Eiji Sugihara

Modulation of glucose metabolism by CD44 contributes to antioxidant status and drug resistance in cancer cells

An increased glycolytic flux accompanied by activation of the pentose phosphate pathway (PPP) is implicated in chemoresistance of cancer cells. In this study, we found that CD44, a cell surface marker for cancer stem cells, interacts with pyruvate kinase M2 (PKM2) and thereby enhances the glycolytic phenotype of cancer cells that are either deficient in p53 or exposed to hypoxia. CD44 ablation by RNA interference increased metabolic flux to mitochondrial respiration and concomitantly inhibited entry into glycolysis and the PPP. Such metabolic changes induced by CD44 ablation resulted in marked depletion of cellular reduced glutathione (GSH) and increased the intracellular level of reactive oxygen species in glycolytic cancer cells. Furthermore, CD44 ablation enhanced the effect of chemotherapeutic drugs in p53-deficient or hypoxic cancer cells. Taken together, our findings suggest that metabolic modulation by CD44 is a potential therapeutic target for glycolytic cancer cells that manifest drug resistance.

Involvement of poly(ADP-Ribose) polymerase 1 and poly(ADP-Ribosyl)ation in regulation of centrosome function.

ABSTRACT The regulatory mechanism of centrosome function is crucial to the accurate transmission of chromosomes to the daughter cells in mitosis. Recent findings on the posttranslational modifications of many centrosomal proteins led us to speculate that these modifications might be involved in centrosome behavior. Poly(ADP-ribose) polymerase 1 (PARP-1) catalyzes poly(ADP-ribosyl)ation to various proteins. We show here that PARP-1 localizes to centrosomes and catalyzes poly(ADP-ribosyl)ation of centrosomal proteins. Moreover, centrosome hyperamplification is frequently observed with PARP inhibitor, as well as in PARP-1-null cells. Thus, it is possible that chromosomal instability known in PARP-1-null cells can be attributed to the centrosomal dysfunction. P53 tumor suppressor protein has been also shown to be localized at centrosomes and to be involved in the regulation of centrosome duplication and monitoring of the chromosomal stability. We found that centrosomal p53 is poly(ADP-ribosyl)ated in vivo and centrosomal PARP-1 directly catalyzes poly(ADP-ribosyl)ation of p53 in vitro. These results indicate that PARP-1 and PARP-1-mediated poly(ADP-ribosyl)ation of centrosomal proteins are involved in the regulation of centrosome function.

C-MYC overexpression with loss of Ink4a/Arf transforms bone marrow stromal cells into osteosarcoma accompanied by loss of adipogenesis

The development of cancer is due to the growth and proliferation of transformed normal cells. Recent evidence suggests that the nature of oncogenic stress and the state of the cell of origin critically affect both tumorigenic activity and tumor histological type. However, this mechanistic relationship in mesenchymal tumors is currently largely unexplored. To clarify these issues, we established a mouse osteosarcoma (OS) model through overexpression of c-MYC in bone marrow stromal cells (BMSCs) derived from Ink4a/Arf (−/−) mice. Single-cell cloning revealed that c-MYC-expressing BMSCs are composed of two distinctly different clones: highly tumorigenic cells, similar to bipotent-committed osteochondral progenitor cells, and low-tumorigenic tripotent cells, similar to mesenchymal stem cells (MSCs). It is noteworthy that both bipotent and tripotent cells were capable of generating histologically similar, lethal OS, suggesting that both committed progenitor cells and MSCs can become OS cells of origin. Shifting mesenchymal differentiation by depleting PPARγ in tripotent MSC-like cells and overexpressing PPARγ in bipotent cells affected cell proliferation and tumorigenic activity. Our findings indicate that differentiation potential has a key role in OS tumorigenic activity, and that the suppression of adipogenic ability is a critical factor for the development of OS.

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.

Complexity of cancer stem cells

Heterogeneity of tumor tissue has been accounted for in recent years by a hierarchy‐based model in which cancer stem cells (CSCs) have the ability both to self‐renew and to give rise to differentiated tumor cells and are responsible for the overall organization of a tumor. Research into CSCs has progressed rapidly and concomitantly with recent advances in the biology of normal tissue stem cells, resulting in the identification of CSCs in a wide range of human tumors. Studies of mouse models of human cancer have provided further insight into the characteristics of CSCs as well as a basis for the development of novel therapies targeted to these cells. However, recent studies have revealed complexities, such as plasticity of stem cell properties and clonal diversity of CSCs, in certain tumor types that have led to revision of the original CSC model. In this review, we summarize the history of the discovery and characterization of CSCs, as well as address recent advances that have revealed the complexity of these cells and their therapeutic implications.

Regulation of MKL1 via actin cytoskeleton dynamics drives adipocyte differentiation

Cellular differentiation is regulated through activation and repression of defined transcription factors. A hallmark of differentiation is a pronounced change in cell shape, which is determined by dynamics of the actin cytoskeleton. Here we show that regulation of the transcriptional coactivator MKL1 (megakaryoblastic leukemia 1) by actin cytoskeleton dynamics drives adipocyte differentiation mediated by peroxisome proliferator-activated receptor γ (PPARγ), a master transcriptional regulator of adipogenesis. Induction of adipocyte differentiation results in disruption of actin stress fibres through downregulation of RhoA-ROCK signalling. The consequent rapid increase in monomeric G-actin leads to the interaction of G-actin with MKL1, which prevents nuclear translocation of MKL1 and allows expression of PPARγ followed by adipogenic differentiation. Moreover, we found that MKL1 and PPARγ act in a mutually antagonistic manner in the adipocytic differentiation programme. Our findings thus provide new mechanistic insight into the relation between the dynamics of cell shape and transcriptional regulation during cellular differentiation.

IGF1 Receptor Signaling Regulates Adaptive Radioprotection in Glioma Stem Cells

Cancer stem cells (CSCs) play an important role in disease recurrence after radiation treatment as a result of intrinsic properties such as high DNA repair capability and antioxidative capacity. It is unclear, however, how CSCs further adapt to escape the toxicity of the repeated irradiation regimens used in clinical practice. Here, we have exposed a population of murine glioma stem cells (GSCs) to fractionated radiation in order to investigate the associated adaptive changes, with the ultimate goal of identifying a targetable factor that regulates acquired radioresistance. We have shown that fractionated radiation induces an increase in IGF1 secretion and a gradual upregulation of the IGF type 1 receptor (IGF1R) in GSCs. Interestingly, IGF1R upregulation exerts a dual radioprotective effect. In the resting state, continuous IGF1 stimulation ultimately induces downregulation of Akt/extracellular‐signal‐regulated kinases (ERK) and FoxO3a activation, which results in slower proliferation and enhanced self‐renewal. In contrast, after acute radiation, the abundance of IGF1R and increased secretion of IGF1 promote a rapid shift from a latent state toward activation of Akt survival signaling, protecting GSCs from radiation toxicity. Treatment of tumors formed by the radioresistant GSCs with an IGF1R inhibitor resulted in a marked increase in radiosensitivity, suggesting that blockade of IGF1R signaling is an effective strategy to reverse radioresistance. Together, our results show that GSCs evade the damage of repeated radiation not only through innate properties but also through gradual inducement of resistance pathways and identify the dynamic regulation of GSCs by IGF1R signaling as a novel mechanism of adaptive radioprotection. STEM CELLS 2013;31:627–640

Hair follicle–derived IL-7 and IL-15 mediate skin-resident memory T cell homeostasis and lymphoma

The skin harbors a variety of resident leukocyte subsets that must be tightly regulated to maintain immune homeostasis. Hair follicles are unique structures in the skin that contribute to skin dendritic cell homeostasis through chemokine production. We demonstrate that CD4+ and CD8+ skin-resident memory T cells (TRM cells), which are responsible for long-term skin immunity, reside predominantly within the hair follicle epithelium of the unperturbed epidermis. TRM cell tropism for the epidermis and follicles is herein termed epidermotropism. Hair follicle expression of IL-15 was required for CD8+ TRM cells, and IL-7 for CD8+ and CD4+ TRM cells, to exert epidermotropism. A lack of either cytokine in the skin led to impaired hapten-induced contact hypersensitivity responses. In a model of cutaneous T cell lymphoma, epidermotropic CD4+ TRM lymphoma cell localization depended on the presence of hair follicle–derived IL-7. These findings implicate hair follicle–derived cytokines as regulators of malignant and non-malignant TRM cell tissue residence, and they suggest that the cytokines may be targeted therapeutically in inflammatory skin diseases and lymphoma.

Myristoylated alanine-rich C kinase substrate phosphorylation promotes cholangiocarcinoma cell migration and metastasis via the protein kinase C-dependent pathway

(Cancer Sci 2010; 101: 658–665)

Loss of p16 expression is associated with the stem cell characteristics of surface markers and therapeutic resistance in estrogen receptor-negative breast cancer

Triple‐negative breast cancer [TNBC, which is negative for the estrogen receptor (ER), progesterone receptor, and human epidermal growth factor receptor 2] is a high‐risk form of the disease without a specific therapy. DNA microarray and immunohistochemical analyses have shown that most TNBCs fall within the basal‐like histological subset of breast cancers, which frequently exhibit inactivation of the retinoblastoma tumor suppressor (Rb) and upregulation of the cyclin‐dependent kinase inhibitor p16INK4a (p16). However, downregulation of p16 expression has been observed in some basal‐like breast cancer cell lines, suggesting that such cells can be divided into two groups according to Rb and p16 status. We now show that cells that are CD44+ and CD24−, a phenotype associated with stem‐like breast cancer cells, are more abundant in ER−/p16− breast cancer cell lines than in ER−/p16+ lines. It was also found that p16 expression was downregulated in mammospheres from an ER‐negative breast cancer cell line. Depletion of p16 by RNA interference in ER‐negative breast cancer cells increased the percentage of CD44+/CD24− cells and increased the expression of mRNA of the ES‐like genes Nanog, Oct4, and Sox2 through an Rb‐independent pathway. Furthermore, such depletion of p16 reduced chemosensitivity. The loss of p16 expression may thus reduce the response of ER‐negative breast cancer cells to chemotherapy by conferring cancer stem cell‐like properties. Consistent with this conclusion, immunohistochemical analysis of the clinical samples suggests that low p16 expression in TNBC is associated with resistance to preoperative chemotherapy.