AMIGO2 enhances the invasive potential of colorectal cancer by inducing EMT.

In our previous studies, we identified amphoterin-inducible gene and open reading frame 2 (AMIGO2) as a driver gene for liver metastasis and found that AMIGO2 expression in cancer cells worsens the prognosis of patients with colorectal cancer (CRC). Epithelial-mesenchymal transition (EMT) is a trigger for CRC to acquire a malignant phenotype, such as invasive potential, leading to metastasis. However, the role of AMIGO2 expression in the invasive potential of CRC cells remains unclear. Thus, this study aimed to examine AMIGO2 expression and elucidate the mechanisms by which it induces EMT and promotes CRC invasion. Activation of the TGFß/Smad signaling pathway was found involved in AMIGO2-induced EMT, and treatment with the TGFß receptor inhibitor LY2109761 suppressed AMIGO2-induced EMT. Studies using CRC samples showed that AMIGO2 expression was highly upregulated in the invasive front, where AMIGO2 expression was localized to the nucleus and associated with EMT marker expression. These results suggest that the nuclear translocation of AMIGO2 induces EMT to promote CRC invasion by activating the TGFß/Smad signaling pathway. Thus, AMIGO2 is an attractive therapeutic target for inhibiting EMT and metastatic CRC progression.

Down-regulating Effect of a Standardized Extract of Cultured Lentinula edodes mycelia on Cortactin in Prostate Cancer Cells Is Dependent on Malignant Potential.

BACKGROUND/AIM: The incidence and mortality rates of prostate cancer have been increasing worldwide. Although prostate cancer cells grow slowly in the local original site, once the cancer cells spread to distant organs they grow rapidly and show very aggressive features. Cortactin is a protein that regulates the actin cytoskeleton and plays crucial roles in cancer metastasis. Up-regulated cortactin is correlated with the metastatic capacity of prostate cancer cells. AHCC®, a standardized extract of cultured Lentinula edodes mycelia, has been previously reported to have cortactin-down-regulating effects on human pancreatic cancer cells. In the present study, the effects of AHCC® treatment on cortactin levels in prostate cancer cells was evaluated. MATERIALS AND METHODS: LNCaP.FGC, DU145, and PC-3 are human prostate cancer cell lines. LNCaP.FGC is well differentiated, androgen-dependent, and poorly metastatic. DU145 is less differentiated, androgen-independent, and moderate metastatic. PC-3 is less differentiated, androgen-independent, and highly metastatic. The effects of AHCC® treatment on cortactin levels in prostate cancer cells was evaluated by western blot. RESULTS: In vitro AHCC® treatment decreased cortactin levels in LNCaP.FGC and DU145 cells but did not change those in PC-3 cells. CONCLUSION: AHCC® treatment down-regulated cortactin expression in poor and moderate metastatic LNCaP.FGC and DU145 cells but showed no effect on cortactin expression in the highly metastatic PC-3 cells. Further studies are required to elucidate the mechanism of the resistance to AHCC® treatment in highly metastatic PC-3 cells.

Plan-view characterization of intergranular precipitates on grain boundaries by combination of FIB lift out method and TEM analyses: A case study in austenitic stainless steel.

A new characterization method is proposed to study intergranular precipitates of polycrystalline material in the planar manner. A dual beam focused ion beam (FIB) - scanning electron microscopy (SEM) was applied to fabricate thin FIB lamella with a grain boundary parallel to the lamella to investigate for transmission electron microscopy (TEM). Distributions, microstructures and compositions of intergranular precipitates of austenitic stainless steel were then examined by TEM, scanning transmission electron microscopy (STEM), and energy dispersive X-ray spectroscopy (EDS). This plan-view microstructural characterization methods would play important roles in the case of materials where the intergranular precipitates play key roles for their physical and chemical properties.

NONO Is a Negative Regulator of SOX2 Promoter.

BACKGROUND/AIM: Sex determining region Y (SRY)-box 2 (SOX2) is a transcription factor essential for the maintenance of proliferation and self-renewal of cancer stem cells and is associated with breast cancer initiation. Regulation of cancer stem cell plasticity by SOX2 requires both positive and negative SOX2 transcription factors, but the negative regulator is still largely unknown. MATERIALS AND METHODS: SOX2 promoter-binding proteins were identified by liquid chromatography-mass spectrometry/mass spectrometry, luciferase assay, and chromatin immunoprecipitation. The effects of one such transcription factor on SOX2 expression was investigated by knockdown and overexpression experiments. RESULTS: Non-POU domain-containing octamer-binding protein (NONO) (also known as 54-kDa nuclear RNA-binding protein, P54NRB) was identified as a SOX2 promoter-binding protein and a negative regulator of SOX2 expression. Its activity was controlled by its coiled-coil domain and the C-terminal domain. CONCLUSION: These results suggest that NONO acts as a key regulator of SOX2 transcription through the repression of SOX2 promoter activity in breast cancer cells.

ADAR2 Regulates Malignant Behaviour of Mesothelioma Cells Independent of RNA-editing Activity.

BACKGROUND/AIM: Malignant pleural mesothelioma (MPM) is an intractable cancer, and causes of its malignant transformation are not well known. Adenosine deaminase acting on RNA (ADAR) is an RNA-editing enzyme that converts adenosine into inosine in double-stranded RNAs potentially involved in malignant development. MATERIALS AND METHODS: To examine the role of ADAR1 and ADAR2 in MPM, small interfering RNAs (siRNAs) against ADAR1 or ADAR2 were used. RESULTS: Transfection of siRNA against ADAR2 suppressed proliferation, motility, and invasiveness of MPM cells expressing both ADAR1 and ADAR2; however, siRNA against ADAR1 did not affect these cellular activities. Overexpression of ADAR2, that was incapable of binding to RNA, suppressed growth, motility, and invasion of MPM cells. However, overexpression of ADAR2 that had no enzyme activity did not alter the malignant properties of MPM cells. CONCLUSION: Enhancement of the malignant characteristics of cultured MPM cells via ADAR2 was independent of RNA-editing activity.

Dominant-negative p53 mutant R248Q increases the motile and invasive activities of oral squamous cell carcinoma cells.

The tumor suppressor gene TP53 (gene) codes for a transcription factor which transactivates its target genes responsible for cell cycle arrest, DNA repair, apoptosis, and senescence. TP53 is well known to be the most frequent target of genetic mutations in nearly half of human cancers including oral squamous cell carcinoma (OSCC). Many p53 mutants including R248Q and R248W not only lose its tumor-suppressor activities, but also interfere with the functions of wild-type p53; this is so-called dominant-negative (DN) mutation. The DN p53 mutation is a predictor of poor outcome in patients with various cancers, and also a risk factor for metastatic recurrence in patients with OSCC. Recently it has been reported that DN p53 mutants acquire new oncogenic activities, which is named gain-of-function (GOF). This study aimed at determining whether R248Q and R248W were involved in OSCC cells' acquiring aggressive phenotypes, using SAS, HSC4 and Ca9-22 cell lines. First, two mutants p53, R248Q and R248W, were respectively transfected into SAS cells harboring recessive-type p53 (E336X). As a result, SAS cells expressing R248Q showed highly spreading, motile and invasive activities compared to parent or mock-transfected cells whereas those expressing R248W did not increase those activities. Secondly, in HSC4 cells harboring R248Q and Ca9-22 cells harboring R248W, expressions of the mutants p53 were inhibited by the transfection with siRNAs targeting p53. The inhibition of the mutants p53 decreased spreading, motile and invasive activities of HSC4 cells whereas it did not affect those activities of Ca9-22 cells. These findings suggest that R248Q p53 mutation, but not R248W p53 mutation, induces more motile and invasive potentials in human OSCC cells.

CUB Domain-containing Protein 1 (CDCP1) Is Down-regulated by Active Hexose-correlated Compound in Human Pancreatic Cancer Cells.

BACKGROUND/AIM: We have previously reported that treatment of pancreatic cancer cells with active hexose-correlated compound (AHCC), an extract of a basidiomycete mushroom, decreases the levels of tumor-associated proteins including heat-shock protein 27 (HSP27), heat shock factor 1 (HSF1) and sex-determining region Y-box 2 (SOX2). The transmembrane glycoprotein, CUB domain-containing protein 1 (CDCP1) has been reported to be up-regulated in various cancers, and be associated with invasion and metastasis. The aim of this study was to examine the effect of AHCC on the expression of CDCP1 in KLM1-R cells. MATERIALS AND METHODS: Gemcitabine-resistant pancreatic cancer cells (KLM1-R) were treated with AHCC (10 mg/ml) for 48 h. Western blot analysis of cell extracts with anti-CDCP1 or anti-actin antibodies was performed to assess the expression of CDCP1. RESULTS: Expression of CDCP1 was reduced by AHCC treatment of KLM1-R cells, whereas expression of actin was not affected. The ratio of intensities of CDCP1/actin in AHCC-treated KLM1-R cells was significantly suppressed (p<0.05) compared to untreated cells. CONCLUSION: AHCC down-regulated CDCP1 expression and inhibited the malignant progression of pancreatic cancer cells.

Tumour endothelial cells in high metastatic tumours promote metastasis via epigenetic dysregulation of biglycan.

Tumour blood vessels are gateways for distant metastasis. Recent studies have revealed that tumour endothelial cells (TECs) demonstrate distinct phenotypes from their normal counterparts. We have demonstrated that features of TECs are different depending on tumour malignancy, suggesting that TECs communicate with surrounding tumour cells. However, the contribution of TECs to metastasis has not been elucidated. Here, we show that TECs actively promote tumour metastasis through a bidirectional interaction between tumour cells and TECs. Co-implantation of TECs isolated from highly metastatic tumours accelerated lung metastases of low metastatic tumours. Biglycan, a small leucine-rich repeat proteoglycan secreted from TECs, activated tumour cell migration via nuclear factor-κB and extracellular signal-regulated kinase 1/2. Biglycan expression was upregulated by DNA demethylation in TECs. Collectively, our results demonstrate that TECs are altered in their microenvironment and, in turn, instigate tumour cells to metastasize, which is a novel mechanism for tumour metastasis.

Fascin regulates chronic inflammation-related human colon carcinogenesis by inhibiting cell anoikis.

By a proteomics-based approach, we identified an overexpression of fascin in colon adenocarcinoma cells (FPCKpP-3) that developed from nontumorigenic human colonic adenoma cells (FPCK-1-1) and were converted to tumorigenic by foreign-body-induced chronic inflammation in nude mice. Fascin overexpression was also observed in the tumors arising from rat intestinal epithelial cells (IEC 6) converted to tumorigenic in chronic inflammation which was induced in the same manner. Upregulation of fascin expression in FPCK-1-1 cells by transfection with sense fascin cDNA converted the cells tumorigenic, whereas antisense fascin-cDNA-transfected FPCKpP-3 cells reduced fascin expression and lost their tumor-forming ability in vivo. The tumorigenic potential by fascin expression was consistent with their ability to survive and grow in the three-dimensional multicellular spheroids. We found that resistance to anoikis (apoptotic cell death as a consequence of insufficient cell-to-substrate interactions), which is represented by the three-dimensional growth of solid tumors in vivo, was regulated by fascin expression through caspase-dependent apoptotic signals. From these, we demonstrate that fascin is a potent suppressor to caspase-associated anoikis and accelerator of the conversion of colonic adenoma cells into adenocarcinoma cells by chronic inflammation.

Enhancement of in vitro cell motility and invasiveness of human malignant pleural mesothelioma cells through the HIF-1α-MUC1 pathway.

In this study, we examined the effects of hypoxia on the malignancy of human malignant pleural mesothelioma (MPM) cell lines, and found (1) hypoxia enhanced motility and invasiveness of human malignant pleural mesothelioma (MPM) cells; (2) this phenomenon resulted from increased expression of sialylated MUC1 through the activation of HIF-1 pathway; (3) two HIF-binding sites located in the promoter region of MUC1 were important for MUC1 transactivation under hypoxia. These findings are useful for better understanding molecular mechanisms of aggressive behavior of MPM cells and for targeting them in the clinical therapies for MPM patients.

Isolation and characterization of human breast cancer cells with SOX2 promoter activity.

Sex determining region Y-box 2 (SOX2) is well known as one of the "stemness" factors and is often expressed in cancers including breast cancer. In this study, we developed a reporter system using fluorescent protein driven by the promoter for SOX2 gene to detect and isolate living SOX2-positive cells. Using this system, we determined that SOX2 promoter activities were well correlated with SOX2 mRNA expression levels in 5 breast cancer cell lines, and that the cell population with positive SOX2 promoter activity (pSp-T(+)) isolated from one of the 5 cell lines, MCF-7 cells, showed a high SOX2 protein expression and high sphere-forming activity compared with very low promoter activity (pSp-T(low/-)). The pSp-T(+) population expressed higher mRNA levels of several stemness-related genes such as CD44, ABCB1, NANOG and TWIST1 than the pSp-T(low/-) population whereas the two populations expressed CD24 at similar levels. These results suggest that the cell population with SOX2 promoter activity contains cancer stem cell (CSC)-like cells which show different expression profiles from those of CSC-marker genes previously recognized in human breast cancers.

Hypoxia affects in vitro growth of newly established cell lines from patients with malignant pleural mesothelioma.

Human malignant pleural mesothelioma (MPM) is highly aggressive, and its prognosis is very poor. For an early diagnosis of MPM and developing new therapeutic strategies against the malignancy, it is necessary to better understand biological characteristics of MPM. In this study, we established two cell lines from pleural effusions derived from patients with MPM. Both cell lines expressed tumor markers of mesothelioma such as mesothelin, podoplanin, WT1, calretinin and keratin 5/6 whereas they did not express either CEA or TTF-1 which are often used as markers of lung adenocarcinoma. The cell lines harboured wild-type TP53, produced hyaluronic acid, and were not infected with SV40. When these two cell lines were cultured under hypoxia (1% O(2)), they showed particular responses to the hypoxic condition, distinct from those to normoxic condition (21% O(2)). Namely, the ability to form a colony originating from a single cell (plating efficiency and cloning efficiency) was stimulated under hypoxia in both cell lines. On the other hand, when the assays of cell growth were started at a relatively high cell density, the growth of both cell lines, regardless of anchorage-dependent or -independent, decreased under hypoxia. The differences of their growth between under hypoxia and under normoxia, and those depending on the cell density, may provide useful hints for developing a new strategy for diagnosis or therapy of MPM.

Differential expressions of matrix metalloproteinases, a disintegrin and metalloproteinases, and a disintegrin and metalloproteinases with thrombospondin motifs and their endogenous inhibitors among histologic subtypes of lung cancers.

Lung cancer is a heterogeneous disease with several histologic subtypes. The two major pathologies, which account for approximately 70% of lung cancers, are adenocarcinoma (AD) and squamous cell carcinoma (SQ). Traditionally, these two subtypes have been categorized as non-small-cell lung cancer and treated similarly. However, they are different not only pathologically, but also functionally. For example, ¹8F-fluorodeoxyglucose positron emission tomography (FDG-PET), which assesses glucose metabolism in tumor tissues, shows that SQ has higher glucose metabolism than does AD. Matrix metalloproteinases (MMPs) and their inhibitors play pleiotropic roles in cancer development, carcinogenesis, apoptosis, angiogenesis, invasion and metastasis. Expression of MMPs and their associated molecules is different among the subtypes of lung cancer. Expression levels of MMP-2, MMP-7, reversion-inducing cysteine-rich protein with Kazal motifs (RECK), tissue inhibitors of metalloproteinase (TIMP)-1, and TIMP-2 are higher in AD than in SQ. In contrast, expression levels of MMP-1, MMP-8, MMP-9 and TIMP-3 are higher in SQ than in AD. Serum levels of a disintegrin and metalloproteinase (ADAM)-8 and ADAM-28 are higher in lung cancer patients than in healthy controls. High expression of ADAM-28 correlates with metastasis and recurrence, but there is no significant difference in ADAM-8 or ADAM-28 expression between AD and SQ. It is necessary to recognize the differential expression patterns of MMPs, their endogenous inhibitors and associated molecules for each subtype of lung cancer in order to develop clinical markers, therapeutic inhibitors and treatment strategies using MMP inhibitors.

Increased expression of the PRL-3 gene in human oral squamous cell carcinoma and dysplasia tissues.

Phosphatase of regenerating liver (PRL) belongs to a class of the protein tyrosine phosphatase family, which is known so far to consist of 3 members, PRL-1, PRL-2, and PRL-3. The aim of this study was to uncover the role of PRL genes in development of oral malignancy. We analyzed expression levels of the 3 PRL genes in 50 human oral squamous cell carcinomas (OSCCs), 11 dysplasia and 12 normal mucosa tissues by a real-time RT-PCR method. PRL-3 but not PRL-1 or PRL-2 expressions were significantly higher in OSCC and dysplasia than in normal mucosa tissues. Additionally, PRL-3 expressions were significantly higher in OSCC tissues harboring dominant-negative p53 or recessive p53 mutation than in those harboring wild-type p53. These results suggest that PRL-3 plays a role in oral cancer development and can be useful as a marker of pre-malignant and malignant lesion of oral mucosa.

Mutant p53 R248Q but not R248W enhances in vitro invasiveness of human lung cancer NCI-H1299 cells.

More than half of all human cancers are associated with mutations of the TP53 gene. In regard to the functional interaction with the remaining wild-type (WT) p53 allele, p53 mutations are classified into two types, recessive and dominant-negative (DN) mutations. The latter mutant protein has a DN activity over the remaining WT allele. We previously showed that the DN p53 mutant was useful as a predictor of poor outcome or a risk factor for metastatic recurrence in patients with some types of cancers, regardless of the presence or absence of loss of heterozygosity (LOH) of WT p53, suggesting that the DN p53 had 'gain-of-function (GOF)' activity besides the transdominance function. In this study, we investigated GOF activity of two DN p53 mutants which had a point mutation at codon 248 (R248Q and R248W), one of the hot spots, by transfecting them respectively into H1299 cells which originally expressed no p53 protein. Growth activity of the transfectants with the two mutants was not different from that of parent or Mock transfectants. Meanwhile, in vitro invasions of Matrigel and type I collagen gel by R248Q-transfectants were significantly higher than those by R248W-transfectants or the control cells. However, there were no differences in cell motile activities, expressions of extracellular matrix-degradative enzymes such as matrix metalloproteinases, urokinase-type plasminogen activator and heparanase, and their inhibitors, between R248Q- and R248W-transfectants. These findings indicate that the p53 mutants have a different quality in GOF activities even if the mutations occurred at the same codon. And detailed information of the status of p53, including transdominancy and GOF activity, is expected to be useful for diagnosis and therapeutic strategy fitting the individual patients.

A multiple primary carcinoma consisting of leukoplakia and SCC: a case report with p53 mutation analysis.

Patients with an oral squamous cell carcinoma (OSCC) often develop multiple malignant lesions. This report examined whether individual tumours developed in a patient show the same genetic alteration, such as p53 mutations. This case study describes three SCCs and three leukoplakias which developed simultaneously in a single 67-year-old Japanese man. A p53 mutation was detected in two of the three SCCs and one of the three leukoplakias. One SCC had a missense mutation at codon 285 (GAG>AAG, Glu>Lys) and the other a nonsense mutation at codon 336, and the leukoplakia had a missense mutation at codon 273 (CGT>CAT, Arg>His). This case showed that individual oral tumours may have different genetic changes even when they develop in a single patient. Therefore, this report provided strong evidence that in cases of multiple tumours it is necessary to design tailor-made therapies for each individual tumour rather than a single standardised therapy for all multiple tumours.

Imatinib mesylate inhibits the proliferation-stimulating effect of human lung cancer-associated stromal fibroblasts on lung cancer cells.

Platelet-derived growth factor (PDGF) is a significant mediator in the proliferation of cancer-associated stromal fibroblasts (CAFs). The inhibition of CAF proliferation by blocking PDGF signaling could lead to a development of novel cancer therapy. We analyzed whether inhibiting proliferation of lung CAFs by imatinib mesylate, which has inhibitory activity on PDGF-receptor tyrosine kinase, could suppress the proliferative activity of lung cancer cells which coexisted in the tumor tissue. First, we established primary cultured fibroblasts from human lung cancer tissues. RT-PCR analysis showed that PDGF-receptors (PDGFRalpha and beta) were more highly expressed in the fibroblasts, whereas PDGFs (PDGF-A, and -B) were more in lung cancer cell lines. Western blotting showed that imatinib treatment inhibited phosphorylation of PDGFRbeta, Akt, and Erk1/2 in the fibroblasts. The treatment also significantly inhibited the proliferative activity of the fibroblasts. The inhibitory effects were exerted more definitely in co-administering imatinib and PDGF-BB, a dimer of the polypeptide chains of B, than in administering imatinib alone. The conditioned media of the fibroblasts significantly increased the proliferative activity of human lung cancer cell line A549 compared to control culture medium. The proliferation-stimulating effect on A549 cells decreased significantly in the conditioned media of the primary cultured fibroblasts that had been treated with imatinib. Our results suggest that imatinib has antitumor activity which is exerted by reducing the proliferation-stimulating effect of CAFs on lung cancer cells, as well as inhibiting the proliferation of CAFs, by way of blocking PDGF signaling.

Aberrant expressions of HOX genes in colorectal and hepatocellular carcinomas.

HOX genes are known as master regulator genes which give cells positional information in embryogenesis. In this study, we compared the expression patterns of 39 HOX genes among human colorectal carcinomas from the right large intestine (cecum, ascending and transverse colon), those from the left large intestine (discending and sigmoid colon, and rectum) and hepatocellular carcinoma. The expression levels of each HOX gene were quantified by analysis based on the real-time RT-PCR. The expression patterns of HOX genes in colorectal and hepatocellular carcinoma tissues differed from those in their normal or non-cancerous tissues. Between the tumor tissues in the right-side large intestine and those in the left-side, different HOX genes were expressed in association with cancer. Further, the expression levels of HOXD8 in liver-metastatic tissues of colorectal carcinomas were as low as in non-cancerous liver tissues, and were significantly lower than those in the primary tissues. These results suggest that dysregulated expressions of HOX genes play an important role in carcinogenesis and malignant progression of colorectal and hepatocellular carcinomas.

Laminin-421 produced by lymphatic endothelial cells induces chemotaxis for human melanoma cells.

Melanoma has a high tendency to metastasize to lymph nodes, which is one of the clinicopathological factors to indicate poor prognosis. Recent investigations have shown the importance of lymphangiogenesis in lymph node metastasis in a variety of human tumors including melanoma. However, molecular mechanism of lymphatic metastasis is still poorly defined. We examined influence of interactions between normal lymphatic endothelial cells (LECs) and melanoma cells on cell migration. Medium conditioned with LEC (LEC-CM) contained chemotactic and chemokinetic activities for human melanoma cell lines. The chemotactic activity was fractionated in more than 100 kDa, and inactivated by heat-treatment. The chemotactic activity of LEC-CM was abolished by immunodepletion with anti-laminin-1 antibody. And immunoprecipitation and Western blot analyses revealed that LEC-CM contained laminin-421. When melanoma C8161 cells were treated with function-blocking antibodies to integrin alpha3 or alpha6, their chemotactic responses to LEC-CM were markedly reduced. Furthermore, the knock-down of tetraspanin CD151 weakened the chemotactic responses of C8161 and MeWo cells to LEC-CM. These data suggest that laminin-421 secreted by LEC possibly facilitates lymphatic metastasis through the induction of chemotaxis of melanoma cells.