Aggressive Cell Behavior Research Articles

Molecular characterization of CD133+ cancer stem-like cells in endometrial cancer

A small subset of cells with CD133 expression is thought to have increased chemoresistance and tumorigenicity, features of cancer stem cells (CSCs); the molecular mechanisms by which these properties arise remain unclear. We characterized CD133+ endometrial cancer cells based on microarray analyses of Ishikawa cells. Of the genes upregulated in CD133+ cells compared with CD133- cells, we noted several key factors involved in the aggressive behavior of cells, including ABCG2 and matrix metalloproteinase (MMP). Flow cytometric analyses identified a side-cell population (SP) with CSC features in Ishikawa cells, and they were found to be more enriched in CD133+ cells than CD133- cells. In particular, CD133+/SP cells exhibited higher proliferative and colony‑forming activity than CD133+/non-SP cells. Matrigel invasion assay revealed that CD133+ cells have enhanced invasive capacity with elevated MT1-MMP expression. siRNA‑based knockdown of MT1-MMP largely abolished the invasive capacity of CD133+ cells, but not CD133- cells due to low levels of constitutive MT1-MMP1 expression. These findings demonstrate that increased chemoresistance and tumorigenic potential of CD133+ cells are at least partly attributed to an enriched SP fraction as well as increased MMP-1 expression. These results will be of assistance in the establishment of molecular target therapy to CSCs in endometrial cancer.

Imatinib sensitizes endometrial cancer cells to cisplatin by targeting CD117-positive growth-competent cells

The use of molecular target therapy has not been established for endometrial cancer. The present study investigated the potential therapeutic strategy of targeting CD117-positive cancer cells as a novel molecular target therapy. FACS-sorted CD117+ cells isolated from endometrial cancer cell lines (Ishikawa or MFE280 cells) exhibited higher proliferative capacity in vitro and colony forming activity on soft agar, and decreased sensitivity to cisplatin, compared to CD117− cells. Immunohistochemical analyses with surgical specimens of endometrial cancers showed that high CD117 expression was tightly linked to advanced FIGO stages, myometrial invasion and histological grade, and was significantly associated with poor overall survival and relapse-free survival (Kaplan–Meier analysis; p<0.001, log-rank test). The Cox-regression hazard model identified high CD117 expression to be an independent prognostic factor for survival (p<0.05). In vitro assay confirmed that stem cell factor (SCF), a ligand of CD117, was produced specifically in CD117+ cells of endometrial cancer, and the colony-forming activity were abrogated by adding anti-SCF antibody, indicating an SCF-dependent growth property. Imatinib was confirmed to selectively target CD117+ cells in vitro, and synergistically enhanced the anti-tumor effect of low dose cisplatin in vivo, which showed only modest effects when used as a single use. These findings suggest that CD117 can be a marker of aggressive behavior of cells as well as an independent prognostic marker in endometrial cancer. Targeting of the SCF/CD117 axis by imatinib sensitized endometrial cancer cells to cisplatin, proposing a novel therapeutic strategy for this tumor type.

Lysophosphatidic acid induces reactive oxygen species generation through protein kinase C in PC‐3 prostate cancer cells

Prostate cancer is one of the most frequently diagnosed cancers in males. PC‐3 cell is a popular cell model for investigating late stage prostate cancer behaviors. Lysophosphatidic acid (LPA) is a lysophospholipid regulates multiple cell processes including proliferation, migration and other aggressive cell behaviors in different cancers. In our previous study, we demonstrated that LPA enhances VEGF‐C expression in PC‐3 cell through activating the generation of reactive oxygen species (ROS), which is known to be important in the development of many different cancers. Using flow cytometry, we measured the relative ROS levels in PC‐3 cells. LPA triggers ROS generation within ten minutes. This ROS production can be suppressed by antioxidants, N‐acetylcysteine, TEMPO and Tiron, respectively. By using LPA1/3 antagonist Ki16425, we confirmed that LPA induced ROS is mediated through LPA1/3‐dependent pathway. Moreover, pretreatment with PKC inhibitors can block the ROS generation stimulated by LPA treatment. Overall, we first demonstrated that LPA induces ROS generation in a PKC dependent manner in PC‐3 prostate cancer cell line.

B7-H1 Molecules on Myeloma Cells Induce Aggressive Cell Behavior

Abstract 474 Introduction:B7-H1 (CD274), a B7 family molecule, is expressed on antigen-presenting cells and plays crucial roles in T-cell regulation in various immune responses. We found that expression of B7-H1 molecules is also detected on some tumor cells and inhibits tumor-specific cytotoxic T lymphocytes (CTLs). Consistent with these data, it was reported that B7-H1 expression on tumor cells was associated with poor prognosis in some cancers, i.e., renal cell carcinoma, breast and ovarian cancer, etc. The expression levels of B7-H1 on plasma cells from multiple myeloma (MM) patients were reported to be higher than on those cells from patients with monoclonal gammopathy of undetermined significance (MGUS) and normal controls, suggesting that B7-H1 expression may be involved in the pathophysiology of MM. In the current study, we investigated the mechanism by which B7-H1 expression is induced on MM cells in the bone marrow (BM) microenvironment and analyzed characteristics of B7-H1+ MM cells in comparison with B7-H1– MM cells, i.e., proliferative potential, drug resistance, and sensitivity to myeloma-specific CTLs and whether B7-H1+ MM cells are associated with patients’ disease status. Methods:We examined 14 human myeloma cell lines (HMCLs) and plasma cells from 40 MM patients, 10 MGUS patients, and 10 hematologically normal controls. B7-H1 expression levels were analyzed by flow cytometry (FCM) and real-time polymerase chain reaction (RT-PCR). Changes in B7-H1 expression levels on MM cells were analyzed after the cells were cultured in the presence of the human BM stromal cell line HS-5, its culture supernatant, various cytokines, or inhibitors of cytokines and transcription factors. Proliferative potential was compared between B7-H1+ and B7-H1– MM cells, including cell cycle status analyzed by propidium iodide (PI) staining, BrdU and Ki67 expression, and cell number in liquid culture. Finally, sensitivity to dexamethasone (DEX) and melphalan (MEL) and expression of apoptosis-related genes were compared between B7-H1+ and B7-H1– MM cells using FCM and RT-PCR, respectively. Results:1) Although B7-H1 mRNA was detected in 9 of 14 HMCLs, only 3 cell lines expressed B7-H1 on their surface in FCM. B7-H1 expression on MM cells was significantly upregulated by co-culture with HS-5 cells or their culture supernatant. 2) Among cytokines present in the HS-5 cell supernatants, i.e., interleukin (IL)-6, IL-8, granulocyte-colony stimulating factor, and macrophage inflammatory protein 1 alpha, IL-6 alone induced B7-H1 expression on MM cells. Moreover, IL-6-neutralizing antibody dose dependently inhibited B7-H1 expression in the HS-5 cell culture supernatant. B7-H1 expression was downregulated by inhibiting STAT3, a transcription factor mediating IL-6 signaling. 3) The proliferative potential was higher in B7-H1+ cells in all assays. 4) Although KMS-27 myeloma cells lacking B7-H1 expression were killed by specific CTLs, KMS-27 cells that had B7-H1 expression induced on their surface resisted CTLs. 5) Nearly 20% of RPMI8226 cells expressed B7-H1, and DEX- and MEL-induced apoptosis was observed in the B7-H1– cell fraction alone. B7-H1 gene transfection in KMS-28PE cells conferred apoptosis resistance. Compared with B7-H1– RPMI8226 cells, B7-H1+ RPMI8226 cells showed higher gene expression levels of Bcl-2 and FasL and lower gene expression levels of caspase-8, caspase-9, and Fas. 6) Plasma cells from MM patients expressed significantly higher levels of B7-H1 than those cells from MGUS patients. One of 10 MM patients with International Scoring System (ISS) stage I, and 8 of 30 MM patients with ISS stage II/III were B7-H1 positive. Moreover, patients with CD45– myeloma, who were reported to have shorter survival, had higher expression levels of B7-H1 compared with CD45+ myeloma patients. In 4 of 8 MM patients, B7-H1 expression levels were upregulated in relapse or refractory status compared with levels at initial diagnosis. Conclusions:Our study indicated that IL-6 derived from BM stromal cells upregulates B7-H1 expression on myeloma cells, giving the cells gain more aggressive intrinsic proliferative potential and resistance to chemotherapeutic drugs and CTLs. The modulating B7-H1 pathway may have therapeutic potential in myeloma. Disclosures:No relevant conflicts of interest to declare.

Epigenetic regulation of HIF-1α in renal cancer cells involves HIF-1α/2α binding to a reverse hypoxia-response element

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene underlies the majority of sporadic clear cell renal cell carcinomas (CCRCCs) and is also responsible for the hereditary VHL cancer syndrome. VHL loss of function results in constitutive stabilization of hypoxia-inducible factors (HIF-1α and HIF-2α) due to insufficient proteolysis in the presence of oxygen. This activates multiple genes relevant to tumorigenesis, allowing cells to acquire further mutations and undergo malignant transformation. However, the specific role of each HIF-α subunit in CCRCC tumorigenesis is not yet well understood. The current paradigm supports that in the first stages of CCRCC formation the stabilization of HIF-1α is dominant and this limits proliferation, but later on HIF-2α increases and this induces a more aggressive cell behavior. Understanding how this transition happens is highly relevant, as it may provide novel ways to treat these cancers. Here, we show that VHL inactivation in CCRCC cells results in HIF-1α/2α-dependent downregulation of HIF-1α mRNA through direct binding of either subunit to a reverse hypoxia-response element in the HIF-1α proximal promoter. This binding activates a series of repressive histone modification marks including histone 3 lysine 27 trimethylation (H3K27me3) to make the changes stable, and if overturned reduces CCRCC cell proliferation due to excessive HIF-1α expression level. Our findings thus help understand how HIF-α subunits influence each other and also reinforce the idea that epigenetic mechanisms are a key step of CCRCC progression.

Factors affecting recurrence in node‐negative advanced gastric cancer

Prognostic factors of lymph node-negative gastric adenocarcinoma after curative resection have been discussed. Recurrent pattern of advanced lymph node-negative gastric cancer after curative resection has rarely been described. Recurrent sites and correlated clinicopathological factors of 372 patients with lymph node-negative advanced gastric adenocarcinoma that underwent R0 resection from 1988 to 2005 were analyzed. Recurrence was noted in 51 (13.7%) patients. Recurrent rates according to site of recurrence were 26 peritoneal seeding (51.0%), 26 locoregional (51.0%), 17 hematogenous (33.3%), and 4 lymph node metastasis (7.8%). Clinicopathological factors to predict peritoneal seeding were serosal exposure, lymphovascular invasion, Lauren's diffuse type differentiation and scirrhous stromal reaction. Serosal exposure, tumor size, microscopic infiltrating growth type predicts locoregional recurrence. Tumor had only lymphovascular invasion predict hematogenous spreading. Node-negative advanced gastric cancer has more peritoneal seeding and locoregional recurrence. Aggressive cell behavior predicted the route of tumor cell spreading.

Overexpression of L1CAM accompanies acquired endocrine resistance and is associated with the development of an aggressive cell phenotype.

Abstract Abstract #3028 Background: Acquired resistance to endocrine therapies is associated with a gain in aggressive features in vitro which may impact on tumour spread in vivo. To identify potential candidate molecules which may promote this adverse cell behaviour, we performed microarray analysis on MCF-7 cell models of fulvestrant resistance ('FasR' cells) applying a commercial algorithm for signalling network discovery. We have subsequently investigated the significance of overexpression of L1CAM, a cell-surface adhesion molecule implicated in a MAPK signalling network and recently associated with tumour metastasis, in FasR cells and in clinical tissue.&amp;#x2028; Materials and Methods: A network analysis algorithm was applied to t-test significant Affymetrix gene lists derived from FasR vs. MCF-7 cells (Almac Diagnostics). Subsequent to revealing a prominent L1CAM network, L1CAM gene and protein expression were assessed using RT-PCR, Western blotting and immunofluorescence. Inhibition of L1CAM was achieved using siRNA transfection. Cell invasion and migration were measured by seeding onto Matrigel- or fibronectin-coated microporous membranes respectively. After 48 hours, invasive/migratory cells were fixed, stained and counted. Analysis of cell signalling molecules was determined using Western blotting with phospho-specific antibodies. Clinical significance of L1CAM expression was determined by analysis of L1CAM expression in tumour (n=101) vs. normal (n=23) breast tissue using Q-PCR.&amp;#x2028; Results: Development of fulvestrant resistance in MCF7 cells was associated with ER loss and a gain in migratory and invasive capacity in vitro. L1CAM was overexpressed at both gene (p&amp;lt;0.05) and protein (p&amp;lt;0.01) level in FasR cells whereas little or no L1CAM was detectable in MCF7 cells confirming microarray findings. Network and ontological analysis predicted L1CAM lies upstream of an ERK2 network (26 genes; p&amp;lt;0.0001) impinging on transcription factors/regulators which may promote aggressive cell behaviour. In agreement, siRNA-mediated inhibition of L1CAM suppressed the activity of nuclear MAP kinase and reduced the intrinsic migratory and invasive nature of FasR cells in vitro. Preliminary analysis indicated L1CAM expression was inversely associated with ER status (p&amp;lt;0.01) and reduced overall survival (p=0.01).&amp;#x2028; Conclusions: These data suggest that L1CAM overexpression associates with emergence of ER-negative fulvestrant resistance in breast cancer, where it may confer a highly motile and invasive phenotype. Additionally, our clinical studies suggest L1CAM may also contribute to inherently aggressive, ER-negative breast cancers. L1CAM signalling may thus provide a new therapeutic target to subvert such aggressive states in vivo. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3028.

Genes Proximal and Distal to MYCN Are Highly Expressed in Human Neuroblastoma as Visualized by Comparative Expressed Sequence Hybridization

MYCN amplification is associated with poor prognosis in neuroblastoma disease. To improve our understanding of the influence of the MYCN amplicon and its corresponding expression, we investigated the 2p expression pattern of MYCN amplified (n = 13) and nonamplified (n = 4) cell lines and corresponding primary tumors (n = 3) using the comparative expressed sequence hybridization technique. All but one MYCN amplified cell line displayed overexpression at 2p. Expression peaks were observed frequently at 2pter and less frequently at 2p24 (MYCN locus), 2p23.3-23.2, and/or 2p23.1. Importantly, cell lines and two corresponding primary tumors displayed expression peaks at similar loci. No significant 2p24 expression level was observed for those cell lines displaying a low amplification rate (n = 3) by comparative genomic hybridization. Only the cell lines with an enhanced peak at 2p23.2-23.3 displayed coamplification of the ALK gene (2p23.2), reported to be associated with unfavorable prognosis. Finally, two of four cell lines without MYCN amplification, both derived from patients with poor outcome, also showed an expression peak at 2p23.2. These data indicate that, besides MYCN, other genes proximal and distal to MYCN are highly expressed in neuroblastoma. The prognostic significance of expression peaks at 2p23.2-23.3, independent of MYCN and ALK status, remains to be investigated.

Biological significance of cancer-associated sialyl-Tn antigen: Modulation of malignant phenotype in gastric carcinoma cells

The activation of an abnormal glycosylation pathway in cancer cells leads to the formation of the sialyl-Tn antigen, blocking regular carbohydrate chain elongation. Sialyl-Tn antigen is rarely expressed in normal tissues but is aberrantly expressed in a variety of carcinomas, where it constitutes a marker of poor prognosis. Although the clinical significance of sialyl-Tn is well characterized, a functional role for this glycan and its contribution to cancer progression remain to be elucidated. This study evaluates the capability of sialyl-Tn to modify processes like cell cycle, apoptosis, actin cytoskeleton dynamics, adhesion and motility on ECM components, cell–cell aggregation and invasion. De-novo expression of sialyl-Tn leads to major morphological and cell behavior alterations in gastric carcinoma cells which were reverted by specific antibody blockage. Sialyl-Tn antigen is able to modulate a malignant phenotype inducing a more aggressive cell behavior, such as decreased cell–cell aggregation and increased ECM adhesion, migration and invasion.

Pro-metastatic signaling by c-Met through RAC-1 and reactive oxygen species (ROS)

Overexpression of the c-Met/hepatocyte growth factor receptor(HGF-R) proto-oncogene and abnormal generation of intracellular oxygen species (reactive oxygen species (ROS)) have been linked, by independent lines of evidence, to cell transformation and to malignant growth. By comparing two subpopulations of the B16 mouse melanoma (B16-F0 and B16-F10) endowed with different lung metastasis capacities (low and high, respectively) we found that both the expression/phosphorylation of c-Met and the steady-state levels of ROS positively correlated with metastatic growth. shRNA-mediated downregulation of c-Met in F10 cells led to a parallel decrease in the generation of oxygen species and in metastatic capacity, suggesting that oxidants may mediate the pro-metastatic activity of the HGF receptor. c-Met activation by a ligand elicits the formation of oxidant species through the oxidase-coupled small GTPase Rac-1, a relevant downstream target of the HGF-R. Moreover, cell treatment with the catalytic ROS scavengers EUK-134 and EUK-189 attenuates Met signaling to ERKs and inhibits the anchorage-independent growth of F10 cells, consistent with a critical role for oxygen species in HGF signaling and in aggressive cell behavior. Finally, genetic manipulation of the Rac-ROS cascade at different levels demonstrated its crucial role in the pro-metastatic activity of c-Met in vivo. Thus, we have outlined a novel cascade triggered by c-Met and mediated by ROS, linked to metastasis and potentially targetable by new antimetastatic, redox-based therapies.