Increasing evidence indicates that several types of solid tumor are hierarchically organized and sustained by a distinct population of cancer stem cells (CSCs). CSCs possess enhanced mechanisms of protection from stress induced by reactive oxygen species (ROS) that render them resistant to chemo- and radiotherapy. Expression of CD44, especially variant isoforms (CD44v) of this major CSC marker, contributes to ROS defense through upregulation of the synthesis of reduced glutathione (GSH), the primary intracellular antioxidant. CD44v interacts with and stabilizes xCT, a subunit of the cystine-glutamate transporter xc(-), and thereby promotes cystine uptake for GSH synthesis. Given that cancer cells are often exposed to high levels of ROS during tumor progression, the ability to avoid the consequences of such exposure is required for cancer cell survival and propagation in vivo. CSCs, in which defense against ROS is enhanced by CD44v are thus thought to drive tumor growth, chemoresistance and metastasis. Therapy targeted to the CD44v-xCT system may therefore impair the ROS defense ability of CSCs and thereby sensitize them to currently available treatments.

Extracellular free amino acids contribute to the interaction between a tumor and its microenvironment through effects on cellular metabolism and malignant behavior. System xc(−) is composed of xCT and CD98hc subunits and functions as a plasma membrane antiporter for the uptake of extracellular cystine in exchange for intracellular glutamate. Here we show that the epidermal growth factor receptor (EGFR) interacts with xCT and thereby promotes its cell surface expression and function in human glioma cells. EGFR-expressing glioma cells manifested both enhanced antioxidant capacity as a result of increased cystine uptake as well as increased glutamate which promotes matrix invasion. Imaging mass spectrometry also revealed that brain tumors formed in mice by human glioma cells stably overexpressing EGFR contained higher levels of reduced glutathione compared with those formed by parental cells. Targeted inhibition of xCT suppressed the EGFR-dependent enhancement of antioxidant capacity in glioma cells as well as tumor growth and invasiveness. Our findings establish a new functional role for EGFR in promoting the malignant potential of glioma cells through interaction with xCT at the cell surface.

Expression of CD44, especially the variant isoforms (CD44v) of this major cancer stem cell marker, contributes to reactive oxygen species (ROS) defense through stabilizing xCT (a cystine–glutamate transporter) and promoting glutathione synthesis. This enhances cancer development and increases chemotherapy resistance. We investigate the role of CD44v in the regulation of the ROS defense system in cholangiocarcinoma (CCA). Immunohistochemical staining of CD44v and p38MAPK (a major ROS target) expression in Opisthorchis viverrini‐induced hamster CCA tissues (at 60, 90, 120, and 180 days) reveals a decreased phospho‐p38MAPK signal, whereas the CD44v signal was increased during bile duct transformation. Patients with CCA showed CD44v overexpression and negative‐phospho‐p38MAPK patients a significantly shorter survival rate than the low CD44v signal and positive‐phospho‐p38MAPK patients (P = 0.030). Knockdown of CD44 showed that xCT and glutathione levels were decreased, leading to a high level of ROS. We examined xCT‐targeted CD44v cancer stem cell therapy using sulfasalazine. Glutathione decreased and ROS increased after the treatment, leading to inhibition of cell proliferation and induction of cell death. Thus, the accumulation of CD44v leads to the suppression of p38MAPK in transforming bile duct cells. The redox status regulation of CCA cells depends on the expression of CD44v to contribute the xCT function and is a link to the poor prognosis of patients. Thus, an xCT inhibitor could inhibit cell growth and activate cell death. This suggests that an xCT‐targeting drug may improve CCA therapy by sensitization to the available drug (e.g. gemcitabine) by blocking the mechanism of the cell's ROS defensive system.

The prognostic role of CD 44v9, a variant isoform of CD 44 and a new cell surface marker of cancer stem cells, remains unclear in bladder cancer ( BC ) patients. Furthermore, limited information is available on the functional role of sulfasalazine ( SSZ ), which could modulate the CD 44v9‐ xCT system in order to enhance cisplatin ( CDDP )‐induced cytotoxicity and inhibit the metastatic potential of BC . CD 44v9 protein expression was examined immunohistochemically in 63 muscle invasive BC ( MIBC ) patients who underwent radical cystectomy. CD 44v9 expression was independently associated with disease recurrence and cancer‐specific death in MIBC . Cytotoxic effects, glutathione levels, and reactive oxygen species production by SSZ and CD 44v9 and phospho‐p38 MAPK protein expression by SSZ with or without CDDP were assessed in MBT ‐2V cells with highly metastatic potential. Sulfasalazine exerted cytotoxic effects against MBT ‐2V cells by inhibiting glutathione levels and inducing the production of reactive oxygen species. Sulfasalazine in combination with CDDP appeared to exert strong cytotoxic effects against MBT ‐2V cells by inhibiting CD 44v9 expression and upregulating phospho‐p38 MAPK expression. The inhibitory effects of SSZ with or without CDDP were also investigated using an MBT ‐2V lung metastatic model. In the murine lung metastatic BC model, SSZ significantly prolonged animal survival. Furthermore, the combination of SSZ with CDDP exerted stronger inhibitory effects on the establishment of lung tumor nodules than SSZ or CDDP alone. CD 44v9 expression could be a clinical biomarker for predicting poor outcomes in MIBC patients. Sulfasalazine in combination with CDDP has potential as a novel therapy against metastatic BC .

The cystine-glutamate antiporter subunit xCT suppresses iron-dependent oxidative cell death (ferroptosis) and is therefore a promising target for cancer treatment. Given that cancer cells often show resistance to xCT inhibition resulting in glutathione (GSH) deficiency, however, we here performed a synthetic lethal screen of a drug library to identify agents that sensitize the GSH deficiency-resistant cancer cells to the xCT inhibitor sulfasalazine. This screen identified the oral anesthetic dyclonine which has been recently reported to act as a covalent inhibitor for aldehyde dehydrogenases (ALDHs). Treatment with dyclonine induced intracellular accumulation of the toxic aldehyde 4-hydroxynonenal in a cooperative manner with sulfasalazine. Sulfasalazine-resistant head and neck squamous cell carcinoma (HNSCC) cells were found to highly express ALDH3A1 and knockdown of ALDH3A1 rendered these cells sensitive to sulfasalazine. The combination of dyclonine and sulfasalazine cooperatively suppressed the growth of highly ALDH3A1-expressing HNSCC or gastric tumors that were resistant to sulfasalazine monotherapy. Our findings establish a rationale for application of dyclonine as a sensitizer to xCT-targeted cancer therapy.

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Targeting the function of membrane transporters in cancer stemlike cells is a potential new therapeutic approach. Cystine‐glutamate antiporter xCT expressed in CD44 variant (CD44v)‐expressing cancer cells contributes to the resistance to oxidative stress as well as cancer therapy through promoting glutathione (GSH)‐mediated antioxidant defense. Amino acid transport by xCT might, thus, be a promising target for cancer treatment, whereas the determination factors for cancer cell sensitivity to xCT‐targeted therapy remain unclear. Here, we demonstrate that high expression of xCT and glutamine transporter ASCT2 is correlated with undifferentiated status and diminished along with cell differentiation in head and neck squamous cell carcinoma (HNSCC). The cytotoxicity of the xCT inhibitor sulfasalazine relies on ASCT2‐dependent glutamine uptake and glutamate dehydrogenase (GLUD)‐mediated α‐ketoglutarate (α‐KG) production. Metabolome analysis revealed that sulfasalazine treatment triggers the increase of glutamate‐derived tricarboxylic acid cycle intermediate α‐KG, in addition to the decrease of cysteine and GSH content. Furthermore, ablation of GLUD markedly reduced the sulfasalazine cytotoxicity in CD44v‐expressing stemlike HNSCC cells. Thus, xCT inhibition by sulfasalazine leads to the impairment of GSH synthesis and enhancement of mitochondrial metabolism, leading to reactive oxygen species (ROS) generation and, thereby, triggers oxidative damage. Our findings establish a rationale for the use of glutamine metabolism (glutaminolysis)‐related genes, including ASCT2 and GLUD, as biomarkers to predict the efficacy of xCT‐targeted therapy for heterogeneous HNSCC tumors.

l‐Type amino acid transporter 1 (LAT1) disulfide linked to CD98 heavy chain (hc) is highly expressed in most cancer cells, but weakly expressed in normal cells. In the present study, we developed novel anti‐LAT1 mAbs and showed internalization activity, inhibitory effects of amino acid uptake and cell growth and antibody‐dependent cellular cytotoxicity, as well as in vivo antitumor effects in athymic mice. Furthermore, we examined the reactivity of mAbs with LAT1 of Macaca fascicularis to evaluate possible side‐effects of antihuman LAT1 mAbs in clinical trials. Antihuman LAT1 mAbs reacted with ACHN human and MK.P3 macaca kidney‐derived cells, and this reactivity was significantly decreased by siRNAs against LAT1. Macaca LAT1 cDNA was cloned from MK.P3, and only two amino acid differences between human and macaca LAT1 were seen. RH7777 rat hepatoma and HEK293 human embryonic kidney cells expressing macaca LAT1 were established as stable transfectants, and antihuman LAT1 mAbs were equivalently reactive against transfectants expressing human or macaca LAT1. Dual (high and low) avidity modes were detected in transfectants expressing macaca LAT1, MK.P3, ACHN and HCT116 human colon cancer cells, and KA values were increased by anti‐CD98hc mAb, suggesting anti‐LAT1 mAbs detect an epitope on LAT1‐CD98hc complexes on the cell surface. Based on these results, LAT1 may be a promising anticancer target and Macaca fascicularis can be used in preclinical studies with antihuman LAT1 mAbs.