CD271 mRNA/hnRNPA2B1 complex promotes proliferation and stemness in oral and head and neck squamous cell carcinoma.

RNAs, such as noncoding RNA, microRNA, and recently mRNA, have been recognized as signal transduction molecules. CD271, also known as nerve growth factor receptor, has a critical role in cancer, although the precise mechanism is still unclear. Here, we show that CD271 mRNA, but not CD271 protein, facilitates spheroid cell proliferation. We established CD271-/- cells lacking both mRNA and protein of CD271, as well as CD271 protein knockout cells lacking only CD271 protein, from hypopharyngeal and oral squamous cell carcinoma lines. Sphere formation was reduced in CD271-/- cells but not in CD271 protein knockout cells. Mutated CD271 mRNA, which is not translated to a protein, promoted sphere formation. CD271 mRNA bound to hnRNPA2B1 protein at the 3'-UTR region, and the inhibition of this interaction reduced sphere formation. In surgical specimens, the CD271 mRNA/protein expression ratio was higher in the cancerous area than in the noncancerous area. These data suggest CD271 mRNA has dual functions, encompassing protein-coding and noncoding roles, with its noncoding RNA function being predominant in oral and head and neck squamous cell carcinoma.

RELA is required for CD271 expression and stem-like characteristics in hypopharyngeal cancer.

CD271 (also referred to as nerve growth factor receptor or p75NTR) is expressed on cancer stem cells in hypopharyngeal cancer (HPC) and regulates cell proliferation. Because elevated expression of CD271 increases cancer malignancy and correlates with poor prognosis, CD271 could be a promising therapeutic target; however, little is known about the induction of CD271 expression and especially its promoter activity. In this study, we screened transcription factors and found that RELA (p65), a subunit of nuclear factor kappaB (NF-κB), is critical for CD271 transcription in cancer cells. Specifically, we found that RELA promoted CD271 transcription in squamous cell carcinoma cell lines but not in normal epithelium and neuroblastoma cell lines. Within the CD271 promoter sequence, region + 957 to + 1138 was important for RELA binding, and cells harboring deletions in proximity to the + 1045 region decreased CD271 expression and sphere-formation activity. Additionally, we found that clinical tissue samples showing elevated CD271 expression were enriched in RELA-binding sites and that HPC tissues showed elevated levels of both CD271 and phosphorylated RELA. These data suggested that RELA increases CD271 expression and that inhibition of RELA binding to the CD271 promoter could be an effective therapeutic target.

Establishment of a monoclonal antibody against glycosylated CD271 specific for cancer cells in immunohistochemistry.

Various proteins are highly expressed in cancer (e.g., epidermal growth factor receptor); however, the majority are also expressed in normal cells, although they may differ in expression intensity. Recently, we reported that CD271 (nerve growth factor receptor), a glycosylated protein, increases malignant behavior of cancer, particularly stemlike phenotypes in squamous cell carcinoma (SCC). CD271 is expressed in SCC and in normal epithelial basal cells. Glycosylation alterations generally occur in cancer cells; therefore, we attempted to establish a cancer-specific anti-glycosylated CD271 antibody. We purified recombinant glycosylated CD271 protein, immunized mice with the protein, and screened hybridomas using an ELISA assay with cancer cell lines. We established a clone G4B1 against CD271 which is glycosylated with O-glycan and sialic acid. The G4B1 antibody reacted with the CD271 protein expressed in esophageal cancer, but not in normal esophageal basal cells. This specificity was confirmed in hypopharyngeal and cervical cancers. G4B1 antibody recognized the fetal esophageal epithelium and Barrett's esophagus, which possess stem cell-like characteristics. In conclusion, G4B1 antibody could be useful for precise identification of dysplasia and cancer cells in SCC.

BEX2 is required for maintaining dormant cancer stem cell in hepatocellular carcinoma.

Cancer stem cells (CSCs) are responsible for therapy resistance and share several properties with normal stem cells. Here, we show that brain-expressed X-linked gene 2 (BEX2), which is essential for dormant CSCs in cholangiocarcinoma, is highly expressed in human hepatocellular carcinoma (HCC) lesions compared with the adjacent normal lesions and that in 41 HCC cases the BEX2high expression group is correlated with a poor prognosis. BEX2 localizes to Ki67-negative (nonproliferative) cancer cells in HCC tissues and is highly expressed in the dormant fraction of HCC cell lines. Knockdown of BEX2 attenuates CSC phenotypes, including sphere formation ability and aldefluor activity, and BEX2 overexpression enhances these phenotypes. Moreover, BEX2 knockdown increases cisplatin sensitivity, and BEX2 expression is induced by cisplatin treatment. Taken together, these data suggest that BEX2 induces dormant CSC properties and affects the prognosis of patients with HCC.

Discovery of a chemical compound that suppresses expression of BEX2, a dormant cancer stem cell-related protein.

Cancer stem cells (CSCs) are believed to cause cancer metastasis and recurrence. BEX2 (brain expressed X-linked gene 2) is a CSC-related gene that is expressed in dormant CSCs in cholangiocarcinoma and induces resistance against chemotherapy. The aim of the present study was to identify small compounds that have activity to inhibit BEX2 expression and result in the attenuation of CSC-related phenotypes. We screened 9600 small chemical compounds in high-throughput screening using cholangiocarcinoma cell line HuCCT1 expressing BEX2 protein fused with NanoLuc, and identified a compound, BMPP (1, 3-Benzenediol, [4-(4-methoxyphenyl)-1H-pyrazol-3-yl]). BMPP was found to exert decreasing effects on BEX2 protein expression and G0 phase population of the tumor cells, and increasing effects on ATP levels and chemotherapeutic sensitivity of the cells. These findings indicate that BMPP is a valuable chemical compound for reducing dormant CSC-related phenotypes. Thus, the identification of BMPP as a potential CSC suppressor provides scope for the development of novel therapeutic modalities for the treatment of cancers with BEX2 overexpressing CSCs.

BEX2 suppresses mitochondrial activity and is required for dormant cancer stem cell maintenance in intrahepatic cholangiocarcinoma.

Cancer stem cells (CSCs) define a subpopulation of cancer cells that are resistant to therapy. However, little is known of how CSC characteristics are regulated. We previously showed that dormant cancer stem cells are enriched with a CD274low fraction of cholangiocarcinoma cells. Here we found that BEX2 was highly expressed in CD274low cells, and that BEX2 knockdown decreased the tumorigenicity and G0 phase of cholangiocarcinoma cells. BEX2 was found to be expressed predominantly in G0 phase and starvation induced the USF2 transcriptional factor, which induced BEX2 transcription. Comprehensive screening of BEX2 binding proteins identified E3 ubiquitin ligase complex proteins, FEM1B and CUL2, and a mitochondrial protein TUFM, and further demonstrated that knockdown of BEX2 or TUFM increased mitochondria-related oxygen consumption and decreased tumorigenicity in cholangiocarcinoma cells. These results suggest that BEX2 is essential for maintaining dormant cancer stem cells through the suppression of mitochondrial activity in cholangiocarcinoma.

CD109 promotes the tumorigenic ability and metastatic motility of pancreatic ductal adenocarcinoma cells.

BACKGROUND: Accumulating evidence indicates that CD109, a glycosylphosphatidylinositol-anchored glycoprotein, is highly expressed in human epithelial carcinomas of multiple organs including the pancreas, but its functional role in carcinoma development has not yet been fully clarified. The aim of this study was to investigate the role of CD109 in the malignancy of pancreatic ductal adenocarcinoma (PDAC). METHODS: PDAC specimens of 145 cases were immunostained for CD109, and correlations between CD109 expression and clinicopathological conditions were analyzed. CD109 expression in PANC-1 cells, a PDAC-derived cell line, was decreased by siRNA or shRNA and its effect on the malignancy of PANC-1 cells was examined. RESULTS: Suppression of CD109 expression in PANC-1 cells resulted in reduction of in vitro cell motility and tumorigenicity in xenografts. Based on these results, we investigated the relationship between CD109 expression and metastasis of PDAC using tumor tissue specimens. Among 106 recurrent cases of 145 PDAC, there was a tendency for CD109-positive cases to be accompanied by distant metastasis. CONCLUSIONS: CD109 plays a critical role in the promotion of tumorigenic ability and cellular motility relating to metastasis of PDAC cells.

Establishment of a Monoclonal Antibody That Recognizes Cysteine-Rich Domain 1 of Human CD271.

CD271 is a common receptor for all neurotrophins that is localized to neurons, endothelial cells, and the basal layer of the epithelium in normal tissue. Recently, we and others reported that CD271 plays essential roles in the development of squamous cell carcinoma, especially in tumor-initiating cells. Since little is known about how CD271 regulates cancer cell initiation and proliferation, antibodies that recognize different domains of CD271 are needed to enable investigation. Therefore, this study aimed to develop an antihuman CD271 antibody by immunizing mice with a CD271 antigen produced by a baculovirus. The antibody was named hCD271mAb#13, and it recognized cysteine-rich domain 1 with a higher affinity than the commercially available antibody ME20.4. We determined that hCD271mAb#13 is suitable for flow cytometry, Western blotting, immunocytochemistry, and immunohistochemistry of formalin-fixed paraffin-embedded tissue. Use of hCD271mAb#13 for CD271 labeling could enable detailed analyses of cancer cell regulation and other biological processes.

Humanized anti-CD271 monoclonal antibody exerts an anti-tumor effect by depleting cancer stem cells.

CD271, known as a neurotrophin receptor, is expressed in various cancers such as hypopharyngeal cancer (HPC) and melanoma. We recently reported that CD271 is a cancer-stem-cell biomarker of HPC, and that its expression is essential for cancer-cell proliferation and is correlated with a poor prognosis in this disease. Here, to develop a therapeutic antibody to CD271, we established a humanized anti-CD271 monoclonal antibody (hCD271 mA b). hCD271 mA b bound to the cysteine-rich domain 1 (CRD1) of human CD271 with high affinity (KD = 1.697 × 10-9 M). In vitro, hCD271 mA b exerted antibody-dependent cell-mediated cytotoxicity (ADCC) activity against SP2/0-CD271 (human CD271-transduced mouse cell line). Treatment with hCD271 mA b also exerted anti-tumor activity in graft models of three cell lines (HPCM2 (patient-derived xenograft cell line of hypopharyngeal cancer), MeWo-Luc (melanoma cell line), and SP2/0-CD271) in mice, resulting in smaller tumors compared to controls and reduced numbers of CD271-positive cells. Collectively, these data suggest that an antibody targeting CD271 is a promising therapeutic strategy.

CD271 is a negative prognostic factor and essential for cell proliferation in lung squamous cell carcinoma.

Squamous cell carcinoma is a major type of cancer in the lung. While several therapeutic target molecules for lung adenocarcinoma have been identified, little is known about lung squamous cell carcinoma (LSCC). We recently reported that CD271 (p75 neurotrophin receptor) serves as a marker for tumor initiation and is a key regulator of cell proliferation in hypopharyngeal squamous cell carcinoma. In this study, we found that CD271 was also expressed in squamous cell carcinoma, but not in adenocarcinoma, of several tissues, including the lung, and the expression of CD271 was associated with a poor prognosis in LSCC. To examine CD271's role in LSCC, we established xenograft cell lines from LSCC patients. Within the sorted live LSCC cell population, the CD271high cells were primarily cycling through the G2/M phase, while the CD271low cells were mostly in the G0 phase. CD271 knockdown in the LSCC cells completely suppressed their proliferation and tumor-formation capability, and increased their cell-cycle arrest in the G0 phase. In the CD271-knockdown cells, ERK-phosphorylation was decreased, while no change was observed in the IκBα-phosphorylation, p65-phosphorylation, or Akt-phosphorylation. Treatment with the MEK inhibitor U0126 decreased the LSCC cells' proliferation capability. Microarray analysis revealed that CD271 knockdown attenuated the RAS-related pathways. The knockdown of TrkB, which forms a heterodimer with CD271 and accelerates its downstream signaling, partially inhibited the LSCC cell proliferation. These results indicated that LSCC exclusively depends on CD271 for cell proliferation, in part through ERK-signaling activation, and CD271 is a promising target for LSCC therapy.

Crucial role of carbonic anhydrase IX in tumorigenicity of xenotransplanted adult T-cell leukemia-derived cells.

Carbonic anhydrase IX (CA9) is a membrane-associated carbonic anhydrase that regulates cellular pH, is upregulated in various solid tumors, and is considered to be a therapeutic target. Here, we describe the essential role of CA9 in the tumorigenicity of cells derived from human adult T-cell leukemia/lymphoma (ATL). We previously established the highly tumorigenic ST1-N6 subline from the ATL-derived ST1 cell line by serial xenotransplantation in NOG mice. In the present study, we first show that CA9 expression is strongly enhanced in ST1-N6 cells. We then sorted ST1 cells by high or low CA9 expression and established ST1-CA9high and ST1-CA9low sublines. ST1-CA9high cells, like ST1-N6 cells, were more strongly tumorigenic than ST1-CA9low or parental ST1 cells when injected into NOG mice. Knockdown of CA9 with shRNAs suppressed the ability of ST1-CA9high cells to initiate tumors, and the tumorigenicity of ST1 cells was significantly enhanced by introducing wild-type CA9 or a CA9 mutant with deletion of an intracytoplasmic domain. However, a CA9 with point mutations in the catalytic site did not increase the tumorigenicity of ST1 cells. Furthermore, we detected a small population of CA9+ CD25+ cells in lymph nodes of ATL patients. These findings suggest that CA9, and particularly its carbonic anhydrase activity, promotes the tumorigenicity of ATL-derived cells and may be involved in malignant development of lymphoma-type ATL.

CD271 regulates the proliferation and motility of hypopharyngeal cancer cells.

CD271 (p75 neurotrophin receptor) plays both positive and negative roles in cancer development, depending on the cell type. We previously reported that CD271 is a marker for tumor initiation and is correlated with a poor prognosis in human hypopharyngeal cancer (HPC). To clarify the role of CD271 in HPC, we established HPC cell lines and knocked down the CD271 expression using siRNA. We found that CD271-knockdown completely suppressed the cells' tumor-forming capability both in vivo and in vitro. CD271-knockdown also induced cell-cycle arrest in G0 and suppressed ERK phosphorylation. While treatment with an ERK inhibitor only partially inhibited cell growth, CDKN1C, which is required for maintenance of quiescence, was strongly upregulated in CD271-depleted HPC cells, and the double knockdown of CD271 and CDKN1C partially rescued the cells from G0 arrest. In addition, either CD271 depletion or the inhibition of CD271-RhoA signaling by TAT-Pep5 diminished the in vitro migration capability of the HPC cells. Collectively, CD271 initiates tumor formation by increasing the cell proliferation capacity through CDKN1C suppression and ERK-signaling activation, and by accelerating the migration signaling pathway in HPC.

Xenotransplantation elicits salient tumorigenicity of adult T-cell leukemia-derived cells via aberrant AKT activation.

The transplantation of human cancer cells into immunodeficient NOD/SCID/IL-2Rγc(null) (NOG) mice often causes highly malignant cell populations like cancer stem cells to emerge. Here, by serial transplantation in NOG mice, we established two highly tumorigenic adult T-cell leukemia-derived cell lines, ST1-N6 and TL-Om1-N8. When transplanted s.c., these cells formed tumors significantly earlier and from fewer initial cells than their parental lines ST1 and TL-Om1. We found that protein kinase B (AKT) signaling was upregulated in ST1-N6 and TL-Om1-N8 cells, and that this upregulation was due to the decreased expression of a negative regulator, INPP5D. Furthermore, the introduction of a constitutively active AKT mutant expression vector into ST1 cells augmented the tumorigenicity of the cells, whereas treatment with the AKT inhibitor MK-2206 attenuated the progression of tumors induced by ST1-N6 cells. Collectively, our results reveal that the AKT signaling pathway plays a critical role in the malignancy of adult T-cell leukemia-derived cells.

Circulating miR-483-3p and miR-21 is highly expressed in plasma of pancreatic cancer.

Several recent studies have revealed that microRNAs (miRNAs) have a role in carcinogenesis and cancer development, and that it is stably detectable in plasma/serum. The aim of this study was to test whether miR­483-3p as well as miR­21 could be plasma biomarkers for PDAC. The plasma samples were obtained from three groups including 32 pancreatic ductal adenocarcinoma (PDAC) patients, 12 patients with intraductal papillary mucinous neoplasm (IPMN) patients and 30 healthy controls (HC). We evaluated the plasma miR­483-3p and miR­21 expression level by quantitative RT-PCR. We compared the differences in the plasma level of these miRNAs among the three groups, and investigated the relevance of their plasma expression level to the clinical factors in PDAC. The expressions of miR­483-3p and miR­21 were detected in all examined plasma samples. The plasma expression levels of these miRNAs were significantly higher in PDAC compared to HC (P<0.01). The plasma miR­483-3p expression was significantly higher in PDAC patients than IPMN patients (P<0.05). The plasma miR­21 level was associated with advanced stage (P<0.05), metastasis to lymph node and liver (P<0.01), and shorter survival (P<0.01) of the PDAC patients. Together, these findings suggest that measurement of the plasma miR­483-3p level is useful for discriminating PDAC from IPMN, and that the plasma miR­21 level predicts outcome of PDAC patients.

Identification of TL-Om1, an adult T-cell leukemia (ATL) cell line, as reference material for quantitative PCR for human T-lymphotropic virus 1.

Quantitative PCR (qPCR) for human T-lymphotropic virus 1 (HTLV-1) is useful for measuring the amount of integrated HTLV-1 proviral DNA in peripheral blood mononuclear cells. Many laboratories in Japan have developed different HTLV-1 qPCR methods. However, when six independent laboratories analyzed the proviral load of the same samples, there was a 5-fold difference in their results. To standardize HTLV-1 qPCR, preparation of a well-defined reference material is needed. We analyzed the integrated HTLV-1 genome and the internal control (IC) genes of TL-Om1, a cell line derived from adult T-cell leukemia, to confirm its suitability as a reference material for HTLV-1 qPCR. Fluorescent in situ hybridization (FISH) showed that HTLV-1 provirus was monoclonally integrated in chromosome 1 at the site of 1p13 in the TL-Om1 genome. HTLV-1 proviral genome was not transferred from TL-Om1 to an uninfected T-cell line, suggesting that the HTLV-1 proviral copy number in TL-Om1 cells is stable. To determine the copy number of HTLV-1 provirus and IC genes in TL-Om1 cells, we used FISH, digital PCR, and qPCR. HTLV-1 copy numbers obtained by these three methods were similar, suggesting that their results were accurate. Also, the ratio of the copy number of HTLV-1 provirus to one of the IC genes, RNase P, was consistent for all three methods. These findings indicate that TL-Om1 cells are an appropriate reference material for HTLV-1 qPCR.

ESCRT-0 protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is targeted to endosomes independently of signal-transducing adaptor molecule (STAM) and the complex formation with STAM promotes its endosomal dissociation.

The ESCRT-0 complex, consisting of the hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) and the signal-transducing adaptor molecule (STAM) proteins, recognizes ubiquitylated cargo during the initial step of endosomal sorting. The endosomal accumulation of overexpressed Hrs has been reported previously to be associated with endosome enlargement. In this study, we have found that co-expressing exogenous STAM1 in Hrs-overexpressing cells leads to a diffuse localization for a large part of the Hrs accumulated on endosomes and a recovery of the impaired cargo protein degradation process, thus suggesting that exogenous STAM abrogates the abnormalities of the Hrs-positive endosomes. A fluorescently labeled Hrs, introduced into the cells by membrane permeabilization, exhibited endosomal localization in the absence of STAM1 and gradually dissociated from the endosomes upon the sequential addition of recombinant STAM1. Furthermore, when microinjected into cells, the fluorescently labeled Hrs also showed endosomal accumulation; however, ESCRT-0 complexes formed prior to the microinjection did not. Analysis of the state of the complex in HeLa cells using blue-native PAGE revealed that the membrane-associated Hrs exists partly as a monomer and not only in the STAM1-bound form. Thus, our data suggest that the membrane binding and dissociation cycle of the ESCRT-0 proteins on the endosomal membrane is a critical step during the cargo sorting process.

OX40 and IL-7 play synergistic roles in the homeostatic proliferation of effector memory CD4⁺ T cells.

T-cell homeostasis preserves the numbers, the diversity and functional competence of different T-cell subsets that are required for adaptive immunity. Naïve CD4(+) T (TN ) cells are maintained in the periphery via the common γ-chain family cytokine IL-7 and weak antigenic signals. However, it is not clear how memory CD4(+) T-cell subsets are maintained in the periphery and which factors are responsible for the maintenance. To examine the homeostatic mechanisms, CFSE-labeled CD4(+) CD44(high) CD62L(low) effector memory T (TEM ) cells were transferred into sublethally-irradiated syngeneic C57BL/6 mice, and the systemic cell proliferative responses, which can be divided distinctively into fast and slow proliferations, were assessed by CFSE dye dilution. We found that the fast homeostatic proliferation of TEM cells was strictly regulated by both antigen and OX40 costimulatory signals and that the slow proliferation was dependent on IL-7. The simultaneous blockade of both OX40 and IL-7 signaling completely inhibited the both fast and slow proliferation. The antigen- and OX40-dependent fast proliferation preferentially expanded IL-17-producing helper T cells (Th17 cells). Thus, OX40 and IL-7 play synergistic, but distinct roles in the homeostatic proliferation of CD4(+) TEM cells.

Suppressive expression of CD274 increases tumorigenesis and cancer stem cell phenotypes in cholangiocarcinoma.

Cholangiocarcinoma is an aggressive malignant tumor originating from intrahepatic or extrahepatic bile ducts. Its malignant phenotypes may be assumed by cancer stem cells (CSC). Here, we demonstrate that CD274 (PD-L1), known as an immunomodulatory ligand, has suppressive effects on CSC-related phenotypes of cholangiocarcinoma. Using two human cholangiocarcinoma cell lines, RBE and HuCCT1, we attempted to isolate the CD274(low) and CD274(high) cells from each cell line, and xenografted them into immunodeficient NOD/scid/γcnull (NOG) mice. We found that the CD274(low) cells isolated from both RBE and HuCCT1 are highly tumorigenic in NOG mice compared with CD274(high) cells. Furthermore, the CD274(low) cells possess several CSC-related characteristics, such as high aldehyde dehydrogenase (ALDH) activity, reduced reactive oxygen species production and a dormant state in the cell cycle. Furthermore, depletion of CD274 expression by shRNA in RBE cells enhances their tumorigenicity and increases ALDH activity. These findings are compatible with our observation that clinical cholangiocarcinoma specimens are classified into low and high groups for CD274 expression, and the CD274 low group shows poorer prognosis when compared with the CD274 high group. These results strongly suggest that CD274 has a novel function in the negative regulation of CSC-related phenotypes in human cholangiocarcinoma, which is distinct from its immunomodulatory actions.

Hepatocyte growth factor regulated tyrosine kinase substrate in the peripheral development and function of B-cells.

Hepatocyte growth factor (HGF)-regulated tyrosine kinase substrate (Hrs) is a vesicular sorting protein that functions as one of the endosomal-sorting proteins required for transport (ESCRT). Hrs, which binds to ubiquitinated proteins through its ubiquitin-interacting motif (UIM), contributes to the lysosomal transport and degradation of ubiquitinated membrane proteins. However, little is known about the relationship between B-cell functions and ESCRT proteins in vivo. Here we examined the immunological roles of Hrs in B-cell development and functions using B-cell-specific Hrs-deficient (Hrs(flox/flox);mb1(cre/)(+):Hrs-cKO) mice, which were generated using a cre-LoxP recombination system. Hrs deficiency in B-cells significantly reduced T-cell-dependent antibody production in vivo and impaired the proliferation of B-cells treated in vitro with an anti-IgM monoclonal antibody but not with LPS. Although early development of B-cells in the bone marrow was normal in Hrs-cKO mice, there was a significant decrease in the number of the peripheral transitional B-cells and marginal zone B-cells in the spleen of Hrs-cKO mice. These results indicate that Hrs plays important roles during peripheral development and physiological functions of B lymphocytes.