Mayumi Kawada

STAT3 is constitutively activated and supports cell survival in association with survivin expression in gastric cancer cells

Signal transduction and activator of transcription 3(STAT3) signaling is constitutively activated in various tumors, and is involved in cell survival and proliferation during oncogenesis. There are few reports, however, on the role of STAT3 signaling in gastric cancer. The aim of the present study was to clarify the role of STAT3 signaling in apoptosis and cellular proliferation in gastric cancer. Here we reported that STAT3 was constitutively activated in various human gastric cancer cells and its inhibition by ectopic dominant-negative STAT3 or Janus kinase inhibitor, tyrphostin AG490, induced apoptosis. Furthermore, STAT3 inhibition markedly decreased survivin expression, and forced expression of survivin rescued AGS cells from apoptosis induced by STAT3 inhibition. Although some reports demonstrated that the PI3K/Akt pathway regulates survivin expression, inhibition of the PI3K/Akt pathway did not affect survivin expression in AGS and MKN1 cells. Finally, activated form of STAT3, Tyr-705 phospho-stat3, was found in the nucleus of cancer cells in 11 of 40 (27.5%) human gastric cancer specimens. These findings suggest that constitutively activated STAT3 signaling supports gastric cancer cell survival in association with survivin expression.

Dclk1 distinguishes between tumor and normal stem cells in the intestine

There is great interest in tumor stem cells (TSCs) as potential therapeutic targets; however, cancer therapies targeting TSCs are limited. A drawback is that TSC markers are often shared by normal stem cells (NSCs); thus, therapies that target these markers may cause severe injury to normal tissues. To identify a potential TSC-specific marker, we focused on doublecortin-like kinase 1 (Dclk1). Dclk1 was reported as a candidate NSC marker in the gut, but recent reports have implicated it as a marker of differentiated cells (for example, Tuft cells). Using lineage-tracing experiments, we show here that Dclk1 does not mark NSCs in the intestine but instead marks TSCs that continuously produce tumor progeny in the polyps of ApcMin/+ mice. Specific ablation of Dclk1-positive TSCs resulted in a marked regression of polyps without apparent damage to the normal intestine. Our data suggest the potential for developing a therapy for colorectal cancer based on targeting Dclk1-positive TSCs.

COX-2 inhibition alters the phenotype of tumor-associated macrophages from M2 to M1 in ApcMin/+ mouse polyps

Macrophages are a major component of tumor stroma. Tumor-associated macrophages (TAMs) show anti- (M1) or protumor (M2) functions depending on the cytokine milieu of the tumor microenvironment. Cyclooxygenase-2 (COX-2) is constitutively expressed in a variety of tumors including colorectal cancer. TAMs are known to be a major source of COX-2 in human and mice intestinal tumors. COX-2 inhibitor reduces the number and size of intestinal adenomas in familial adenomatous polyposis patients and Apc(Min/+) mice. Although COX-2 inhibitor is thought to regulate cancer-related inflammation, its effect on TAM phenotype remains unknown. Here, we examined the effects of COX-2 inhibition on TAM phenotype and cytokine expression both in vivo and in vitro. Firstly, the selective COX-2 inhibitor celecoxib changed the TAM phenotype from M2 to M1, in proportion to the reduction in number of Apc(Min/+) mouse polyps. Concomitantly, the expression of M1-related cytokine interfron (IFN)-γ was significantly upregulated by celecoxib, although the M2-related cytokines interleukin (IL)-4, IL-13 and IL-10 were not significantly altered. Secondly, IFN-γ treatment attenuated M2 phenotype of mouse peritoneal macrophages and oriented them to M1 even in the presence of M2-polarizing cytokines such as IL-4, IL-13 and IL-10. Thus, our results suggest that COX-2 inhibition alters TAM phenotype in an IFN-γ-dependent manner and subsequently may reduce intestinal tumor progression.

Growth arrest‐specific gene 6 and Axl signaling enhances gastric cancer cell survival via Akt pathway

Activation of tyrosine kinases is an important factor during cancer development. Axl, one of the receptor tyrosine kinases, binds to the specific ligand growth arrest‐specific gene 6 (Gas6), which encodes a vitamin K‐dependent γ‐carboxyglutamyl protein. Although many receptor tyrosine kinases and their ligands are involved in gastric carcinogenesis, whether Gas6‐Axl signaling is involved in gastric carcinogenesis has not been elucidated. The aim of this study was to investigate the expression of Gas6 and Axl in gastric cancer and also their roles during gastric carcinogenesis. mRNA and protein of Gas6 and Axl were highly expressed in a substantial proportion of human gastric cancer tissue and cell lines, and Gas6 expression was significantly associated with lymph node metastasis. With recombinant Gas6 and a decoy‐receptor of Axl in vitro, we demonstrated that Gas6‐Axl signaling pathway enhanced cellular survival and invasion and suppressed apoptosis via Akt pathway. Our results suggests that Gas6‐Axl signaling plays a role during gastric carcinogenesis, and that targeting Gas6‐Axl signaling could be a novel therapeutic for gastric cancer.

Chitinase 3-like 1 promotes macrophage recruitment and angiogenesis in colorectal cancer.

Chitinase 3-like 1 (CHI3L1), one of the mammalian members of the chitinase family, is expressed in several types of human cancer, and elevated serum level of CHI3L1 is suggested to be a biomarker of poor prognosis in advanced cancer patients. However, the overall biological function of CHI3L1 in human cancers still remains unknown. Studies were performed to characterize the role of CHI3L1 in cancer pathophysiology utilizing human colorectal cancer samples and human cell lines. Plasma protein and tissue mRNA expression levels of CHI3L1 in colorectal cancer were strongly upregulated. Immunohistochemical analysis showed that CHI3L1 was expressed in cancer cells, and CHI3L1 expression had a significant association with the number of infiltrated macrophages and microvessel density (MVD). By utilizing transwell migration and tube-formation assays, overexpression of CHI3L1 in SW480 cells (human colon cancer cells) enhanced the migration of THP-1 cells (human macrophage cells) and HUVECs (human endothelial cells), and the tube formation of HUVECs. The knockdown of CHI3L1 by RNA interference or the neutralization of CHI3L1 by anti-CHI3L1 antibody displayed strong suppression of CHI3L1-induced migration and tube formation. Cell proliferation assay showed that CHI3L1 overexpression significantly enhanced the proliferation of SW480 cells. Enzyme-linked immunosorbent assay (ELISA) analysis showed that CHI3L1 increased the secretion of inflammatory chemokines, IL-8 and monocyte chemoattractant protein-1 (MCP-1), from SW480 cells through mitogen-activated protein kinase (MAPK) signaling pathway. Both neutralization of IL-8 or MCP-1 and inhibition or knockdown of MAPK in SW480 cells significantly inhibited CHI3L1-induced migration and tube formation. In a xenograft mouse model, overexpression of CHI3L1 in HCT116 cells (human colon cancer cells) enhanced the tumor growth as well as macrophage infiltration and MVD. In conclusion, CHI3L1 expressed in colon cancer cells promotes cancer cell proliferation, macrophage recruitment and angiogenesis. Thus, the inhibition of CHI3L1 activity may be a novel therapeutic strategy for human colorectal cancer.

The role of CXCR3 and CXCR4 in colorectal cancer metastasis

Chemokines and their receptors play key roles in leukocyte trafficking and are also implicated in cancer metastasis. We previously demonstrated that forced expression of CXCR3 promotes colon cancer metastasis preferentially to the draining lymph nodes (LNs), with poor prognosis. Using clinical colorectal cancer (CRC) samples, here, we show that expressions of CXCR3 and CXCR4 are significantly higher in metastatic foci within LNs and liver compared to primary tumors, whereas ligands for CXCR3 and CXCR4 are not. We also have demonstrated that some human CRC cell lines constitutively express both CXCR3 and CXCR4, and that activation of CXCR3 strengthens the CXCR4‐mediated cell migration in vitro in a synergistic manner. By constructing SW620 cell lines with reduced expression of CXCR3 and/or CXCR4 using microRNA, we investigated in vivo metastatic activities in a mouse rectal transplantation model. Six weeks after inoculation, CXCR3‐, CXCR4‐, and CXCR3/CXCR4 double‐knockdowns significantly reduced metastasis to LNs, liver and lungs, compared to the control (p < 0.05). Importantly, its suppressive effect on LN metastasis was significantly stronger in CXCR3‐ and CXCR3/CXCR4 double‐knockdowns. In addition, CXCR3‐ and CXCR3/CXCR4 double‐knockdowns significantly decreased the dissemination of cancer cells to liver and lungs, even after 2 weeks. These results indicate that targeting CXCR3 and CXCR4 can be a promising therapy against CRC metastasis.

Signal transducers and activators of transcription 3 activation is involved in nuclear accumulation of β-catenin in colorectal cancer

Nuclear accumulation of beta-catenin is a key event for the development of colorectal cancer. Little is known, however, about the mechanisms underlying translocation of beta-catenin from the cytoplasm or the membrane to the nucleus. The present study examined whether signal transducers and activators of transcription 3 (STAT3) activation is involved in the nuclear accumulation of beta-catenin in colorectal cancer cells. Of the 90 primary colorectal cancer tissues, 40 (44.4%) were positive for nuclear staining of p-STAT3 and 63 (70.0%) were positive for nuclear staining of beta-catenin. The nuclear staining of both p-STAT3 and beta-catenin were observed predominantly in the periphery of the cancer tissues. Importantly, of the 40 tumors with p-STAT3 nuclear staining, 37 (92.5%) were also positive for nuclear beta-catenin staining and there was a significant correlation between p-STAT3 and beta-catenin nuclear staining (P < 0.01). Coexpression of nuclear p-STAT3 and beta-catenin was associated with lower patient survival (P < 0.01). In an in vitro study using a human colon cancer cell line, SW480, inhibition of STAT3 by dominant negative STAT3 or the Janus kinase inhibitor, AG490, induced translocation of beta-catenin from the nucleus to the cytoplasm or membrane. Luciferase assays revealed that STAT3 inhibition resulted in significant suppression of beta-catenin/T-cell factor transcription in association with significant inhibition of cell proliferation (P < 0.05). These findings suggest that in colorectal cancer, STAT3 activation is involved in the nuclear accumulation of beta-catenin, resulting in poor patient survival.

Relationship between 18F-Fluorodeoxyglucose Accumulation and KRAS/BRAF Mutations in Colorectal Cancer

Purpose: Positron emission tomography (PET) with 18F-fluorodeoxyglucose (FDG) has been widely used in the management of colorectal cancer (CRC). However, the relationship between FDG accumulation and KRAS/BRAF mutations has not yet been investigated. The purpose of this study was to investigate whether KRAS/BRAF mutations affect FDG accumulation in CRC. Experimental Design: Retrospective analysis was conducted in 51 patients with CRC who underwent FDG-PET/computed tomographic (CT) scans for staging before primary tumor resection. The maximum standardized uptake value (SUVmax) for the primary tumor and the tumor-to-liver ratio (TLR) were calculated from FDG accumulation and compared between KRAS/BRAF mutated and wild-type groups. Expression levels of glucose transporter-1 (GLUT1) and hexokinase type-II (HXK-II) were assessed by immunohistochemical analysis. Results: Both SUVmax and TLR were significantly higher in the KRAS/BRAF-mutated group compared with the wild-type group (P = 0.006 and 0.001, respectively). Multivariate analysis indicated that SUVmax and TLR remained significantly associated with KRAS/BRAF mutations (P = 0.016 and 0.01, respectively). KRAS/BRAF status could be predicted with an accuracy of 75% when a SUVmax cutoff value of 13 or 14 was used. GLUT1 expression in cancer cells was positively correlated with FDG accumulation and KRAS/BRAF status whereas HXK-II expression was not. Conclusion: FDG accumulation was higher in CRC with KRAS/BRAF mutations. FDG-PET/CT scans may be useful for predicting the KRAS/BRAF status of patients with CRC and thus aid in determination of therapeutic strategies for patients with CRC. Clin Cancer Res; 18(6); 1696–703. ©2012 AACR.

Nardilysin and ADAM proteases promote gastric cancer cell growth by activating intrinsic cytokine signalling via enhanced ectodomain shedding of TNF‐α

Nardilysin (NRDc), a metalloendopeptidase of the M16 family, promotes ectodomain shedding of the precursor forms of various growth factors and cytokines by enhancing the protease activities of ADAM proteins. Here, we show the growth‐promoting role of NRDc in gastric cancer cells. Analyses of clinical samples demonstrated that NRDc protein expression was frequently elevated both in the serum and cancer epithelium of gastric cancer patients. After NRDc knockdown, tumour cell growth was suppressed both in vitro and in xenograft experiments. In gastric cancer cells, NRDc promotes shedding of pro‐tumour necrosis factor‐alpha (pro‐TNF‐α), which stimulates expression of NF‐κB‐regulated multiple cytokines such as interleukin (IL)‐6. In turn, IL‐6 activates STAT3, leading to transcriptional upregulation of downstream growth‐related genes. Gene silencing of ADAM17 or ADAM10, representative ADAM proteases, phenocopied the changes in cytokine expression and cell growth induced by NRDc knockdown. Our results demonstrate that gastric cancer cell growth is maintained by autonomous TNF‐α–NF‐κB and IL‐6–STAT3 signalling, and that NRDc and ADAM proteases turn on these signalling cascades by stimulating ectodomain shedding of TNF‐α.

Clinical Performance of 2 Dedicated PET Scanners for Breast Imaging: Initial Evaluation

The purpose of this study was to investigate the diagnostic performance of 2 newly developed dedicated breast PET scanners in patients with known or suspected breast cancer. Methods: Two types of scanner were evaluated, an O-shaped scanner and a C-shaped scanner. The O scanner was designed for imaging patients who were prone, and the C scanner was designed for those patients positioned leaning forward. Sixty-nine women with known or suspected breast carcinoma (80 lesions: 72 invasive carcinomas, 4 noninvasive carcinomas [ductal carcinoma in situ, or DCIS], 1 case of adenomatous ductal hyperplasia, and 3 benign lesions) were enrolled in this study. All patients underwent a conventional whole-body PET/CT scan, followed by breast scanning using both dedicated devices. The diagnostic performance of each scanner was assessed. Results: The maximal diameter of invasive tumors ranged from 4 to 112 mm, with an average of 26 mm. With the O scanner, 62 of 76 malignant lesions (including 3 DCIS) were detected, 5 lesions were not detected, and the remaining 9 lesions were outside the field of view. With the C scanner, 63 of 76 malignant lesions (including 2 DCIS) were detected, 7 lesions were not detected, and the remaining 6 lesions were outside the field of view. The lesion-based sensitivities of the O and C scanners were 82% (62/76) and 83% (63/76), respectively; sensitivities excluding lesions outside the field of view were 93% (62/67) and 90% (63/70), respectively. The sensitivity of conventional PET/CT was 92% (70/76). All lesions outside the field of view were close to the chest wall. The breast-based specificities of the O, C, and conventional scanners were 98% (48/49), 98% (56/57), and 100% (70/70), respectively. Conclusion: Our preliminary study indicates that both dedicated breast PET scanners are clinically feasible and yield reasonably high sensitivity. More detailed information was obtained with these scanners than with the conventional scanner.