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Supplemental Figure S1. Parietal cells loss induced by tamoxifen injection in WT mouse.

Supplementary Methods and Figure legend.

Loss of parietal cells initiates the development of spasmolytic polypeptide–expressing metaplasia (SPEM), a precancerous lesion in stomach. CD44 variant (CD44v) that enhances the ability to defend against reactive oxygen species (ROS) in epithelial cells is expressed de novo in SPEM of K19-Wnt1/C2mE mice, a transgenic model of gastric tumorigenesis, and is required for the efficient development of SPEM and gastric tumor in these animals. The role of ROS and its downstream signaling in CD44-dependent gastric tumorigenesis has remained unknown, however. With the use of the K19-Wnt1/C2mE mouse, we now show that parietal cells in the inflamed stomach are highly sensitive to oxidative stress and manifest activation of p38MAPK signaling by ROS. Oral treatment with the antioxidant ascorbic acid or genetic ablation of the Ink4a/Arf locus, a major downstream target of ROS-p38MAPK signaling, inhibited parietal cell loss and the subsequent gastric tumorigenesis. Our results indicate that signaling activated by oxidative stress in parietal cells plays a key role in CD44-dependent gastric tumorigenesis. Cancer Prev Res; 8(6); 492–501. ©2015 AACR.

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PDF file - 70K, Gene list and SAM P values of GSEA analysis in Figure S4.

PDF file - 688K, A,B and C. The effect of retinoic acid and sulfasalazine on HNSCC cells. D. The effect of CD44 RNAi on retinoic acid sensitivity. E.The effect of CDDP and sulfasalazine on tumor growth formed by HSC-2 cells.

PDF file - 79K, Clinicopathological characteristics of HNSCC patients in Figure 1.

PDF file - 156K, GSEA of microarray data for OSC19/HSC-4 cell (GSE41495) and cetuximab-resistant/cetuximab-sensitive HNSCC (GSE21483) profiles.

Supplementary Table S1, Supplementary legends for Figures S1-S3, Supplementary Methods and References

PDF file - 695K, A, B, C and D. The effect of CD44 RNAi on xCT expression and GSH level in HNSCC cells. E. Immunoprecipitation analysis for XCT and CD44v interaction in HNSCC cells.

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EGFRvIII promotes glutamate release by glioma cells in a kinase-independent manner.

EGFR interacts with the central portion of xCT and thereby promotes surface expression of xCT.

Sulfasalazine does not affect intracellular ROS levels in Becker or U87MG cells.

Individuals who used skin‐whitening cosmetics (quasi‐drugs) containing 2% rhododendrol‐containing agents, developed leukoderma at a higher frequency than those who have used other skin‐whitening cosmetics. The Rhododenol Research Team (RD‐Team) was formed and commissioned by Kanebo Cosmetics Inc. to conduct research in treatments of rhododendrol‐induced leukoderma (RDL), to evaluate effective treatment options from a medical standpoint, and provide information to a wide range of people. In this study, we evaluated the efficacy of various treatments for RDL from a medical perspective, based on the information published in the literature as original or review articles. We searched the PubMed (international) and the Igaku Chuo Zasshi (ICHUSHI) (Japanese) databases using the keywords “Rhododenol” and “rhododendrol”, for articles published between July 2013 and November 2020. We discuss the main clinical findings and treatments (topical, oral, phototherapy, and surgical) of this condition based on the literature review. We found that ultraviolet light therapy is the most effective treatment for RDL. We have also summarized reports of the efficacy of oral vitamin D3 in RDL. A topical prostaglandin derivative has been reported in a new study to be effective. We have provided guidance for patients using self‐tanning and skin‐whitening agents to improve their quality of life. Finally, we have highlighted the importance of providing patients with information on contact dermatitis and instructing them to discontinue product use immediately if they develop any symptoms of contact dermatitis while using skin‐whitening agents.

A small proportion of individuals utilizing cosmetics containing rhododendrol developed leukoderma with various pathological conditions, in some cases indistinguishable from vitiligo. In this review, we investigate and evaluate the major considerations for developing rhododendrol‐induced leukoderma based on data from original or review articles published in the literature to provide a wide range of information regarding the pathophysiology, mechanisms, risk evaluation, and possible mechanism‐based treatments. We compile and discuss the latest information, including data related to the cytotoxicity of rhododendrol, cytoprotective functions, and involvement of the immune system, and consider the possibility of novel treatments based on the differences between individual patients and on the mechanism underlying the onset of the condition. Understanding the pathophysiology of rhododendrol‐induced leukoderma helps not only elucidate the mechanisms of non‐segmental vitiligo onset and progression, but also suggests prevention and treatment.

This study investigated a case of spindle cell carcinoma (SpCC) in tongue pathological lesions. The patient experienced a local recurrence and distant metastasis after surgical intervention. Although standard chemotherapy was administered, a granulomatous mass continued to develop. This aggressive growth led to survival of the tumor. Secondary debulking surgery was performed to improve the patient’s quality of life at the request of the patient. Using a tissue sample derived from the secondary debulking surgery, we performed an analysis of the tumor’s cell surface antigens, differentiation potential, metastatic ability, and inhibition potential by anticancer reagents. In vitro analysis revealed that the cell population grown under adherent culture conditions expressed the mesenchymal stem cell (MSC) markers CD73, CD90, and CD105. The cell line established from this SpCC contained colony-forming unit fibroblasts (CFU-Fs) and exhibited multipotent differentiation into several mesenchymal lineages, including bone, cartilage, and fat. The SpCC cells also displayed vigorous mobilization. These characteristics suggested that they had the differentiation potential of mesenchymal cells, especially MSCs, rather than that of epithelial cells. The surgical specimen analyzed in this study resisted the molecular target reagent cetuximab, which is an epidermal growth factor receptor inhibitor. This clinical insight revealed that chemotherapy-resistant SpCC cells have different characteristics compared to most other cancer cells, which are sensitive to cetuximab. Our cell death assay revealed that SpCC cell death was induced by the anticancer drug imatinib, which is known to inhibit protein tyrosine kinase activity of ABL, platelet-derived growth factor receptor α (PDGFRα), and KIT. Here, we report recurrent SpCC with characteristics of MSCs and potential for treatment with imatinib.

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.

Eventual relapse of tumor growth is commonly observed in head and neck cancer patients, following treatment with platinum-based chemotherapies. This occurrence is believed to be related to the failure to eradicate drug resistant, cancer stem cell (CSC) niches, thereby enriching their population in tumors after treatment. In this study, we show that in contrast to free cisplatin (CDDP), the polymer micelle-based nanomedicine incorporating cisplatin (CDDP/m), can eradicate both the undifferentiated cell and the differentiated cancer cell populations within a head and neck tumor model. Immunohistochemistry of treated tumors showed that opposing to CDDP treatment, CDDP/m could reduce tumor growth without concentrating the CSC-like population. We further showed that CDDP/m, but not CDDP, can localize into hypoxic regions, possibly CSC-rich areas, in the tumors, and can overcome their detoxification mechanism based-on high cellular expression of glutathione to successfully deliver Pt to nuclear DNA. Our data suggests CDDP/m to be a replacement for current platinum therapies, for its ability to eradicate both bulk and CSC-like populations, and in turn to prevent recurrence of tumor growth.

Objective In any immediate reconstruction of the maxillofacial defect after total maxillectomy, the defect is often covered with a flap, however, dentures cannot be maintained stably by the flap, because of the high mobility and large amount of displacement of the flap under pressure. Therefore, we developed a method in which a muscle flap is transplanted without creating a skin island in the oral cavity and a denture is immediately fixed with titanium screws, and obtained favorable results. Patients and methods The subjects were 9 patients who underwent resection for maxillary sinus cancer. In all patients, a free rectus abdominis muscle flap was used for the reconstruction and the raw surface of the muscle tissue was left in the oral cavity without creating a skin island. Dentures that had been made preoperatively were fixed on the remaining palate and alveolus with titanium screws for one month, so that they covered the muscle tissue. Results Good palatal morphology was formed without hanging down after epithelialization of the surface of the muscle tissue. In comparison with the preoperative levels, the masticatory and conversational functions recovered to 77% and 90%, on average. Conclusions Our method is considered to be one of the useful reconstruction methods for maxillofacial defects.

Background: Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth most common cancer in the world. Despite the development of various treatments, the five-year survival rate for HNSCC is about 50-60% and has not been improved in recent years. Immune checkpoint inhibitors are promising, but the effect of those is limited. The development of new reagents having different mechanisms against tumor progression is still needed. From the result of drug screening, Pimozide (PMZ), an antipsychotic drug used for the treatment of schizophrenia and chronic psychosis, demonstrated a strong antitumor ability to HNSCC cells expressing high level of CD44v. In this study, we examined the combined effects of PMZ and paclitaxel (PTX) for HNSCC cells. Methods: Using HNSCC cell lines (FaDu, Detroit562, HSC-2, OSC-19), the antitumor effects of PTX, PMZ, and the combination of PTX and PMZ were evaluated by MTS assay. Moreover, the reactive oxygen species (ROS) expression level under each drug administration was measured by flow cytometry. Moreover, PTX, PMZ (0.45mg/kg), PMZ (1mg/kg), PTX+PMZ were administered to the xenograft model by OSC-19. The tumor was resected after those treatments. The expression of CD44v, ALDH3A1, and NRF2 was evaluated by immunohistochemistry (IHC) of FFPE tissues from those extracted tumors. Results: PMZ single-agent inhibited tumor growth in various tumor cell lines, and the antitumor effect was enhanced by concomitant use of PTX. PMZ, which significantly increased ROS of tumor cells, was more effective than PTX in OSC-19, which was a chemoresistant cell line expressing high CD44v. Xenograft model exhibited that tumor growth was suppressed in PTX+PMZ group than in PTX and PMZ single-agent group. IHC staining revealed that PMZ administration restrained CD44v expression, and ALDH3A1 expression markedly decreased in PMZ treated tumors, but not in PTX treated tumors. Conclusions: PMZ dramatically induced ROS production in tumor cells. These data suggest that aldehyde produced by ROS might suppress HNSCC tumor growth. The results from this study indicated that PMZ could be a novel therapeutic reagent for HNSCC in combination with existing anticancer drugs. Citation Format: Shintaro Nakamura, Hiroyuki Ozawa, Juntaro Yamasaki, Shogo Okazaki, Momoko Yoshikawa, Tomoya Soma, Seiji Asoda, Osamu Nagano, Mariko Sekimizu, Shin Saito, Keisuke Yoshihama, Takuya Mikoshiba, Kaoru Ogawa, Hideyuki Saya. Pimozide combined with paclitaxel demonstrates a significant antitumor effect in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3579.

Background Rhododendrol (RD), a skin-whitening agent, is believed to be associated with cases of cosmetics-related leukoderma that have been reported in Japan. Recently, we have shown that RD is catalyzed by tyrosinase to produce putative toxic metabolites RD-catechol and RD-cyclic catechol. Objective To examine the cytotoxicity and production of reactive oxygen species (ROS) in melanocytic cells by RD and its metabolic products. Methods The growth inhibitory effect of RD or its metabolite on the normal human epidermal melanocyte (NHEM) and B16F1 cells was assessed by cell counting or WST assay. ROS production was detected by flow cytometry and confocal microscopy after cells were treated with 2′,7′-dichlorofluorescein and RD or its metabolite. Results Growth of NHEM derived from African American (NHEMb) and B16F1 cells was suppressed by 300 μM or more RD. Growth inhibitory activity of RD (IC50 of B16F1: 671 μM) was weaker than hydroquinone (IC50 of B16F1: 28.3 μM) or resveratrol (IC50 of B16F1: 27.1 μM). Flow cytometric analysis detected ROS production in the NHEMb and B16F1 cells exposed to RD. However, neither RD nor H 2 O 2 increased the subG1 fraction of these melanocytic cells. RD-catechol and RD-cyclic catechol inhibited growth of NHEMb and B16F1 cells much more strongly than did RD. RD-catechol, as well as RD, produced ROS detected by both flow cytometry and immunostaining, while RD-cyclic catechol produced a hardly detectable amount of ROS in B16F1 cells. Conclusions These results suggest that RD exerts the cytotoxicity in melanocytic cells through its oxidative metabolites and that ROS plays a role in RD-mediated cytotoxicity.

Rhododendrol-induced leukoderma is an acquired depigmentation that develops mainly at the contact site after repeated use of skin-whitening cosmetics containing rhododendrol. In most cases, cessation of further depigmentation or occurrence of repigmentation is observed after discontinuing the use of cosmetics. However, some patients develop vitiligo vulgaris through the spread of depigmentation into the non-exposed areas. Our study aims to investigate the patient-specific factors that may affect the extent of depigmentation or repigmentation, as well as development of vitiligo vulgaris. The degree of depigmentation of the face, neck and hands where exposed to rhododendrol was scored using photographs over time. The relationships between depigmentation score at first visit/improvement rate of depigmentation score and patient demographics were evaluated and three important clinical observations were made. First, repigmentation of the face was superior compared with that of the hands and neck, suggesting a possible role for the migration and differentiation of melanocyte stem cells from hair follicles, as a mechanism of repigmentation. Second, the intensity of rhododendrol exposure did not contribute to differences in the severity of depigmentation. This suggested a possibility of underlying genetic susceptibility to melanocyte cytotoxicity or immune reaction. Third, depigmentation score at first visit and past history of atopic dermatitis were significantly high in patients who developed vitiligo vulgaris. This suggested that severe chemical damage of melanocytes by rhododendrol leads to a higher risk of developing vitiligo vulgaris through the possible involvement of an immune reaction. These clinical observations may help to further understand the pathogenesis of rhododendrol-induced leukoderma.

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 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. Cancer Res; 76(10); 2954–63. ©2016 AACR.

Loss of parietal cells initiates the development of spasmolytic polypeptide–expressing metaplasia (SPEM), a precancerous lesion in stomach. CD44 variant (CD44v) that enhances the ability to defend against reactive oxygen species (ROS) in epithelial cells is expressed de novo in SPEM of K19-Wnt1/C2mE mice, a transgenic model of gastric tumorigenesis, and is required for the efficient development of SPEM and gastric tumor in these animals. The role of ROS and its downstream signaling in CD44-dependent gastric tumorigenesis has remained unknown, however. With the use of the K19-Wnt1/C2mE mouse, we now show that parietal cells in the inflamed stomach are highly sensitive to oxidative stress and manifest activation of p38MAPK signaling by ROS. Oral treatment with the antioxidant ascorbic acid or genetic ablation of the Ink4a/Arf locus, a major downstream target of ROS-p38MAPK signaling, inhibited parietal cell loss and the subsequent gastric tumorigenesis. Our results indicate that signaling activated by oxidative stress in parietal cells plays a key role in CD44-dependent gastric tumorigenesis. Cancer Prev Res; 8(6); 492–501. ©2015 AACR.

Malignant glioma such as glioblastoma is a cancer still difficult to treat. 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, which has been reported to be involved in malignant phenotypes of glioma cells. However, the underlying mechanism of xCT regulation in glioma is not elucidated. 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 extracellular glutamate which promotes glioma 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 propose that xCT is a promising therapeutic target in EGFR-overexpressing malignant glioma. Citation Format: Kenji Tsuchihashi, Shogo Okazaki, Momoko Yoshikawa, Ryo Seishima, Oltea Sampetrean, Nobuyuki Onishi, Hiroaki Wakimoto, Frank Furnari, Eishi Baba, Koichi Akashi, Hideyuki Saya, Osamu Nagano. xCT promotes malignant phenotypes in EGFR-expressing glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-334. doi:10.1158/1538-7445.AM2017-LB-334

CD44 is frequently overexpressed in a wide variety of epithelial malignancies including gastrointestinal cancer and causes resistance to currently available treatments. MicroRNAs (miRNAs) are non-coding RNAs that regulate molecular pathways in cancer by targeting various genes. The aim of this study was to investigate the regulation of CD44 expression by miRNAs and to develop new molecular targets in gastrointestinal cancer. We performed miRNA screening in six human gastrointestinal cancer cell lines and identified three candidate miRNAs that could regulate CD44 expression in gastrointestinal cancer. Among these, we focused on miR-328 and examined its functional relevance using growth assays and cytotoxicity assays. CD44 expression was reduced in gastrointestinal cancer cell lines forced to express miR-328, leading to inhibition of cancer cell growth in vitro and in vivo, and impaired resistance to chemotherapeutic drugs and reactive oxygen species (ROS). In contrast, induction of CD44 expression by miR-328 inhibitor led to promotion of cancer cell growth. Furthermore, we revealed that ROS produced by macrophages triggered CD44 expression through suppression of miR-328 in gastric cancer cells. Finally, tumor-infiltrating macrophages (CD68 and CD163) were closely related to both miR-328 downregulation and CD44 upregulation in 63 patients with surgically resected gastric cancer. These findings suggest that macrophages in the tumor microenvironment may cause increased CD44 expression through miR-328 suppression, resulting in tumor progression by enhancing ROS defense. miR-328-CD44 signaling mediated by macrophages may thus represent a potential target for the treatment of gastrointestinal cancer.

Cancer development is often preceded by the appearance of preneoplastic lesions. In gastric carcinogenesis, chronic inflammation and histopathologic progression of the stomach epithelium lead to the development of metaplasia and eventually adenocarcinoma. The cell surface protein CD44, especially its variant isoforms (CD44v), has been implicated in metaplasia-carcinoma sequence progression in the stomach. We recently found that CD44v interacts with and stabilizes xCT, a subunit of the cystine transporter system xc(-), in cancer cells and thereby increases cystine uptake and confers resistance to various types of cellular stress in vivo. The functional relevance of CD44v and xCT in the development of preneoplastic lesions, however, has remained unknown. We have now examined the role of the CD44v-xCT system in the development of spasmolytic polypeptide-expressing metaplasia (SPEM) in mouse models of gastric carcinogenesis. CD44v was found to be expressed de novo in SPEM, and CD44v(+) metaplastic cells manifested upregulation of xCT expression compared with CD44v(-) cells. Genetic ablation of CD44 or treatment with sulfasalazine, an inhibitor of xCT-dependent cystine transport, suppressed the development of SPEM and subsequent gastric tumor growth. Therapy targeted to CD44v-xCT could thus prove effective for prevention or attenuation of the CD44v-dependent development of preneoplastic lesions and cancer.

The targeting of antioxidant systems that allow stem-like cancer cells to avoid the adverse consequences of oxidative stress might be expected to improve the efficacy of cancer treatment. Here, we show that head and neck squamous cell carcinoma (HNSCC) cells that express variant isoforms of CD44 (CD44v) rely on the activity of the cystine transporter subunit xCT for control of their redox status. xCT inhibition selectively induces apoptosis in CD44v-expressing tumor cells without affecting CD44v-negative differentiated cells in the same tumor. In contrast to CD44v-expressing undifferentiated cells, CD44v-negative differentiated cells manifest EGF receptor (EGFR) activation and rely on EGFR activity for their survival. Combined treatment with inhibitors of xCT-dependent cystine transport and of EGFR resulted in a synergistic reduction of EGFR-expressing HNSCC tumor growth. Thus, xCT-targeted therapy may deplete CD44v-expressing undifferentiated HNSCC cells and concurrently sensitize the remaining differentiating cells to available treatments including EGFR-targeted therapy. Cancer Res; 73(6); 1855–66. ©2012 AACR.

The in vitro and in vivo electrochemical detection of the reduced form of glutathione (L-γ-glutamyl-L-cysteinyl-glycine, GSH) using boron doped diamond (BDD) microelectrode for potential application in the assessment of cancerous tumors is presented. Accurate calibration curve for the determination of GSH could be obtained by the in vitro electrochemical measurements. Additionally, it was shown that it was possible to separate the detection of GSH from the oxidized form of glutathione (GSSG) using chronoamperometry measurements. In vivo GSH detection measurements have been performed in human cancer cells inoculated in immunodeficient mice. These measurements have shown that the difference of GSH level between cancerous and normal tissues can be detected. Moreover, GSH detection measurements carried out before and after X-ray irradiation have proved that it is possible to assess in vivo the decrease in GSH concentration in the tumor after a specific treatment.

Cachexia, a major cause of cancer-related death, is characterized by depletion of muscle and fat tissues, anorexia, asthenia, and hypoglycemia. Recent studies indicate that secretions of proinflammatory cytokines such as interleukin-6 (IL-6) play a crucial role in cachexia development, and that these cytokines are secreted from not only cancer cells but also host cells such as macrophages. In this study, we investigated the therapeutic effects of hochuekkito, a Kampo formula, on cachexia induced by colon 26 adenocarcinoma in mice. Hochuekkito treatment did not inhibit tumor growth, but significantly attenuated the reduction in carcass weight, food and water intake, weight of the gastrocnemius muscle and fat tissue around the testes, and decrease of serum triglyceride level compared with controls. Furthermore, hochuekkito treatment significantly reduced serum IL-6 level and IL-6 expression level in macrophages in tissues surrounding the tumor. In vitro studies showed that hochuekkito suppressed the production of IL-6 by THP-1 or RAW264.7 macrophage cells, although it did not affect IL-6 production by colon 26 carcinoma cells. These results suggest that hochuekkito inhibits the production of proinflammatory cytokines, particularly IL-6, by host cells such as macrophages. Therefore, hochuekkito may be a promising anticachectic agent for the treatment of patients with cancer.

In cancer metastasis, various environmental stressors attack the disseminating cells. The successful colonization of cancer cells in secondary sites therefore requires the ability of the cells to avoid the consequences of such exposure to the stressors. Here we show that orthotopic transplantation of a CD44 variant isoform-expressing (CD44v(+)) subpopulation of 4T1 breast cancer cells, but not that of a CD44v(-) subpopulation, in mice results in efficient lung metastasis accompanied by expansion of stem-like cancer cells. Such metastasis is dependent on the activity of the cystine transporter xCT, and the stability of this protein is controlled by CD44v. We find that epithelial splicing regulatory protein 1 regulates the expression of CD44v, and knockdown of epithelial splicing regulatory protein 1 in CD44v(+) cells results in an isoform switch from CD44v to CD44 standard (CD44s), leading to reduced cell surface expression of xCT and suppression of lung colonization. The epithelial splicing regulatory protein 1-CD44v-xCT axis is thus a potential therapeutic target for the prevention of metastasis.

Cancer metastasis is established by the seeding and successful colonization of stem-like cancer cells which show tumor-initiating ability at distant organs. CD44 is a cell surface marker protein which is highly expressed in stem-like cells of several human cancers including breast cancer. CD44 exists in various isoforms produced through alternative splicing of mRNA. Increased expression of CD44 variant isoform (CD44v) has been shown to correlate with metastatic potential and aggressive clinical behavior of various human cancers. We recently showed that CD44v contributes to reactive oxygen species (ROS)-defense in cancer cells by enhancing cystine uptake by the cystine transporter xCT. However, the functional relevance of CD44v and xCT in the metastatic spread of stem-like cancer cells has remained elusive. In the present study, we show that metastatic breast cancer 4T1 cells consist of CD44v+ and CD44v- cells. The orthotopic transplantation of CD44v+ population but not that of CD44v-, in mice resulted in efficient lung metastasis. We found that CD44v expressing metastatic cancer cells highly express xCT and thereby maintain high reduced glutathione (GSH) level. A specific xCT inhibitor sulfasalazine suppressed lung metastatsis of CD44v+ cells. Alternative splicing of CD44mRNA in such metastatic cancer cells is regulated by Epithelial Splicing Related Protein1 (ESRP1) and short hairpin RNA (shRNA) targeting ESRP1 switched isoform from CD44v to CD44 standard (CD44s), leading to the suppression of lung metastasis. Furthermore, we found that ESRP1 expression is regulated epigenetically through histone modification by performing chromatin immunoprecipitation (ChIP) sequencing analysis of the ESRP1 locus in CD44v+ and CD44v- cells. These findings establish a novel role of ESRP1-regulated alternative splicing program in the regulation of antioxidant status of stem-like cancer cells which is crucial for the metastatic ability. Citation Format: Kenji Tsuchihashi, Osamu Nagano, Toshifumi Yae, Takatsugu Ishimoto, Takeshi Motohara, Momoko Yoshikawa, Go J Yoshida, Takeyuki Wada, Takashi Masuko, Kaoru Mogushi, Hiroshi Tanaka, Tsuyoshi Osawa, Yasuharu Kanki, Takashi Minami, Hiroyuki Aburatani, Mitsuyo Ohmura, Akiko Kubo, Makoto Suematsu, Kazuhisa Takahashi, Eishi Baba, Koichi Akashi, Hideyuki Saya. ESRP1 regulated alternative splicing of CD44mRNA enhances lung colonization of metastatic cancer cell. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4967. doi:10.1158/1538-7445.AM2014-4967