FAXC interacts with ANXA2 and SRC in mitochondria and promotes tumorigenesis in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is one of the most difficult malignancies to treat as the therapeutic options are limited. Although several driver genes have been identified, most remain unknown. In this study, we identified a failed axon connection homolog (FAXC), whose function is unknown in mammals, by analyzing serially passaged CCA xenograft models. Knockdown of FAXC reduced subcutaneous tumorigenicity in mice. FAXC was bound to annexin A2 (ANXA2) and c-SRC, which are tumor-promoting genes. The FAXC/ANXA2/c-SRC complex forms in the mitochondria. FAXC enhances SRC-dependent ANXA2 phosphorylation at tyrosine-24, and the C-terminal amino acid residues (351-375) of FAXC are required for ANXA2 phosphorylation. Transcriptome data from a xenografted CCA cell line revealed that FAXC correlated with epithelial-mesenchymal transition, hypoxia, and KRAS signaling genes. Collectively, these findings advance our understanding of CCA tumorigenesis and provide candidate therapeutic targets.

Simultaneous determination of all aminobutyric acids by chiral derivatization and liquid chromatography-tandem mass spectrometry.

Aminobutyric acids include eight structural or stereoisomers that exhibit a wide range of biological activities. Recent evidence on some low abundant isomers have increased the demand for highly selective analysis of all the isomers; however, simultaneous separation of all the aminobutyric acid isomers has not been successful yet, except for a specialized method that uses multiple separation columns and a split of samples. In this study, we developed a new analytical method using chiral derivatization and liquid chromatography-tandem mass spectrometry to separate all the aminobutyric acid isomers in a single separation column. All the diastereomeric derivatives were resolved in a C18 column, and the derivatives showed characteristic fragmentation patterns in tandem mass spectrometry. By using the method, we analyzed the isomers in the Arabidopsis thaliana seeds and revealed the existence of three low abundant isomers, i.e., D-, L-ß-aminoisobutyric acid, and D-ß-aminobutyric acid. The proposed method uses a commercially available chiral derivatizing reagent and a broadly used column; therefore, it can be widely used in biological and food analyses.

Precolumn derivatization LC/MS method for observation of efficient hydrogen sulfide supply to the kidney via d-cysteine degradation pathway.

d-Cysteine (d-Cys) is metabolized to hydrogen sulfide (H2S) by d-amino acid oxidase (DAO)/3-mercaptopyruvate sulfurtransferase pathway. The pathway is required for H2S supplementation that ameliorates acute kidney injury after the oral administration of d-Cys in mice. However, whether the rate-limiting activity of DAO regulates the tissue-selectivity or the extent of d-Cys degradation and H2S supplementation remains unclear. Here, to analyze the levels of d-Cys and H2S, we use two derivatization methods, a new method with no detectable isomerization of Cys and an established method for H2S. The derivatives were determined by LC/MS using a C18 column. With the methods, we show that inhibition of DAO significantly suppresses the H2S supplementation and d-Cys degradation in the mouse kidney. Additionally, we found that d-Cys is more efficiently metabolized into H2S than l-Cys in the kidney. Our results reveal the utility of the method and support the advantage of d-Cys administration in improving the supply of H2S to the kidneys.

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.

Periostin is a negative prognostic factor and promotes cancer cell proliferation in non-small cell lung cancer.

Periostin is a matricellular protein that is secreted by fibroblasts and interacts with various cell-surface integrin molecules. Although periostin is known to support tumor development in human malignancies, little is known about its effect on lung-cancer progression. We here demonstrate that periostin is a negative prognostic factor that increases tumor proliferation through ERK signaling in non-small cell lung carcinoma. We classified 189 clinical specimens from patients with non-small cell lung-cancer according to high or low periostin expression, and found a better prognosis for patients with low rather than high periostin, even in cases of advanced-stage cancer. In a syngenic implantation model, murine Ex3LL lung-cancer cells formed smaller tumor nodules in periostin-/- mice than in periostin+/+ mice, both at the primary site and at metastatic lung sites. An in vitro proliferation assay showed that stimulation with recombinant periostin increased Ex3LL-cell proliferation. We also found that periostin promotes ERK phosphorylation, but not Akt or FAK activation. These findings suggest that periostin represents a potential target in lung-cancer tumor progression.

Periostin attenuates tumor growth by inducing apoptosis in colitis-related colorectal cancer.

Inflammatory bowel diseases, which are multifactorial autoimmune colitis diseases, are occurring with increasing prevalence. One of the most serious complications of these diseases is colorectal cancer. Here we investigated the role of periostin (Postn), a matricellular protein that interacts with various integrin molecules on the cell surface, in colitis-induced colorectal cancer. Immunohistochemistry of mouse and human colorectal cancer samples revealed that Postn was expressed in the stroma and was upregulated in close proximity to the cancer cells. The colonic tumorigenesis in an inflammation-related colon carcinogenesis mouse model was increased in Postn knock-out (Postn-/-) mice compared to Postn+/+ mice. Although no difference was found in the degree of colitis between Postn+/+ and Postn-/- mice, Postn inhibited tumor growth and induced the apoptosis of mouse rectal cancer cells in vitro. Furthermore, fewer apoptotic colorectal cancer cells were observed in Postn-/- than in Postn+/+ mice. These data suggested that Postn has an anti-tumor effect on colitis-induced colorectal cancer.

Long non-coding RNA HOTAIR up-regulates chemokine (C-C motif) ligand 2 and promotes proliferation of macrophages and myeloid-derived suppressor cells in hepatocellular carcinoma cell lines.

Accumulating evidence demonstrated that Hox antisense intergenic RNA (HOTAIR) serves essential roles in the development and metastasis of several types of cancer. In hepatocellular carcinoma (HCC), high expression of HOTAIR is associated with poor prognosis, and HOTAIR regulates cell migration and proliferation. However, the downstream molecular targets of HOTAIR depend on the cancer cell types, and little is known about the precise molecular mechanisms of HOTAIR involved in cancer development. The present study investigated the role of HOTAIR in HCC cell lines. Notably, the overexpression of HOTAIR in HCC cell lines did not affect cell migration and proliferation capability. In the microarray analysis, C-C motif chemokine ligand (CCL)2 was identified to be differentially expressed in HOTAIR-overexpressing cells, and it was confirmed that HOTAIR promotes the secretion of CCL2. Furthermore, it was revealed that the proportion of macrophages and myeloid-derived suppressor cells (MDSCs) were increased when peripheral blood mononuclear cells were co-cultured with HOTAIR-overexpressing cells. Collectively, these data suggest that HOTAIR regulates CCL2 expression, which may be involved in the recruitment of macrophages and MDSCs to the tumor microenvironment.

Long non-coding RNA HOTAIR promotes cell migration by upregulating insulin growth factor-binding protein 2 in renal cell carcinoma.

Renal cell carcinoma (RCC) is one of the most lethal urologic cancers. About one-third of RCC patients already have distal metastasis at the time of diagnosis. There is growing evidence that Hox antisense intergenic RNA (HOTAIR) plays essential roles in metastasis in several types of cancers. However, the precise mechanism by which HOTAIR enhances malignancy remains unclear, especially in RCC. Here, we demonstrated that HOTAIR enhances RCC-cell migration by regulating the insulin growth factor-binding protein 2 (IGFBP2) expression. HOTAIR expression in tumors was significantly correlated with nuclear grade, lymph-node metastasis, and lung metastasis. High HOTAIR expression was associated with a poor prognosis in both our dataset and The Cancer Genome Atlas dataset. Migratory capacity was enhanced in RCC cell lines in a HOTAIR-dependent manner. HOTAIR overexpression accelerated tumorigenicity and lung metastasis in immunodeficient mice. Microarray analysis revealed that IGFBP2 expression was upregulated in HOTAIR-overexpressing cells compared with control cells. The enhanced migration activity of HOTAIR-overexpressing cells was attenuated by IGFBP2 knockdown. IGFBP2 and HOTAIR were co-expressed in clinical RCC samples. Our findings suggest that the HOTAIR-IGFBP2 axis plays critical roles in RCC metastasis and may serve as a novel therapeutic target for advanced RCC.

Enhanced expression of semaphorin 3E is involved in the gastric cancer development.

Semaphorins and their receptors are abnormally expressed in various cancers, but little is known about the expression and function of semaphorin 3E (SEMA3E) and its receptor, plexin D1 (PLXND1), in gastric cancer development or metastasis. We evaluated SEMA3E and PLXND1 expression by quantitative RT-PCR in gastric tissues from 62 patients who underwent gastrectomy and analyzed the correlation between their expression and clinicopathological variables. To assess the function of SEMA3E, we generated human gastric cancer cell lines with suppressed or increased SEMA3E expression. The expression level of SEMA3E, but not PLXND1, was correlated with lymph node involvement and metastatic progression in gastric cancer. A significant association was observed between a high level of SEMA3E expression and poor differentiation or poor survival in the intestinal type of gastric cancer. SEMA3E knockdown in gastric cancer cells attenuated cell proliferation and metastatic ability in vitro and in vivo. Moreover, SEMA3E caused cell proliferation and anchorage-independent cell growth in the intestinal type of gastric cancer. These results suggested that SEMA3E is likely to be involved in the development of gastric cancer and might also be a therapeutic target for its treatment.

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.

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.

Heme oxygenase-1 inhibits cytokine production by activated mast cells.

Heme oxygenase-1 (HO-1) is thought to contribute to host defense reactions against various stresses. In addition, recent reports have suggested that HO-1 modulates immunocyte activation and functions. HO-1 suppresses mast cell degranulation, but whether HO-1 suppresses cytokine synthesis as well is not yet known. We examined whether rat HO-1 cDNA transfected rat basophilic leukemia (RBL)-2H3 cells have altered cytokine production in response to stimulation with anti-ovalbumin (OA) serum/OA compared to Mock transfected RBL-2H3 cells. HO-1 inhibited anti-OA serum/OA-induced IL-3 and TNF-alpha production. Inhibition of HO-1 activity by Zn (II) protoporphyrin IX, a specific HO-1 inhibitor, prevented the suppression of TNF-alpha production. The cytokine inhibition by HO-1 was associated with selective suppression of the DNA-binding activity of AP-1 transcription factors. The suppression of mast cell cytokine production by HO-1 may be an important aspect of the processes that lead to resolution of allergic inflammation.