Taeko Suzuki

Comparison of Fixation Methods for Preservation of Morphology, RNAs, and Proteins From Paraffin-Embedded Human Cancer Cell-Implanted Mouse Models

Xenograft transplantation of human tumor cells into immunodeficient mice is an important method to clarify the roles of specific molecules or chemicals in vivo. Recently, this method has been reported as a definitive examination to identify tumor stem cells. In this study, the authors compared the morphology and the quality and quantity of ribonucleic acid (RNA) and protein in paraffin-embedded tissues of nude mice implanted with human uterine cervical cancer cells, followed by fixation with commonly used fixatives, including 4% paraformaldehyde (PFA), 10% neutral buffered formalin (NBF), 20% NBF, and 99% ethanol (EtOH). The quality of the isolated RNA from PFA- and NBF-fixed paraffin-embedded tissues was high, while EtOH-fixed tissues showed degradation of RNA. NBF-fixed tissues showed excellent quality of morphology, but EtOH-fixed tissues showed contraction of cells. Immunohistochemical results showed differences depending on fixations. The 99% EtOH-fixed samples showed decreases of Ki-67 and VEGF-A immunoreactivities, but improved cytokeratin immunoreactivity. This study indicated that formalin fixation is better than alcohol fixation for RNA preservation in paraffin-embedded cancer cell implantation models. Immunohistochemical results differed markedly depending on fixation materials and antibodies; therefore, suitable fixations are needed to quantify and compare the results of immunohistochemical staining on cancer cell implanted nude mice tissues.

Defined localization of nestin-expressing cells in L-arginine-induced acute pancreatitis.

Objective: Nestin is a stem cell marker originally described as an intermediate filament protein expressed in neuroepithelial stem cells. In the pancreas, a small number of nestin-expressing cells, which are believed to represent either stem cells or progenitor cells, are known to be present in islets, as well as in some stellate cells, pericytes, and endothelial cells. We monitored pancreatic nestin expression to delineate the location of stem cells/progenitor cells in the pancreas after l-arginine-induced pancreatitis. Methods: Male Wistar rats received 2 intraperitoneal injections of l-arginine, each consisting of 250 mg/100 g of body weight, and were killed 3, 6, and 12 hours and 1, 4, 7, and 14 days later. Results: Serum amylase and lipase levels increased after l-arginine injection, maximal levels occurring at 3 and 12 hours postinjection, respectively. Six hours after l-arginine injection, interstitial edema was observed in the pancreas, whereas on day 4 postinjection, there was severe pancreatic necrosis. Neovascularization and ductal-ductular proliferation were also present in the pancreas. Immunohistochemical analysis revealed increased Ki-67 labeling in acinar cells and capillary endothelial cells. Immunoblotting using antinestin antibody revealed increased nestin expression after l-arginine injection. In the control rat pancreas, nestin immunoreactivity was detected in a few capillary endothelial cells in some islets. After l-arginine injection, nestin was expressed in proliferating capillary endothelial cells, in stellate cells surrounding ductular structures and in submesothelial cells. Conclusions: Transient nestin expression occurs in specific cell types during the proliferative stage after recovery from l-arginine-induced pancreatitis and may represent the contribution of stem cells and/or progenitor cells to the regenerative capacity of the pancreas.

Inhibition of fibroblast growth factor receptor 2 attenuates proliferation and invasion of pancreatic cancer

The alternative splicing of the extracellular domain of fibroblast growth factor receptor (FGFR)‐2 generates the IIIb and IIIc isoforms. Expression of FGFR‐2 IIIb correlates with vascular endothelial growth factor‐A (VEGF‐A) expression and venous invasion of pancreatic ductal adenocarcinoma (PDAC). By contrast, FGFR‐2 IIIc expression correlates with faster development of liver metastasis after surgery, and increased proliferation rates and invasion of the cancer. In this study, we analyzed the expression and roles of total FGFR‐2 (both isoforms) to determine the effectiveness of FGFR‐2‐targeting therapy for PDAC. Immunohistochemically, FGFR‐2 was highly expressed in 25/48 (52.1%) PDAC cases, and correlated with advanced stage cancer. In FISH analysis, FGFR2 was amplified in 3/7 PDAC cell lines. We stably transfected an FGFR‐2 shRNA targeting the IIIb and IIIc isoforms into FGFR2‐amplified PDAC cells. The proliferation rates, migration, and invasion of FGFR‐2‐shRNA‐transfected cells were lower than those of control cells in vitro. In response to FGF‐2, FGFR‐2‐shRNA‐transfected cells showed decreased phosphorylation of ERK compared with control cells. The FGFR‐2‐shRNA‐transfected cells also expressed lower levels of vascular endothelial growth factor‐A than control cells, and formed smaller s.c. tumors in nude mice. These findings suggest that FGFR‐2 is a therapeutic target for inhibition in PDAC.

Nestin: Neural Stem/Progenitor Cell Marker in Brain Tumors

Glioblastomas are the most common primary malignant neoplasms in the adult brain, and have the characteristics of glial cells [1]. The WHO histopathological classification guidelines categorize gliomas according to their histopathological grades; low-grade gliomas are not anaplastic and are associated with a favorable patient prognosis, while high-grade gliomas exhibit increased cellularity, nuclear atypia, mitotic activity, microvascular proliferation, and necrosis. Glioblastomas are the highest grade of the gliomas. Surgical treatment is the main therapy used for glioblastomas, with radiotherapy and chemotherapy performed as adjuvant care. Despite intensive research and recent advances in treatment, the prognosis for patients with glioblastoma remains poor, with a five-year survival rate of approximately 3% [2, 3]. In addition to having rapid growth rates, glioblastomas aggressively invade the adjacent normal brain tissues, they are often surgically unresectable, and recurrent glioblas‐ tomas are resistant to conventional radiotherapy and chemotherapy.

Abstract 5203: Long non-coding RNA H19 as a novel therapeutic target for pancreatic cancer

Long non-coding RNAs (lncRNAs), non-protein coding transcripts longer than 200 nucleotides, have been recently reported to play important roles in carcinogenesis and cancer metastasis through epigenetic regulation. In the present study, we examined the expression levels and roles of lncRNA in pancreatic cancer to elucidate whether lncRNA could be a novel candidate for pancreatic cancer therapy. First, we injected PANC-1 and PK-45H, pancreatic ductal adenocarcinoma cells into the spleens of NOD/Shi-scid, IL-2Rγnull (NOG) mice, and then established novel cell lines from liver and lung metastatic nodules. Microarray analysis revealed that H19, a member of lncRNA, and CTAG1A and CTAG2, cancer-testis antigens, were the most increased RNAs in PANC-lung-1A cells from lung metastasis as compared to the parental cells (82.4, 84.4 and 62.2-fold, respectively). Quantitative RT-PCR (qRT-PCR) confirmed higher levels of H19 mRNA in the metastatic cell lines from PANC-1 and PK-45H cells than in PANC-Skin cells from subcutaneous tumors. H19 is an imprinted non-coding RNA, and the H19 gene produces a 2.3 kb lncRNA during embryogenesis. Its expression is low or non-existent in normal human tissues. H19 is abundantly expressed in liver, breast, endometrial and bladder cancers, but the roles of H19 that have been reported in these cancers are controversial. In six of 10 pancreatic carcinoma cell lines, the H19 levels determined by qRT-PCR were higher than those of immortalized pancreatic ductal epithelial cell lines (HPDE 4 and 6). Established pancreatic cancer cells from liver metastasis of the same patient (PK-45H) showed higher H19 mRNA levels than those from primary tumor (PK-45P). In situ hybridization analysis using branched DNA probe for H19 mRNA showed that H19 was expressed in ductal cells, but not in acinar and islet cells in normal pancreatic tissues. On the other hand, H19 was strongly expressed in pancreatic cancer cells in 6 out of 38 (16%) pancreatic ductal adenocarcinoma tissues. siRNA targeting H19 was transfected to PANC-lung-1A cells to clarify the roles of H19 in the metastatic cells. The cell proliferation rate was not altered in the siRNA transfected metastatic cells, but cell migration, as observed using a Boyden chamber assay, and sphere formation were significantly decreased in the cells. H19 mRNA in PANC-1 cells was more abundantly detected in the spheres than the non-sphere cells. These findings suggest that H19 lncRNA plays important roles in the metastasis and cancer stem cell functions of pancreatic cancer. H19 is a novel candidate as a therapeutic target for pancreatic cancer metastasis. Note: This abstract was not presented at the meeting. Citation Format: Hisashi Yoshimura, Yoko Matsuda, Taeko Suzuki, Zenya Naito, Toshiyuki Ishiwata. Long non-coding RNA H19 as a novel therapeutic target for pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5203. doi:10.1158/1538-7445.AM2014-5203

Abstract 3750: Inhibition of phosphorylation of nestin decrease pancreatic cancer cell growth.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Nestin is a class VI intermediate filament, first described as a central nervous system (CNS) progenitor/stem cell marker and recently reported as a tumor stem cell marker in several malignant tumors, including glioblastoma and melanoma. In the normal pancreas, nestin is expressed in exocrine progenitor cells. Previously, we have reported that 30% of pancreatic cancer cases expressed nestin in cancer cells, and its expression correlated with nerve invasion and the presence of cancer cells in the tumor resection margins (Kawamoto, et al. Hum Pathol 2009). Knockdown of nestin exhibited a sheet-like appearance with tight cell-cell adhesion, increased expression of filamentous-actin and E-cadherin, and attenuated migration and invasion, both of which recovered following nestin re-expression (Matsuda, et al. Cancer Biol Ther 2011). Furthermore, knockdown of nestin suppressed sphere-forming ability, and altered the expression levels of stemness-related genes such as NANOG and transcription factor 4 (Matsuda, et al. AACR 2012). Therefore, nestin is considered to be a novel therapeutic target for pancreatic cancer via suppression of cell migration, invasion, metastasis and stemness. To develop nestin-targeting therapy, we focused on the phosphorylation of nestin protein. Phosphorylation of nestin at two different sites of progenitor cells of the CNS and myocytes has been reported to regulate the cell cycle via activation of cyclin-dependent kinase 5 and cdc2 kinase (Sahlgren, et al. Mol Cell Biol 2003). In this study, we analyzed the expression and roles of nestin phosphorylation in pancreatic cancer cells. Methods: Expression levels of phosphorylated nestin in pancreatic cancer cells were determined by flow cytometer, fluorescent staining and Western blot analyses. To assess the functions of phosphorylated nestin, we prepared three mutated nestin expression vectors which possess mutations at two different phosphorylated sites, both in nestin. A human pancreatic cancer cell line, MIA PaCa-2, which express very low levels of nestin was transfected with the mutated or wild-type nestin-expression vector. Results: Expression level of phosphorylated nestin was higher in M-phase cells than S- or G0/G1-phase cells. Phosphorylated nestin was strongly and diffusely localized in M-phase cells, while it was weakly detected in the nuclei of cells in other phases. Mutated nestin in phosphorylated site-transfected cells showed lower levels of cell growth, migration and invasion than wild-type nestin-transfected cells. Furthermore, mutated nestin-transfected cells had a tendency towards increasesed multinuclear cells. Conclusion: Phosphorylation of nestin affects the cell growth, migration and invasion of pancreatic cancer cells, and phosphorylated nestin may play important roles in the regulation of the cell cycle. Inhibition of phosphorylated nestin may be a novel target for pancreatic cancer treatment. Citation Format: Yoko Matsuda, Hisashi Yoshimura, Taeko Suzuki, Zenya Naito, Kiyoko Kawahara, Yuji Yanagisawa, Yoko Kawamoto, Toshiyuki Ishiwata. Inhibition of phosphorylation of nestin decrease pancreatic cancer cell growth. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3750. doi:10.1158/1538-7445.AM2013-3750

Abstract 249: Inhibition of nestin using siRNA as a novel therapeutic option for pancreatic cancer.

Introduction: Nestin is a member of the class VI intermediate filaments and it was first described as a central nervous system (CNS) progenitor/stem cell marker. Nestin is highly expressed in various cancers and was recently reported as a tumor stem cell marker for glioblastoma and melanoma. Previously,we have reported that 30% of pancreatic cancer cases expressed nestin in cancer cells, and its expression correlated with nerve invasion and the presence of cancer cells in the tumor resection margins (Kawamoto, et al. Hum Pathol 2009). Knockdown of nestin exhibited a sheet-like appearance with tight cell-cell adhesion, increased expression of filamentous actin and E-cadherin, and attenuated migration and invasion (Matsuda, et al. Cancer Biol Ther 2011). These findings suggest that nestin is a novel therapeutic target for pancreatic cancer. In this study, we examined the effects of siRNA targeting nestin on pancreatic cancer cell growth migration, and invasion in vitro and in vivo. Methods: Two types of siRNA targeting nestin, which binds to different sites of nestin mRNA, were prepared and transfected to PANC-1 and PK-45H cells. Cell growth, migration, invasion and sphere formation abilities of these cells were analyzed in vitro. We also determined the synergistic effects of gemcitabine in pancreatic cancer cells under treatment with siRNA targeting nestin. For an in vivo study, we constructed luciferase-gene-transfected pancreatic cancer cells and orthotopically implanted the cells into the pancreas of NOD/Shi-scid/IL-2Rγ null (NOG) mice. We injected siRNA into the tail vein of NOG mice once per week with Invivofectamine. Every week, luciferin was injected into the peritoneal cavity of NOG mice and images of primary and metastases were taken using IVIS. After 6 weeks, animals were sacrificed, and the primary and metastatic tumors were macro and microscopically examined. Results: siRNA targeting nestin inhibited the growth, migration, invasion and sphere-forming ability of both PANC-1 and PK-45H cells. Treatment with gemcitabine, in addition to the treatment with siRNA targeting nestin in pancreatic cancer cells, decreased cell viability as compared with only gemcitabine, siRNA targeting nestin or control siRNA- treated pancreatic cancer cells. In the orthotopic implantation model, siRNA targeting nestin significantly decreased primary tumor formation in the pancreas, as compared with negative control siRNA groups. Furthermore, nestin siRNA markedly suppressed the formation of metastatic nodules in the liver and lungs. Conclusion: In pancreatic cancer cells, siRNA targeting nestin inhibited cell growth, migration, invasion and sphere formation in vitro, and primary tumor growth and metastatic tumor formation in vivo. Furthermore, siRNA targeting nestin had synergistic effects with gemcitabine. Inhibition of nestin using siRNA may be a novel therapeutic optiont for pancreatic cancer treatment. Citation Format: Toshiyuki Ishiwata, Hisashi Yoshimura, Taeko Suzuki, Yuji Yanagisawa, Yoko Kawamoto, Kiyoko Kawahara, Zenya Naito, Murray Korc, Yoko Matsuda. Inhibition of nestin using siRNA as a novel therapeutic option for pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 249. doi:10.1158/1538-7445.AM2013-249

Abstract 5200: Nestin regulates stem cell functions of pancreatic cancer cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: Nestin, a class VI intermediate filament, is expressed in pancreatic exocrine progenitor cells. Previously, we reported that 30% of PDAC cases expressed nestin in cancer cells, and its expression was correlated with invasion (Kawamoto, et al. Hum Pathol 2009). Knockdown of nestin using shRNA in human PDAC cell lines inhibited cell migration and invasion in vitro and liver metastasis in vivo. Restoring the expression level of nestin returned the inhibition of cell migration and invasion to the previous level (Matsuda, et al. Cancer Biol Ther 2011). In this study we analyzed the expression and roles of nestin in pancreatic cancer stem cells. Methods: Expressions of stem cell markers and nestin were analyzed using immunocytochemistry and real-time PCR. To assess cancer stem cells, a sphere formation assay was performed as follows: cells were grown in an ultra-low attachment surface plate supplemented with epithelial growth factor and fibroblast growth factor 2. Nestin-regulating molecules were clarified by DNA microarray analysis. Results: The expression level of nestin was high in sphere-forming PDAC cells, and its expression levels correlated with sphere-forming ability. In DNA microarray analysis using nestin shRNA-transfected clones and nestin-expression vector transfected-clones, at a 2-fold cut-off, only transcriptional factor 4 (TCF4) showed a compatible change coincident with nestin expression. It has been reported that TCF4 is involved in the Wnt pathway, and negatively regulates the transcription of NANOG. In real-time PCR, expression levels of TCF4 and NANOG correlated with nestin expression level. Conclusion: Nestin expression levels correlated with sphere-forming ability and expression levels of TCF4 and NANOG. These results indicate that nestin regulate stemness via TCF4 and NANOG.The expression of nestin is associated with highly metastatic cancer stem-like cells, and nestin regulates migration, invasion, and metastasis by altering the pancreatic cancer stem cell function. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5200. doi:1538-7445.AM2012-5200

Abstract 412: Expression of cancer stem cell markers in pancreatic ductal adenocarcinomas and pancreatic intraepithelial neoplasias

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with a high incidence of distant metastasis. Recent studies have shown that cancer stem cells (CSCs) are important in cancer cell growth, invasion, metastasis, and recurrence. Several studies have revealed some CSC-specific markers for PDAC, such as CD133, CD24, CD44, CXCR4, ESA, and nestin. Some evidence also suggests that PDAC progresses through a multistep process comprised of noninvasive precursor lesions known as pancreatic intraepithelial neoplasias (PanINs). The reports of CSCs in PanINs are limited. We performed a comprehensive analysis of the expression of CSC markers in PDACs, PanINs, and normal pancreatic tissues. Materials & Methods: CD24, CD44, ESA, CD133, CXCR4, and nestin were used as CSC markers. Human PDACs (n=67), PanIN-1 (n=35), PanIN-2 (n=52), PanIN-3 (n=18), and normal pancreatic tissues (n=54), which were obtained from patients who underwent surgical operations in the surgical department of Nippon Medical School, were used for immunohistochemical analysis. Human PDAC cell lines, PANC-1, KLM-1, and MIA PaCa-2, were used for flow cytometric assays and quantitative RT-PCR analysis. Results: Regarding the clinicopathological features, the positivity of CD44 and CD133 showed significant correlations for UICC classifications and histological features In the survival analysis, CD133-positive PDACs led to a significantly poorer prognosis. In the immunohistochemical analysis, CD24, CD44, ESA, CXCR4, and nestin showed a gradual increase of positivity along with the grades of PanINs. Flow cytometric assays and RT-PCR analysis confirmed the expression profile in PDAC. Conclusion: CSC markers were expressed to various extents in PDAC, and the expression of CD133 and CD44 was correlated with the prognosis and stage. Furthermore, CSC markers showed a gradual increase along with the grades of PanINs; therefore, CSC may be involved in the carcinogenesis of PDAC. ![Figure][1] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 412. doi:1538-7445.AM2012-412 [1]: pending:yes

Abstract 3316: The stem cell marker nestin inhibits growth and invasion of malignant melanoma

BACKGROUND: Nestin, a class VI intermediate filament protein, was first described as a neural stem cell/progenitor cell marker. Nestin was detected in various neoplasms, and its expression level has relevance to the poor prognosis in some tumors, including malignant melanomas. Recently, nestin was reported as one of the cancer stem cell markers in melanomas. Furthermore, nestin is expressed in circulating tumor cells of melanoma patients, and its expression was found to be correlated with the prognosis. These reports indicate that nestin plays important roles in melanomas, and has the possibility of becoming a therapeutic target in melanomas. In the present study, we used a silencing strategy of nestin to clarify the effectiveness of nestin-targeting therapy in malignant melanomas. METHODS: We constructed a plasmid vector of short hairpin RNA (shRNA) tageting nestin and scramble vector as a negative control. Human malignant melanoma cells, A375 cells, were stably transfected with these vectors. Using nestin-shRNA-transfected cells, scramble vector-transfected cells, and wild-type cells, we analyzed cell growth, sphere formation, and invasion in vitro. Activation of the AKT/MAPK pathway was analyzed using a phosphorylated protein array. To determine the effect of nestin-knockdown in vivo, we performed tail vein injection of transfected and wild-type A375 cells into nude mice, and analyzed the metastatic tumor formation in 5 weeks. RESULTS: Nestin shRNA transfection suppressed cell growth, invasion, and sphere formation of A375 cells in vitro. In the protein array, the suppression of nestin decreased the phosphorylation of AKT and p53. The incidence of liver metastasis in nude mice was 100% in wild-type cell-injected mice, 75% in scramble vector-transfected cell-injected mice, and 16.7% in nestin-shRNA-transfected cell-injected mice. CONCLUSION: These findings suggest that nestin regulates cell growth, stemness, invasion, and metastasis of melanomas, in part, by alterling the phosphorylation of the MAPK/AKT pathway. Nestin may be a candidate therapeutic target in malignant melanomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3316. doi:1538-7445.AM2012-3316