永野 修

Over 50% of patients with high-grade serous carcinoma (HGSC) are homologous recombination proficient, making them refractory to platinum-based drugs and poly (ADP-ribose) polymerase (PARP) inhibitors. These patients often develop progressive resistance within 6 months after primary treatment and tend to die early, thus new therapies are urgently needed. In this study, we comprehensively investigated this tumor type by leveraging a combination of machine learning analysis of a large published dataset and newly developed genetically engineered HGSC organoid models from murine fallopian tubes. Aberrant activation of RAS/PI3K signaling was a signature of poor prognosis in BRCA1/2 wild-type ovarian cancer, and mTOR-induced elevated p62 expression was a robust marker of chemotherapy-induced mTOR-p62-NRF2 signal activation. mTOR inhibition with everolimus decreased p62 and enhanced sensitivity to conventional chemotherapy, indicating that p62 serves as an important biomarker for therapeutic intervention. Combination therapy with conventional chemotherapy and mTOR inhibitors is a promising therapeutic strategy for refractory HGSC, with p62 as a biomarker.

Developmental toxicity testing is essential to identify substances that may harm embryonic development. This study aimed to establish a protocol for evaluating developmental toxicity using human induced pluripotent stem cells (iPSCs) by analyzing cellular activity and gene expression changes. Two ICH S5(R3) positive substances, valproic acid (VPA), which is a substance previously detected as positive by other test methods, and thalidomide (Thalido), were examined during early trichoderm differentiation without fetal bovine serum. RNA-seq analysis identified seven candidate genes, including TP63, associated with altered expression following exposure to VPA or Thalido. These genes were implicated in pathways related to tissue development, cell growth, and molecular interactions. While the assay effectively detected VPA and Thalido, its limitations include testing only soluble substances and focusing on early differentiation stages. Nevertheless, the protocol demonstrates potential for the classification and evaluation of emerging modality drugs based on physical properties such as solubility, polarity, and pH. Integration with AI analysis may enhance its capacity to uncover genetic variations and evaluate previously uncharacterized substances. This study provides a foundation for alternative developmental toxicity testing methods, with further refinements in the culture method expected to improve accuracy and applicability in regulatory toxicology.

Cell cycle progression requires periodic gene expression through splicing control. However, the splicing factor that directly controls this cell cycle-dependent splicing remains unknown. Cell cycle-dependent expression of the AURKB (aurora kinase B) gene is essential for chromosome segregation and cytokinesis. We previously reported that RNPS1 is essential to maintain precise splicing in AURKB intron 5. Here we show that RNPS1 plays this role in PSAP complex with PNN and SAP18, but not ASAP complex with ACIN1 and SAP18. Whole-transcriptome sequencing of RNPS1- and PNN-deficient cells indicated that RNPS1, either alone or as PSAP complex, is an essential splicing factor for a subset of introns. Remarkably, protein expression of RNPS1, but not PNN, is coordinated with cyclical splicing in PSAP-controlled introns including AURKB intron 5. The ubiquitin-proteasome pathway is involved in the periodic decrease of RNPS1 protein level. RNPS1 is a key factor that controls periodic splicing during the cell cycle.

Diffuse-type gastric cancer (DGC) is a subtype of gastric cancer that is prone to peritoneal dissemination, with poor patient prognosis. Although intercellular adhesion loss between cancer cells is a major characteristic of DGCs, the mechanism underlying the alteration in cell-to-extracellular matrix (ECM) adhesion is unclear. We investigated how DGCs progress and cause peritoneal dissemination through interactions between DGC cells and the tumour microenvironment (TME). p53 knockout and KRASG12V-expressing (GAN-KP) cells and Cdh1-deleted GAN-KP (GAN-KPC) cells were orthotopically transplanted into the gastric wall to mimic peritoneal dissemination. The GAN-KPC tumour morphology was similar to that of human DGCs containing abundant stroma. RNA sequencing revealed that pathways related to Rho GTPases and integrin-ECM interactions were specifically increased in GAN-KPC cells compared with GAN-KP cells. Notably, we found that Rac Family Small GTPase 1 (RAC1) induces Integrin Subunit Alpha 6 (Itga6) trafficking, leading to its enrichment on the GC cell membrane. Fibroblasts activate the FAK/AKT pathway in GC cells by mediating extracellular matrix (ECM)-Itga6 interactions, exacerbating the malignant phenotype. In turn, GC cells induce abnormal expression of fibroblast collagen and its transformation into cancer-associated fibroblasts (CAFs), resulting in DGC-like subtypes. These findings indicate that Cdh1 gene loss leads to abnormal expression and changes in the subcellular localization of ITGA6 through RAC1 signalling. The latter, through interactions with CAFs, allows for peritoneal dissemination.

Abstract Refractory high-grade serous carcinoma (HGSC) is one of the most severe clinical problems in gynecology, but we have barely grasped the underlying mechanism of aggressiveness and chemo-resistance. Previous study has shown that homologous recombination genes, BRCA1/2-unmutated HGSC patients possess the potential to be in refractory condition, and their primary response to chemotherapy was heterogenous; thus, there can be subtype-specific determinant factors hatching refractoriness in BRCA1/2-unmutated HGSC patients. To explore the determinant factors, we performed gene set variation analysis and comprehensive log-rank tests on TCGA advanced serous ovarian cancer datasets. As a result, regulation of small GTPase signaling prominently correlated with poor prognosis uniquely in BRCA1/2-unmutated HGSC patients (p-value < 0.001), and eventually, pathway clustering analysis by variational Bayesian Gaussian mixture model discovered aberrant RAS/PI3K crosstalk as peculiar poor prognostic signature of BRCA1/2-unmutated HGSC patients. To elucidate the mechanism of poor progression triggered by RAS/PI3K, we utilized our syngeneic HGSC organoid models newly established from murine fallopian tube epithelium by making Myc overexpressing, and knocking out Trp53 and Rb1; as a supplement, the propriety of these genetic alterations to develop HGSC models was backed up by genetic profiles of high-grade serous ovarian cancer patients in AACR Project GENIE. After that, we knocked out Nf1 and Pten whose deletions were detected relatively with high frequency in HGSC to induce aberrant RAS/PI3K crosstalk in the primitive HGSC model. In-vivo evaluation and growth comparison indicated that aberrant RAS/PI3K crosstalk transformed Brca1/2-unmutated HGSC modeling cells into aggressive phenotypes such as poor survival (p-value < 0.01) and high growth rate (p-value < 0.001). Chemo-sensitivity assay also showed that the genetic manipulation made Brca1/2-unmutated HGSC modeling cells more tolerant to anti-tumor agents like carboplatin (p-value < 0.001), paclitaxel (p-value < 0.01), and olaparib (p-value < 0.01). To investigate biological processes that induce the aggressive and chemo-resistant phenotype, we performed gene set enrichment analysis among our HGSC models. The result showed that aberrant RAS/PI3K crosstalk induces cell cycle acceleration and downregulation of apoptosis in the HGSC cells, which backed up aggressive phenotype and chemo-resistance in our HGSC models. In conclusion, aberrant RAS/PI3K crosstalk transforms Brca1/2-unmutated HGSC modeling cells more aggressive and chemo-resistant, which was well recapitulated by our HGSC modeling organoids. The integration of our computational approach and experimental models will lead us to detect peculiar therapeutic targets against refractory HGSC. Citation Format: Tomohiro Tamura, Kenta Masuda, Shimpei Nagai, Keiyo Imaeda, Juntaro Yamasaki, Eiji Sugihara, Hiroyuki Nobusue, Yuji Otsuki, Rui Yamaguchi, Kazuhiro Sakurada, Wataru Yamagami, Hideyuki Saya, Osamu Nagano. Integration of human omics analysis and new syngeneic tumor organoid models reveals that aberrant RAS/PI3K crosstalk triggers refractoriness in high-grade serous carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1254.

Malignant ascites accompanied by peritoneal dissemination contain various factors and cell populations as well as cancer cells; however, how the tumor microenvironment is shaped in ascites remains unclear. Single-cell proteomic profiling and a comprehensive proteomic analysis are conducted to comprehensively characterize malignant ascites. Here, we find defects in immune effectors along with immunosuppressive cell accumulation in ascites of patients with gastric cancer (GC) and identify five distinct subpopulations of CD45(-)/EpCAM(-) cells. Mesothelial cells with mesenchymal features in CD45(-)/EpCAM(-) cells are the predominant source of chemokines involved in immunosuppressive myeloid cell (IMC) recruitment. Moreover, mesothelial-mesenchymal transition (MMT)-induced mesothelial cells strongly express extracellular matrix (ECM)-related genes, including tenascin-C (TNC), enhancing metastatic colonization. These findings highlight the definite roles of the mesenchymal cell population in the development of a protumorigenic microenvironment to promote peritoneal dissemination.

Glycolysis is highly enhanced in Pancreatic ductal adenocarcinoma (PDAC) cells; thus, glucose restrictions are imposed on nontumor cells in the PDAC tumor microenvironment (TME). However, little is known about how such glucose competition alters metabolism and confers phenotypic changes in stromal cells in the TME. Here, we report that cancer-associated fibroblasts (CAFs) with restricted glucose availability utilize lactate from glycolysis-enhanced cancer cells as a fuel and exert immunosuppressive activity in the PDAC TME. The expression of lactate dehydrogenase A (LDHA), which regulates lactate production, was a poor prognostic factor for PDAC patients, and LDHA depletion suppressed tumor growth in a CAF-rich murine PDAC model. Coculture of CAFs with PDAC cells revealed that most of the glucose was taken up by the tumor cells and that CAFs consumed lactate via monocarboxylate transporter 1 to enhance proliferation through the TCA cycle. Moreover, lactate-stimulated CAFs upregulated IL6 expression and suppressed cytotoxic immune cell activity synergistically with lactate. Finally, the LDHA inhibitor FX11 reduced tumor growth and improved antitumor immunity in CAF-rich PDAC tumors. Our study provides new insights into crosstalk among tumor cells, CAFs, and immune cells mediated by lactate and offers therapeutic strategies for targeting LDHA enzymatic activity in PDAC cells.

OBJECTIVE: Many cancers engage embryonic genes for rapid growth and evading the immune system. SOX9 has been upregulated in many tumours, yet the role of SOX9 in mediating immunosuppressive tumour microenvironment is unclear. Here, we aim to dissect the role of SOX9-mediated cancer stemness attributes and immunosuppressive microenvironment in advanced gastric adenocarcinoma (GAC) for novel therapeutic discoveries. METHODS: Bulk RNAseq/scRNA-seq, patient-derived cells/models and extensive functional studies were used to identify the expression and functions of SOX9 and its target genes in vitro and in vivo. Immune responses were studied in PBMCs or CD45+ immune cells cocultured with tumour cells with SOX9high or knockout and the KP-Luc2 syngeneic models were used for efficacy of combinations. RESULTS: SOX9 is one of the most upregulated SOX genes in GAC and highly expressed in primary and metastatic tissues and associated with poor prognosis. Depletion of SOX9 in patient-derived GAC cells significantly decreased cancer stemness attributes, tumour formation and metastases and consistently increased CD8+ T cell responses when cocultured with PBMCs/CD45+ cells from GAC patients. RNA sequencing identified the leukaemia inhibitory factor (LIF) as the top secreted molecule regulated by SOX9 in tumour cells and was enriched in malignant ascites and mediated SOX9-induced M2 macrophage repolarisation and inhibited T cell function. CONCLUSION: Epithelial SOX9 is critical in suppressing CD8+ T cell responses and modified macrophage function in GAC through the paracrine LIF factor. Cotargeting LIF/LIFR and CSF1R has great potential in targeting SOX9-mediated cancer stemness, T cell immunosuppression and metastases suggesting the novel combination therapy against advanced GAC.

Epithelial-mesenchymal plasticity (EMP) refers to the reversible cellular transition between epithelial and mesenchymal status. Spontaneous EMP is also reported in breast and prostate cancer, leading to the acquisition of stem-cell properties and chemoresistance. However, the presence of spontaneous EMP is still not reported in esophageal cancer. We screened 11 esophageal squamous cancer cell (ESCC) cell lines by CD44 isoform expression. KYSE520 was found to comprise heterogenous populations consisting of CD44v+ and CD44v- subpopulations. CD44v+ and CD44v- cells showed the expression of epithelial and mesenchymal markers, respectively. Single-cell sorting of CD44v+ and CD44v- cells revealed both cells gave rise to cell populations consisting of CD44v+ and CD44v- cells, indicating CD44v+ epithelial-like and CD44v- mesenchymal-like cells can generate counterparts, respectively. The ablation of Epithelial splicing regulatory protein 1 (ESRP1), a major regulator of CD44 mRNA splicing, resulted in the shift from CD44v+ to CD44v- cells in KYSE520. However, the expression of epithelial-mesenchymal transition (EMT)-related markers or transcriptional factors were almost not affected, suggesting ESRP1 functions downstream of EMP. Our results revealed the presence of spontaneous EMP in esophageal cancer and KYSE520 is useful model to understand spontaneous EMP.

PURPOSE: Angiopoietin-like 4 (ANGPTL4) was recently shown to be associated with cancer progression but little is known about its contribution to cancer metabolism. The purpose of this study was to elucidate the role of ANGPTL4 in glucose metabolism in colorectal cancer (CRC). METHODS: Immunohistochemical staining of CRC specimens classified 84 patients into two groups according to ANGPTL4 expression. Clinicopathological characteristics, gene mutation status obtained by next-generation sequencing, and fluorodeoxyglucose (FDG) uptake measured by positron emission tomography/computed tomography (PET/CT) were compared between the two groups. Furthermore, the impact of ANGPTL4 expression on cancer metabolism was investigated by a subcutaneous xenograft mouse model using the ANGPTL4 knockout CRC cell line, and glucose transporter (GLUT) expression was evaluated. RESULTS: There were significantly more cases of T3/4 tumours (94.3% vs. 57.1%, P < 0.001) and perineural invasion (42.9% vs. 22.4%, P = 0.046) in the ANGPTL4-high group than in the low group. Genetic exploration revealed a higher frequency of KRAS mutation (54.3% vs. 22.4%, P = 0.003) in the ANGPTL4-high tumours. All the FDG uptake parameters were significantly higher in ANGPTL4-high tumours. In vivo analysis showed a significant reduction in tumour size due to ANGPTL4 knockout with lower expression of GLUT1 and GLUT3, and suppression of AKT phosphorylation. CONCLUSION: ANGPTL4 regulates the expression of GLUTs by activating the PI3K-AKT pathway and thereby promoting glucose metabolism in CRC. These findings establish a new functional role of ANGPTL4 in cancer progression and lay the foundation for developing a novel therapeutic target.

Metastatic progression of tumors is driven by genetic alterations and tumor-stroma interaction. To elucidate the mechanism underlying the oncogene-induced gastric tumor progression, we have developed an organoid-based model of gastric cancer from GAstric Neoplasia (GAN) mice, which express Wnt1 and the enzymes COX2 and microsomal prostaglandin E synthase 1 in the stomach. Both p53 knockout (GAN-p53KO) organoids and KRASG12V -expressing GAN-p53KO (GAN-KP) organoids were generated by genetic manipulation of GAN mouse-derived tumor (GAN wild-type [WT]) organoids. In contrast with GAN-WT and GAN-p53KO organoids, which manifested Wnt addiction, GAN-KP organoids showed a Wnt-independent phenotype and the ability to proliferate without formation of a Wnt-regulated three-dimensional epithelial architecture. After transplantation in syngeneic mouse stomach, GAN-p53KO cells formed only small tumors, whereas GAN-KP cells gave rise to invasive tumors associated with the development of hypoxia as well as to liver metastasis. Spatial transcriptomics analysis suggested that hypoxia signaling contributes to the metastatic progression of GAN-KP tumors. In particular, such analysis identified a cluster of stromal cells located at the tumor invasive front that expressed genes related to hypoxia signaling, angiogenesis, and cell migration. These cells were also positive for phosphorylated extracellular signal-regulated kinase (ERK), suggesting that mitogen-activated protein kinase (MAPK) signaling promotes development of both tumor and microenvironment. The MEK (MAPK kinase) inhibitor trametinib suppressed the development of GAN-KP gastric tumors, formation of a hypoxic microenvironment, tumor angiogenesis, and liver metastasis. Our findings therefore establish a rationale for application of trametinib to suppress metastatic progression of KRAS-mutated gastric cancer.

Fibroblast activation protein (FAP) generally shows low or undetectable expression in most normal tissues but is highly expressed in fibroblasts in almost all carcinomas. FAP is one of the potential molecules to detect activated fibroblasts and has multiple roles in tumour progression. We generated transgenic mice that specifically expressed tdTomato along with FAP promoter activity. Coculturing a mouse gastric cancer cell line and FAP-tdTomato transgenic mouse-derived fibroblasts showed that tdTomato expression was elevated in the cocultured fibroblasts. Moreover, stomach wall transplanted tumours in mice also showed FAP-tdTomato expression in fibroblasts of the stomach and each metastatic legion. These results indicated that FAP-tdTomato expression in fibroblasts was elevated by stimulation through the interaction with cancer cells. Functionally, collagen production was increased in FAP/tdTomato-positive fibroblasts cocultured with mouse cancer cells. These FAP-tdTomato transgenic mice have the potential to be used to investigate real-time FAP dynamics and the importance of FAP expression in tumour development.

The major cellular antioxidant glutathione (GSH) protects cancer cells from oxidative damage that can lead to the induction of ferroptosis, an iron-dependent form of cell death triggered by the aberrant accumulation of lipid peroxides. Inhibitors of the cystine-glutamate antiporter subunit xCT, which mediates the uptake of extracellular cystine and thereby promotes GSH synthesis, are thus potential anticancer agents. However, the efficacy of xCT-targeted therapy has been found to be diminished by metabolic reprogramming that affects redox status in cancer cells. Identification of drugs for combination with xCT inhibitors that are able to overcome resistance to xCT-targeted therapy might thus provide the basis for effective cancer treatment. We have now identified the vasodilator oxyfedrine (OXY) as a sensitizer of cancer cells to GSH-depleting agents including the xCT inhibitor sulfasalazine (SSZ). Oxyfedrine contains a structural motif required for covalent inhibition of aldehyde dehydrogenase (ALDH) enzymes, and combined treatment with OXY and SSZ was found to induce accumulation of the cytotoxic aldehyde 4-hydroxynonenal and cell death in SSZ-resistant cancer cells both in vitro and in vivo. Microarray analysis of tumor xenograft tissue showed cyclooxygenase-2 expression as a potential biomarker for the efficacy of such combination therapy. Furthermore, OXY-mediated ALDH inhibition was found to sensitize cancer cells to GSH depletion induced by radiation therapy in vitro. Our findings thus establish a rationale for repurposing of OXY as a sensitizing drug for cancer treatment with agents that induce GSH depletion.

© 2019 American Association for Cancer Research. Molecular characterization of lung squamous cell carcinoma (LUSC), one of the major subtypes of lung cancer, has not sufficiently improved its nonstratified treatment strategies over decades. Accumulating evidence suggests that lineage-specific transcriptional regulators control differentiation states during cancer evolution and underlie their distinct biological behaviors. In this study, by investigating the super-enhancer landscape of LUSC, we identified a previously undescribed "neural" subtype defined by Sox2 and a neural lineage factor Brn2, as well as the classical LUSC subtype defined by Sox2 and its classical squamous partner p63. Robust protein–protein interaction and genomic cooccupancy of Sox2 and Brn2, in place for p63 in the classical LUSC, indicated their transcriptional cooperation imparting this unique lineage state in the "neural" LUSC. Forced expression of p63 downregulated Brn2 in the "neural" LUSC cells and invoked the classical LUSC lineage with more squamous/ epithelial features, which were accompanied by increased activities of ErbB/Akt and MAPK–ERK pathways, suggesting differential dependency. Collectively, our data demonstrate heterogeneous cell lineage states of LUSC featured by Sox2 cooperation with Brn2 or p63, for which distinct therapeutic approaches may be warranted. Significance: Epigenomic profiling reveals a novel subtype of lung squamous cell carcinoma with neural differentiation.

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.

Nanomedicine modification with ligands directed to receptors on tumor blood vessels has the potential for selectively enhancing nanomedicine accumulation in malignant tissues by overcoming the vascular barrier of tumors. Nevertheless, the development of broadly applicable ligand approaches capable of promoting the transvascular transport of nanomedicines in a wide spectrum of tumors has been elusive so far. By considering the indispensable and persistent glycolytic fueling of tumors, we developed glucose-installed polymeric micelles loading cisplatin (Gluc-CDDP/m) targeting the glucose transporter 1 (GLUT1), which is overexpressed in most tumors and present on vascular endothelial cells, toward improving the delivery efficiency and therapeutic efficacy. The design of the glucose ligands on Gluc-CDDP/m was engineered to control the conjugation via the carbon 6 of the glucose moieties, as well as the ligand density on the poly (ethylene glycol) (PEG) shell of the micelles. The series of micelles was then studied in vitro and in vivo against GLUT1-high human squamous cell carcinoma of the head and neck OSC-19 cells and GLUT1-low human glioblastoma-astrocytoma U87MG cells. Our results showed that precisely tuning the micelles to have glucose ligands on 25% of their PEG chains increased the efficacy against the tumors by significantly enhancing the tumor accumulation, even in GLUT1-low U87MG tumors. The enhancement of the intratumoral levels of these micelles was hindered by concomitant administration of glucose, or the GLUT1 inhibitor STF-31, confirming a GLUT1/glucose-mediated increment of the accumulation. Intravital confocal laser scanning microscopy imaging of tumor tissues further demonstrated the rapid extravasation and penetration of Gluc-CDDP/m in OSC-19 tumors compared to non-targeted CDDP/m. These findings indicate GLUT1-targeting as a promising approach for overcoming the vascular barrier and boosting the delivery of nanomedicine in tumors.

© 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association. Autocrine and paracrine factors, including glutamate and epidermal growth factor (EGF), are potent inducers of brain tumor cell invasion, a pathological hallmark of malignant gliomas. System xc(–) consists of xCT and CD98hc subunits and functions as a plasma membrane antiporter for the uptake of extracellular cystine in exchange for intracellular glutamate. We previously showed that the EGF receptor (EGFR) interacts with xCT and thereby promotes the activity of system xc(–) in a kinase-independent manner, resulting in enhanced glutamate release in glioma cells. However, the molecular mechanism underlying EGFR-mediated glioma progression in a glutamate-rich microenvironment has remained unclear. Here we show that the GluN2B subunit of the N-methyl-d-aspartate–sensitive glutamate receptor (NMDAR) is a substrate of EGFR in glioma cells. In response to EGF stimulation, EGFR phosphorylated the COOH-terminal domain of GluN2B and thereby enhanced glutamate-NMDAR signaling and consequent cell migration in EGFR-overexpressing glioma cells. Treatment with the NMDAR inhibitor MK-801 or the system xc(–) inhibitor sulfasalazine suppressed EGF-elicited glioma cell migration. The administration of sulfasalazine and MK-801 also synergistically suppressed the growth of subcutaneous tumors formed by EGFR-overexpressing glioma cells. Furthermore, shRNA-mediated knockdown of xCT and GluN2B cooperatively prolonged the survival of mice injected intracerebrally with such glioma cells. Our findings thus establish a central role for EGFR in the signaling crosstalk between xCT and GluN2B-containing NMDAR in glioma cells.

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.

© 2018 The Author(s). Gold deposition with diagonal angle towards boehmite-based nanostructure creates random arrays of horse-bean-shaped nanostructures named gold-nanofève (GNF). GNF generates many electromagnetic hotspots as surface-enhanced Raman spectroscopy (SERS) excitation sources, and enables large-area visualization of molecular vibration fingerprints of metabolites in human cancer xenografts in livers of immunodeficient mice with sufficient sensitivity and uniformity. Differential screening of GNF-SERS signals in tumours and those in parenchyma demarcated tumour boundaries in liver tissues. Furthermore, GNF-SERS combined with quantum chemical calculation identified cysteine-derived glutathione and hypotaurine (HT) as tumour-dominant and parenchyma-dominant metabolites, respectively. CD44 knockdown in cancer diminished glutathione, but not HT in tumours. Mechanisms whereby tumours sustained HT under CD44-knockdown conditions include upregulation of PHGDH, PSAT1 and PSPH that drove glycolysis-dependent activation of serine/glycine-cleavage systems to provide one-methyl group for HT synthesis. HT was rapidly converted into taurine in cancer cells, suggesting that HT is a robust anti-oxidant for their survival under glutathione-suppressed conditions.

The low turnover rate of thyroid follicular cells and the lack of a long-term thyroid cell culture system have hampered studies of thyroid carcinogenesis. We have now established a thyroid organoid culture system that supports thyroid cell proliferation in vitro. The established mouse thyroid organoids performed thyroid functions including thyroglobulin synthesis, iodide uptake, and the production and release of thyroid hormone. Furthermore, transplantation of the organoids into recipient mice resulted in the formation of normal thyroid–like tissue capable of iodide uptake and thyroglobulin production in vivo. Finally, forced expression of oncogenic NRAS (NRASQ61R) in thyroid organoids established from p53 knockout mice and transplantation of the manipulated organoids into mouse recipients generated a model of poorly differentiated thyroid cancer. Our findings suggest that this newly developed thyroid organoid culture system is a potential research tool for the study of thyroid physiology and pathology including thyroid cancer.

A previous dose-escalation study of sulfasalazine (SSZ), an inhibitor of cystine-glutamate exchange transporter xc (-), in the variant form of CD44 (CD44v)-positive cancer stem cells (CSCs) suggested that administration of SSZ induces the reduction of CD44v-positive cells and intracellular reduced glutathione (GSH) levels in patients with advanced gastric cancer (AGC). Here we report a study to evaluate SSZ in combination with cisplatin in patients with CD44v-expressing AGC refractory to cisplatin. SSZ was given by oral administration four times daily with 2 weeks on and 1 week off. Cisplatin at 60 mg/m(2) was administered every 3 weeks. Of the 15 patients who underwent prescreening of CD44v expression, 8 patients were positive, and 7 patients were treated with the dose level of SSZ at 6 g/day. One patient experienced dose-limiting toxicity (DLT) as grade 3 anorexia. Although no other patients experienced DLT, 4 patients required dose interruption or reduction of SSZ; thus, we terminated further dose escalation. No patient achieved objective response, but 1 patient completed six cycles with stable disease for more than 4 months as well as reduction of intratumoral GSH level. The combination of SSZ plus cisplatin was manageable, although dose modification was frequently required during a short observational period.

&lt;p&gt;Objectives The regulation of redox balance in cancer cells is an important factor in tumor development and chemoresistance, with oncogene activation having been shown to induce the generation of reactive oxygen species (ROS). Activating mutations of the epidermal growth factor receptor gene (EGFR) are oncogenic drivers in non–small cell lung cancer (NSCLC), but it has remained unknown whether ligand-independent EGFR signaling conferred by EGFR mutation triggers ROS generation in NSCLC cells. Materials and Methods HEK293T cells were transfected with an expression vector for mutant EGFR. The expression of CD44 variant (CD44v) isoforms in NSCLC cell lines was evaluated by flow cytometry. Cells were depleted of CD44v by RNA interference and assayed for ROS and glutathione (GSH) levels. The effect of CD44v on cisplatin sensitivity was evaluated in vitro with the MTS assay. Results EGFR signaling due to EGFR mutation increased ROS levels in transfected HEK293T cells. The expression of CD44v isoforms was found to be inversely correlated with basal ROS levels in EGFR mutation–positive NSCLC cell lines. Knockdown of CD44v induced depletion of intracellular GSH and increased ROS level

Spliced variant isoforms of CD44 (CD44v) are a marker of cancer stem cells in solid tumors. They stabilize the xCT subunit of the transporter system xc(-) and thereby promote synthesis of the antioxidant glutathione. Salazosulfapyridine (SASP) is an inhibitor of xCT and suppresses the proliferation of CD44v-positive cancer cells. Chemotherapy-naive patients with advanced non-squamous non-small-cell lung cancer were enrolled in a dose-escalation study (standard 3 + 3 design) of SASP in combination with cisplatin and pemetrexed. The primary end-point was the percentage of patients who experience dose-limiting toxicity. Fifteen patients were enrolled in the study. Dose-limiting toxicity was observed in one of six patients at a SASP dose of 1.5 g/day (elevation of aspartate and alanine aminotransferase levels, each of grade 3), two of five patients at 3 g/day (hypotension or pneumonitis, each of grade 3), and two of three patients at 4.5 g/day (anorexia of grade 3). The maximum tolerated dose was thus 3 g/day, and the recommended dose was 1.5 g/day. The overall response rate was 26.7% and median progression-free survival was 11.7 months, much longer than that for cisplatin-pemetrexed alone in previous studies. Exposure to SASP varied markedly among individuals according to ABCG2 and NAT2 genotypes. The serum concentration of free CD44v protein was increased after the first cycle of treatment, possibly reflecting death of cancer stem cells. Salazosulfapyridine was thus given safely in combination with cisplatin-pemetrexed, with the addition of SASP tending to prolong progression-free survival. This trial is registered in the UMIN Clinical Trials Registry as UMIN000017854.

Recalcitrant head and neck squamous cell carcinoma (HNSCC) usually relapses after therapy due to the enrichment of drug resistant cancer stem-like cells (CSCs). Nanomedicines have shown potential for eradicating both cancer cells and CSCs by effective intratumoral navigation for reaching particular cell populations and controlling drug delivery. The installation of ligands on nanomedicines is an attractive approach for improving the delivery to CSCs within tumors, though the development of CSC-selective ligand-receptor systems has been challenging. Herein, we found that the CSC subpopulation in HNSCC cells overexpresses alpha(v)beta(5) integrins, which is preferentially expressed in tumor neovasculature and cancer cells, and can be effectively targeted by using cyclic Arg-Gly-Asp (cRGD) peptide. Thus, in this study, we propose installing cRGD peptide on micellar nanomedicines incorporating cisplatin for improving their activity against CSCs and enhancing survival. Both cisplatin-loaded micelles (CDDP/m) and cRGD-installed CDDP/m (cRGD-CDDP/m) were effective against HNSCC SAS-L1-Luc cells in vitro, though cRGD-installed CDDP/m was more potent than CDDP/m against the CSC fraction. In vivo, the cRGD-CDDP/m also showed significant antitumor activity against HNSCC orthotopic tumors, i.e. SAS-L1 and HSC-2. Moreover, cRGD-CDDP/m rapidly accumulated into the lymph node metastasis of SAS-L1 tumors, effectively inhibiting their growth, and prolonging mice survival. These findings indicate cRGD-installed nanomedicines as an advantageous strategy for targeting CSCs in HNSCC, and particularly, cRGD-CDDP/m as a significant therapeutic strategy against regionally advanced HNSCC.

Epithelial ovarian cancer is a highly lethal malignancy; moreover, overcoming chemoresistance is the major challenging in treating ovarian cancer patients. The cancer stem cell (CSC) hypothesis considers CSCs to be the main culprits in driving tumor initiation, metastasis, and resistance to conventional therapy. Although growing evidence suggest that CSCs are responsible for chemoresistance, the contribution of CSC marker EpCAM to resistance to chemotherapy remains unresolved. Here we have demonstrated that ovarian cancers containing high levels of EpCAM have a significantly much lower probability of achieving overall responsive rates after first-line chemotherapy. In addition, multivariate analysis revealed that EpCAM expression is an independent risk factor for chemoresistance, indicating that EpCAM expression is a predictive biomarker of chemotherapeutic response. Consistent with these clinical observations, in vitro assays, we found that the subpopulation of EpCAM-positive ovarian cancer cells shows a significantly higher viability compared with EpCAM-negative cells in response to cisplatin treatment by preventing chemotherapy-induced apoptosis, which is regulated by EpCAM-Bcl-2 axis. Furthermore, in an in vivo mouse model, platinum agents preferentially eliminated EpCAM-negative cells in comparison with EpCAM-positive cells, suggesting that the remaining subpopulation of EpCAM-positive cells contributes to tumor recurrence after chemotherapy. Finally, we also found that an increased expression of EpCAM is associated with poor prognosis in ovarian cancer patients. Our findings highlight the clinical significance of EpCAM in the resistance to chemotherapy and provide a rationale for EpCAM-targeted therapy to improve chemoresistance. Targeting EpCAM should be a promising approach to effectively extirpate the CSCs as the putative root of ovarian cancer.

Acute lymphoblastic leukemia (ALL) with mixed lineage leukemia (MLL) gene rearrangements (MLL+ALL) has a dismal prognosis and is characterized by high surface CD44 expression. Known that CD44 has the specific binding sites for a natural ligand hyaluronan ( HA), we investigated biological effects of HA with different molecular sizes on MLL+ALL cell lines, and found that the addition of ultra-low-molecular-weight (ULMW)-HA strongly suppressed their thymidine uptakes. The MLL+ALL cell line lacking surface CD44 expression established by genome editing showed no suppression of thymidine uptake. Surface CD44-high B-precursor ALL cell lines other than MLL+, but not T-ALL cell lines, were also suppressed in their thymidine uptakes. The inhibition of thymidine uptakes was because of induction of cell death, but dead cells lacked features of apoptosis on cytospin smears and flow cytometric analysis. The cell death was neither blocked by pan-caspase inhibitor nor autophagy inhibitor, but was completely blocked by necrosis inhibitor necrostatin-1. Necrotic cell death was further supported by a marked release of a high-mobility protein group B1 and morphological changes on transmission electron microscopy. Elevation of intracellular reactive oxygen species production suggested a role for inducing this necrotic cell death. ULMW-HA-triggered cell death was similarly demonstrated in surface CD44-high primary B-precursor leukemia cells. Assuming that ULMW-HA is abundantly secreted at the site of infection and inflammation, this study sheds light on understanding the mechanism of a transient inflammation-associated remission of leukemia. Further, the CD44-targeting may become an effective approach in future for the treatment of refractory B-precursor ALL by its capability of predominantly eradicating CD44-high leukemia-initiating cells.

Cancer stem cells (CSCs) have enhanced mechanisms of protection from oxidative stress. A variant form of CD44 (CD44v), a major CSC marker, was shown to interact with xCT, a subunit of cystine-glutamate transporter, which maintains high levels of intracellular reduced glutathione (GSH) which defend the cell against oxidative stress. Sulfasalazine (SSZ) is an inhibitor of xCT and was shown to suppress the survival of CD44v-positive stem-like cancer cells both in vitro and in vivo. To find the dose of SSZ which can safely reduce the population of CD44v-positive cells in tumors, a dose-escalation study in patients with advanced gastric cancer was conducted. SSZ was given four times daily by oral administration with 2 weeks as one cycle. Tumor biopsies were obtained before and after 14 days of administration of SSZ to evaluate expression of CD44v and the intratumoral level of GSH. Eleven patients were enrolled and received a dosage from 8 to 12 g/day. Safety was confirmed up to a dosage of 12 g/day, which was considered the maximum tolerated dose. Among the eight patients with CD44v-positive cells in their pretreatment biopsy samples, the CD44v-positive cancer cell population appeared to be reduced in the posttreatment biopsy tissues of four patients. Intratumoral GSH levels were also decreased in two patients, suggesting biological effectiveness of SSZ at 8 g/day or greater. This is the first study of SSZ as an xCT inhibitor for targeting CSCs. Reduction of the levels of CD44v-positive cells and GSH was observed in some patients, consistent with the mode of action of SSZ in CSCs.

Background Autoimmune gastritis (AIG) and adenocarcinoma-associated chronic atrophic gastritis (CAG) are both associated with oxyntic atrophy, but AIG patients demonstrate an increased risk of carcinoid tumors rather than the elevated risk of adenocarcinoma observed with CAG. We therefore sought to compare the characteristics of the metaplastic mucosa in AIG and CAG patients. Methods We examined markers for metaplasia (spasmolytic polypeptide expressing metaplasia (SPEM) and intestinal metaplasia) as well as proliferation (Ki67) and immune cell populations (neutrophils, macrophages, and eosinophils) in gastric sections from 16 female patients with autoimmune thyroiditis and AIG and 17 patients with CAG associated with gastric adenocarcinoma. Results Both AIG and CAG patients demonstrated prominent SPEM and intestinal metaplasia. However, AIG patients displayed significantly lower numbers of infiltrating macrophages and significantly reduced mucosal cell proliferation as compared to CAG patients. Conclusions These findings indicate that, while both AIG and CAG patients display prominent oxyntic atrophy and metaplasia, the AIG patients do not show proliferative metaplastic lineages that would predispose to adenocarcinoma.