Kazuharu Kai

CD44 Variant Regulates Redox Status in Cancer Cells by Stabilizing the xCT Subunit of System xc− and Thereby Promotes Tumor Growth

CD44 is an adhesion molecule expressed in cancer stem-like cells. Here, we show that a CD44 variant (CD44v) interacts with xCT, a glutamate-cystine transporter, and controls the intracellular level of reduced glutathione (GSH). Human gastrointestinal cancer cells with a high level of CD44 expression showed an enhanced capacity for GSH synthesis and defense against reactive oxygen species (ROS). Ablation of CD44 induced loss of xCT from the cell surface and suppressed tumor growth in a transgenic mouse model of gastric cancer. It also induced activation of p38(MAPK), a downstream target of ROS, and expression of the gene for the cell cycle inhibitor p21(CIP1/WAF1). These findings establish a function for CD44v in regulation of ROS defense and tumor growth.

Microfluidics separation reveals the stem-cell-like deformability of tumor-initiating cells

Here we report a microfluidics method to enrich physically deformable cells by mechanical manipulation through artificial microbarriers. Driven by hydrodynamic forces, flexible cells or cells with high metastatic propensity change shape to pass through the microbarriers and exit the separation device, whereas stiff cells remain trapped. We demonstrate the separation of (i) a mixture of two breast cancer cell types (MDA-MB-436 and MCF-7) with distinct deformabilities and metastatic potentials, and (ii) a heterogeneous breast cancer cell line (SUM149), into enriched flexible and stiff subpopulations. We show that the flexible phenotype is associated with overexpression of multiple genes involved in cancer cell motility and metastasis, and greater mammosphere formation efficiency. Our observations support the relationship between tumor-initiating capacity and cell deformability, and demonstrate that tumor-initiating cells are less differentiated in terms of cell biomechanics.

CD44+ slow‐cycling tumor cell expansion is triggered by cooperative actions of Wnt and prostaglandin E2 in gastric tumorigenesis

(Cancer Sci 2010; 101: 673–678)

Breast cancer stem cells

Since the initial discovery of leukemia stem cells nearly a decade ago, a great deal of cancer research has focused on the identification of cancer stem cells (CSCs) in many types of solid tumors, including breast cancer. Through analysis of cell surface markers and xenotransplant models, a subpopulation of putative human breast cancer stem cells (BCSCs) that is CD24-negative/CD44-positive (CD24−/CD44+) and bears high aldehyde dehydrogenase 1 activity has been isolated in clinical samples of breast cancer tissues. Human BCSCs are considered to be derived from basal cells that reside in the basal membranes of alveolar units in human adult mammary glands. Furthermore, BCSCs have been shown to express higher levels of oxidative stress-responsive genes, which could confer part of their ability to resist anti-cancer therapy, than non-CSCs. The emerging picture of the biological properties of BCSCs would contribute for devising innovative therapies for breast cancer, targeting the intrinsic and extrinsic factors that maintain the BCSCs.

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.

Maintenance of HCT116 colon cancer cell line conforms to a stochastic model but not a cancer stem cell model

The cancer stem cell (CSC) model, in which a small population of cells within a tumor possesses the ability to self‐renew and reconstitute the phenotype of primary tumor, has gained wide acceptance based on evidence over the past decade. It has also been reported that cancer cell lines contain a CSC subpopulation. However, phenotypic differences between CSCs and non‐CSCs in cancer cell lines are not better defined than in primary tumors. Furthermore, some cell lines do not have a CSC population, revealed as a side population and expression of CD133. Thus, the identification of CSCs in cancer cell lines remains elusive. Here, we investigated the CSC hierarchy within HCT116 colon cancer cells, which do not have a CD133‐positive subpopulation. We examined the expression of alternative CSC markers epithelial specific antigen (ESA) and CD44 in floating‐sphere‐derived cells, which are known to be the cells of enriching CSCs. Sphere‐derived HCT116 cells exhibited heterogeneous expression of ESA and CD44. The two major subpopulations of HCT116 sphere cells (ESAlowCD44−/low and ESAhighCD44high) exhibited a biological/proliferative hierarchy of sphere‐forming and soft agar colony‐forming activity. However, there was no difference between the two subpopulations in the incidence of xenograft tumors. When ESAlowCD44−/low cells were allowed to aggregate and re‐form floating‐spheres, the biological/proliferative hierarchy of parental HCT116 spheres was reconstituted, in terms of ESA and CD44 expression. Thus, HCT116 cells have plasticity when they are set in floating‐spheres, suggesting that maintenance of the HCT116 cell line conforms to a stochastic model, not a CSC model. (Cancer Sci 2009; 100: 2275–2282)

p53 expression status is a significant molecular marker in predicting the time to endocrine therapy failure in recurrent breast cancer: A cohort study

BackgroundHormone receptor status has been one of the most important factors in predicting the response to endocrine therapy in breast cancer patients. However, half of those patients with estrogen receptor-positive tumors do not respond to endocrine therapy. There have been no universal factors for predicting resistance to endocrine therapy in this population. Recently, p53 status has been extensively used as a predictive factor for response to systemic therapy, because tumor cells lacking p53 function do not respond to systemic therapy due to a failure in apoptosis. We therefore studied the relationship between the efficacy of endocrine therapy and biological factors, including p53.MethodsThe expression of p53, Ki67, and human epidermal growth factor receptor (HER)2 was examined by immunostaining in the primary tumors of 53 patients who received endocrine therapy for recurrent or advanced breast cancer. The following clinical factors were also analyzed: site treated, disease-free interval, and response to first-line endocrine therapy. To evaluate the significance of these factors, time to endocrine therapy failure (TTEF), or the total duration of sequential endocrine therapies was adopted as representing the clinical outcome.ResultsThe median TTEF was 16.1 months (range, 2.5–89.9 months). Multivariate analysis showed significantly reduced TTEF associated with no response to first-line endocrine therapy (P = 0.006 and P = 0.002 in all patients and in recurrent patients, respectively) and associated with positive p53 expression (P = 0.066 and P = 0.004, respectively).Conclusionp53 expression status was a significant molecular marker as well as the response to first-line endocrine therapy for predicting TTEF in recurrent breast cancer with hormone-sensitive disease.

Comparison of molecular subtype distribution in triple-negative inflammatory and non-inflammatory breast cancers

IntroductionBecause of its high rate of metastasis, inflammatory breast cancer (IBC) has a poor prognosis compared with non-inflammatory types of breast cancer (non-IBC). In a recent study, Lehmann and colleagues identified seven subtypes of triple-negative breast cancer (TNBC). We hypothesized that the distribution of TNBC subtypes differs between TN-IBC and TN-non-IBC. We determined the subtypes and compared clinical outcomes by subtype in TN-IBC and TN-non-IBC patients.MethodsWe determined TNBC subtypes in a TNBC cohort from the World IBC Consortium for which IBC status was known (39 cases of TN-IBC; 49 cases of TN-non-IBC). We then determined the associations between TNBC subtypes and IBC status and compared clinical outcomes between TNBC subtypes.ResultsWe found the seven subtypes exist in both TN-IBC and TN-non-IBC. We found no association between TNBC subtype and IBC status (P = 0.47). TNBC subtype did not predict recurrence-free survival. IBC status was not a significant predictor of recurrence-free or overall survival in the TNBC cohort.ConclusionsOur data show that, like TN-non-IBC, TN-IBC is a heterogeneous disease. Although clinical characteristics differ significantly between IBC and non-IBC, no unique IBC-specific TNBC subtypes were identified by mRNA gene-expression profiles of the tumor. Studies are needed to identify the subtle molecular or microenvironmental differences that contribute to the differing clinical behaviors between TN-IBC and TN-non-IBC.

Ink4a and Arf are crucial factors in the determination of the cell of origin and the therapeutic sensitivity of Myc-induced mouse lymphoid tumor

The cell of origin of tumors and the factors determining the cell of origin remain unclear. In this study, a mouse model of precursor B acute lymphoblastic leukemia/lymphoma (pre-B ALL/LBL) was established by retroviral transduction of Myc genes (N-Myc or c-Myc) into mouse bone marrow cells. Hematopoietic stem cells (HSCs) exhibited the highest susceptibility to N-Myc-induced pre-B ALL/LBL versus lymphoid progenitors, myeloid progenitors and committed progenitor B cells. N-Myc was able to induce pre-B ALL/LBL directly from progenitor B cells in the absence of Ink4a and Arf. Arf was expressed higher in progenitor B cells than Ink4a. In addition, N-Myc induced pre-B ALL/LBL from Arf−/− progenitor B cells suggesting that Arf has a predominant role in determining the cell of origin of pre-B ALL/LBL. Tumor cells derived from Ink4a/Arf−/− progenitor B cells exhibited a higher rate of proliferation and were more chemoresistant than those derived from wild-type HSCs. Furthermore, the Mdm2 inhibitor Nutlin-3 restored p53 and induced massive apoptosis in mouse pre-B ALL/LBL cells derived from Ink4a/Arf−/− cells and human B-ALL cell lines lacking Ink4a and Arf expression, suggesting that Mdm2 inhibition may be a novel therapeutic approach to the treatment of Ink4a/Arf−/− B-ALL/LBL, such as is frequently found in Ph+ ALL and relapsed ALL. Collectively, these findings indicate that Ink4a and Arf are critical determining factors of the cell of origin and the therapeutic sensitivity of Myc-induced lymphoid tumors.

Loss of heterozygosity at the ATBF1-A locus located in the 16q22 minimal region in breast cancer

BackgroundLoss of heterozygosity (LOH) on the long arm of chromosome 16 is one of the most frequent genetic events in solid tumors. Recently, the AT-motif binding factor 1 (ATBF1)-A gene, which has been assigned to chromosome 16q22.3-23.1, was identified as a plausible candidate for tumor suppression in solid tumors due to its functional inhibition of cell proliferation and high mutation rate in prostate cancer. We previously reported that a reduction in ATBF1-A mRNA levels correlated with a worse prognosis in breast cancer. However, the mechanisms regulating the reduction of ATBF1-A mRNA levels (such as mutation, methylation in the promoter region, or deletion spanning the coding region) have not been fully examined. In addition, few studies have analyzed LOH status at the ATBF1-A locus, located in the 16q22 minimal region.MethodsProfiles of ATBF1-A mRNA levels that we previously reported for 127 cases were used. In this study, breast cancer specimens as well as autologous blood samples were screened for LOH using 6 polymorphic microsatellite markers spanning chromosome band 16q22. For mutational analysis, we selected 12 cases and analyzed selected spots in the ATBF1-A coding region at which mutations have been frequently reported in prostate cancer.ResultsForty-three cases that yielded clear profiles of LOH status at both D16S3106 and D16S3018 microsatellites, nearest to the location of the ATBF1-A gene, were regarded as informative and were classified into two groups: LOH (22 cases) and retention of heterozygosity (21 cases). Comparative assessment of the ATBF1-A mRNA levels according to LOH status at the ATBF1-A locus demonstrated no relationship between them. In the 12 cases screened for mutational analysis, there were no somatic mutations with amino acid substitution or frameshift; however, two germ line alterations with possible polymorphisms were observed.ConclusionThese findings imply that ATBF1-A mRNA levels are regulated at the transcriptional stage, but not by genetic mechanisms, deletions (LOH), or mutations.