Kenji Tsuchihashi

Alternative splicing of CD44 mRNA by ESRP1 enhances lung colonization of metastatic cancer cell

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.

xCT inhibition depletes CD44v-expressing tumor cells that are resistant to EGFR-targeted therapy in head and neck squamous cell carcinoma

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.

Functional role of CD44v‐xCT system in the development of spasmolytic polypeptide‐expressing metaplasia

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 EGF receptor promotes the malignant potential of glioma by regulating amino acid transport system xc(

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.

Impacts of CD44 knockdown in cancer cells on tumor and host metabolic systems revealed by quantitative imaging mass spectrometry

CD44 expressed in cancer cells was shown to stabilize cystine transporter (xCT) that uptakes cystine and excretes glutamate to supply cysteine as a substrate for reduced glutathione (GSH) for survival. While targeting CD44 serves as a potentially therapeutic stratagem to attack cancer growth and chemoresistance, the impact of CD44 targeting in cancer cells on metabolic systems of tumors and host tissues in vivo remains to be fully determined. This study aimed to reveal effects of CD44 silencing on alterations in energy metabolism and sulfur-containing metabolites in vitro and in vivo using capillary electrophoresis-mass spectrometry and quantitative imaging mass spectrometry (Q-IMS), respectively. In an experimental model of xenograft transplantation of human colon cancer HCT116 cells in superimmunodeficient NOG mice, snap-frozen liver tissues containing metastatic tumors were examined by Q-IMS. As reported previously, short hairpin CD44 RNA interference (shCD44) in cancer cells caused significant regression of tumor growth in the host liver. Under these circumstances, the CD44 knockdown suppressed polyamines, GSH and energy charges not only in metastatic tumors but also in the host liver. In culture, HCT116 cells treated with shCD44 decreased total amounts of methionine-pool metabolites including spermidine and spermine, and reactive cysteine persulfides, suggesting roles of these metabolites for cancer growth. Collectively, these results suggest that CD44 expressed in cancer accounts for a key regulator of metabolic interplay between tumor and the host tissue.

Development of an ErbB4 monoclonal antibody that blocks neuregulin-1-induced ErbB4 activation in cancer cells

The use of monoclonal antibodies (mAbs) for cancer therapy is one of the most important strategies for current cancer treatment. The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, which regulates cancer cell proliferation, survival, and migration, is a major molecular target for antibody-based therapy. ErbB4/HER4, which contains a ligand-binding extracellular region, is activated by several ligands, including neuregulins (NRGs), heparin-binding EGF-like growth factor, betacellulin and epiregulin. Although there are clinically approved antibodies for ErbB1 and ErbB2, there are no available therapeutic mAbs for ErbB4, and it is not known whether ErbB4 is a useful target for antibody-based cancer therapy. In this study, we developed an anti-ErbB4 mAb (clone P6-1) that suppresses NRG-dependent activation of ErbB4 and examined its effect on breast cancer cell proliferation in the extracellular matrix.

Ink4a/Arf-Dependent Loss of Parietal Cells Induced by Oxidative Stress Promotes CD44-Dependent Gastric Tumorigenesis.

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.

Pancreatic acinar cell carcinoma presenting with panniculitis, successfully treated with FOLFIRINOX: A case report

Pancreatic acinar cell carcinoma (PACC) is a rare tumor of the exocrine pancreas, representing only 1% of all pancreatic malignancies. A 50-year-old man presented with edema of the thumb joints bilaterally, followed by an appearance of masses in the bilateral lower extremities and fever (38°C). The masses were diagnosed as panniculitis by skin biopsy, and multiple intraperitoneal masses were incidentally detected on pelvic magnetic resonance imaging performed to investigate the leg abnormalities. The patient was referred to the Kyushu University Hospital for further investigation, and fluorodeoxyglucose-positron emission tomography/computed tomography (CT) revealed high-uptake tumors in the pancreatic tail, in the periphery of the liver, and in the pelvis. Laboratory examinations revealed high serum concentrations of pancreatic exocrine enzymes, such as lipase, trypsin, elastase 1 and pancreatic phospholipase A2. Histological examination of a bioptic specimen obtained from a hepatic lesion revealed proliferation of atypical cells arranged in a tubular or glandular pattern. Immunohistochemical staining revealed that the atypical cells were positive for cytokeratin (CK)7, CK19 and lipase, but negative for CK20 and thyroid transcription factor-1, leading to a final diagnosis of acinar cell carcinoma of the pancreatic tail (T4bN0M1, stage IV according to the 7th edition of the TNM Classification of Malignant Tumors). Combined chemotherapy with oxaliplatin, irinotecan and fluorouracil (FOLFIRINOX) was administered and fever was soon alleviated. The serum levels of lipase also declined and panniculitis completely resolved. As of the start of the 8th course of chemotherapy, the levels of the pancreatic exocrine enzymes were within normal ranges and CT revealed partial response. Therefore, the severe lipase hypersecretion syndrome was well controlled by the FOLFIRINOX regimen and shrinkage of the mass was also achieved. Thus, the FOLFIRINOX regimen may represent an effective treatment option for advanced PACC.

Eradication of CD44-variant positive population in head and neck tumors through controlled intracellular navigation of cisplatin-loaded nanomedicines

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.

Programmed death-ligand 1 expression is associated with fibrosarcomatous transformation of dermatofibrosarcoma protuberans

Dermatofibrosarcoma protuberans (DFSP) is a locally invading tumor, characterized by the presence of the collagen type I α 1 (COL1A1)-platelet-derived growth factor (PDGF) β fusion gene. We herein report the case of a 31-year-old man with a history of resection of an abdominal wall DFSP. The patient presented with chest pain and a computed tomography scan revealed a large mass in the posterior mediastinum and another mass in the right lung. The mediastinal mass was a sarcomatous lesion expressing the COL1A1-PDGFβ fusion gene, suggesting that it represented a metastasis of the DFSP following fibrosarcomatous (FS) transformation. Following resection of the mediastinal metastasis and subsequent radiotherapy, the mass in the right lung was also resected. Due to the emergence of pleural and pancreatic tail metastases, the patient was treated with a combination therapy of adriamycin and ifosfamide. After five courses, the disease progressed and the patient was subsequently treated with pazopanib for ~2 months until further progression. Three years after the diagnosis of the mediastinal metastasis of DFSP, the patient was referred to another hospital for palliative care. The expression of programmed cell death 1 ligand (PD-L1) in the primary and metastatic tumors was investigated: PD-L1 expression was detected in the metastasis but not in the primary tumor. Given that the metastatic tumor exhibited FS transformation (DFSP-FS), PD-L1 expression may be induced by FS transformation, contributing to the metastasis through escape from immune surveillance. Further investigation of the PD-L1 pathway in DFSP and DFSP-FS in primary as well as metastatic sites is required to evaluate the clinical efficacy of therapies targeting the PD-L1 signaling cascade.