regulated ß-catenin's subcellular localization and transcriptional activity. OBJECTIVE: The objectives of this investigation were to identify the putative O-GlcNAc sites of ß-catenin and the relevance of identified sites in the regulation of ß-catenin's localization and transcriptional activity. METHOD: Missense mutations were introduced to potential O-GlcNAc sites of pEGFP-C2-N-Terminal- or pEGFP-C2-Wild Type-ß-catenin by site-directed mutagenesis. We determined the levels of O-GlcNAc-ß-catenin, subcellular localization, interaction with binding partners and transcriptional activity of the various constructs. RESULTS: Serine 23 of ß-catenin was determined as a site for O-GlcNAc modification which regulated its subcellular distribution, its interactions with cellular partners and consequently its transcriptional activity. SIGNIFICANCE: O-GlcNAcylation of Serine 23 is a novel regulatory modification for ß-catenin's subcellular localization and transcriptional activity. This study is the first report to characterize site specific regulation of ß-catenin by the O-GlcNAc modification.