bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5284: The Oncogenic Role of MALL in NSCLC
Authors: Lai Yen-Chun
Published: February 2011

MALL (T-cell differentiation protein-like) located on the human chromosome 2q13 locus encodes a 17.5KDa protein enriched in glycolipid and cholesterol enriched membrane raft (GEM). It is involved in the raft-mediated trafficking machinery in endothelial cells. Previous study showed that MALL can interact with Caveolin-1. However, Caveolin-1 can be phosphorylated by FAK and then promotes cell proliferation and colony formation in NSCLC (non-small cell lung cancer) cell lines. However, the role of MALL in cancer progression is totally unknown. In this study, we found that the expression of MALL is upregulated up to 250-fold in the high invasive lung cancer cells (CL1-5) than low invasive cells (CL1-0). To investigate the biological function of MALL, we first generated a constitutively MALL-expressed lung cancer cell line. We found that MALL can significantly enhance cancer invasiveness by the Boyden chamber matrigel assay (2.12 fold, p<0.01). MALL also promote cell migration by the transwell assay (1.53 fold, p<0.01) and wound healing assay. Moreover, MALL increased the colonogenesis of lung cancer cells by using an anchorage-dependent colony formation assay (1.73 fold, p=0.035) but did not affect the rate of cell proliferation by MTT assay. These findings indicated that MALL can promote tumor metastasis and tumorigenesis. To determine the underlying mechanisms by which MALL increases tumorigenesis and invasion Affymetrix oligonucleotide analysis was performed. The genes differentially expressed between MALL and mock transfectants with greater than two-fold change was identified by ANOVA analysis (FDR< 0.05). The most relevant networks involved were analyzed using MetaCore and IPA (ingenuity pathway analysis) software including cytoskeleton, ECM signaling, and other networks. Taken together, MALL may be a potential oncogene which might provide new insights into the future treatment of lung cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5284.
Abstract 5285: PERK regulates autophagy and survival of luminal cells during mammary acinar morphogenesis
Authors: Avivar-Valderas Alvaro
Published: February 2011

During normal mammary gland development both alveoli and lactiferous ducts develop a hollow lumen. Integrin-mediated cell attachment to the extracellular matrix (ECM) is crucial for proper growth and survival of mammary epithelial cells. Improper engagement to the surrounding ECM lead to the adhesion-dependent programmed cell death, or anoikis. However, in early breast cancer these hollow ducts become filled with anoikis-resistant cells, a hallmark of ductal carcinoma in situ (DCIS). The endoplasmic reticulum (ER) kinase PERK is a critical mediator of the translational repression during ER stress and is activated in matrix-deprived cells. Novel findings showed that ECM detachment robustly induces autophagy which transiently promote survival of luminal cells. Autophagy is a tightly regulated anabolic pro-survival process but if not reversed can ultimately lead to cell death. ER stress signaling and in particular PERK can activate autophagy. Furthermore, autophagy has been proposed as a mechanism to enhance the survival of early transformed cells lacking proper ECM contact. Using constitutive inhibition of PERK, we previously found that in ECM bound cells this kinase has an anti-proliferative function. However, this strategy precluded us from testing the precise regulation of PERK during lumen formation. Using MCF10A-Fv2E-PERK cells, where PERK phosphorylation is induced by the divalent molecule AP20187 (AP), we have now found that PERK promotes survival of intraluminal cells via autophagy. During mammary acinar morphogenesis PERK phosphorylation becomes detectable by day 4, peaks at day 6 and after day 8 it is extinguished. This coincided with the timing of autophagy induction in luminal cells. AP-mediated activation of PERK in Fv2E-PERK 3D cultures beyond day 8, induced ATG7 and Beclin1 expression, suggesting that accumulation of luminal cells is linked to autophagy. PERK induction of autophagy might block apoptosis because AP treatment resulted in a downregulation of BimEL expression and an overall reduction in the percentage of apoptotic cells. Moreover, mechanistic analysis revealed that PERK induction of autophagy is dependent on mTOR->p70S6K signaling inhibition. We propose that PERK mediated inhibition of mTOR protects basement membrane detached cells from apoptosis via autophagy allowing for proper morphogenesis. We conclude that PERK activation in luminal cells leads to increased survival and luminal cell accumulation. This is associated with increased autophagy gene expression/processing and reduced expression of the pro-apoptotic factor BimEL.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5285.
Abstract 5288: S100PBP is ubiquitously expressed and modulates the function of S100 calcium-binding proteins in pancreatic cancer
Authors: Lines Kate E. , Audi Namaa , Chelala Claude , Kocher Hemant , Wijesuriya Nilu , Hurst Helen , Crnogorac-Jurcevic Tatjana
Published: February 2011

We have recently discovered a 37kDa novel S100P-binding protein, S100PBP, which shows no homology to any currently known protein; we have therefore explored its expression in a wide range of normal and cancer tissues, its cellular localization and performed several in vitro functional assays to define its putative roles.

In order to determine S100PBP localization in human samples immunohistochemistry was performed on a multi-organ tissue array (Pantomics) and on an in-house made pancreatic ductal adenocarcinoma tissue array including normal, cancer and metastatic lesions. To determine S100PBP function cells with both silenced and stably over-expressing S100PBP were established and investigated by Affymetrix gene expression arrays and a battery of functional assays including adhesion, migration and invasion.

We show that this protein is almost ubiquitously expressed, in varying levels, between normal and tumor specimens, which is both cancer- and tissue-type dependent. In normal pancreas it is widely expressed in both exocrine and endocrine compartments while in pancreatic adenocarcinoma its expression is absent in the stroma and retained in some of the cancer cells but with significantly lower intensity.

We further show that in addition to S100P, in pancreatic cancer cells S100PBP binds, in a calcium-dependant fashion, several other S100 proteins, namely S100A2, S100A4 and S100A6. Over-expression of S100PBP led to changes in the expression levels of S100P and S100A9, which were down-regulated, and S100A4, which was up-regulated. Furthermore, manipulation of S100PBP expression led to phenotypic changes affecting the adhesive, migratory and invasive capacity of pancreatic cancer cells, that varied depending on the cellular context and constellation of the different S100 proteins present. Both over-expression and silencing of S100PBP also led to an increase/decrease in Cathepsin Z levels, respectively.

In summary, S100PBP is ubiquitously expressed and is deregulated in many different cancer types, and appears to play a role in the adhesive, migratory and invasive capabilities of pancreatic cancer cells.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5288.
Abstract 5289: HO-3867 inhibits cell migration, invasion and tumor growth of ovarian cancer through alterations in fatty acid synthase (FASN) and focal adhesion kinase (FAK) signaling
Authors: Selvendiran Karuppaiyah , Ahmed S. , Dayton A. , Bratasz A. , Kuppusamy ML. , Rivera BK. , Kuppusamy K. Hideg and P.
Published: February 2011

Fatty acid synthase (FAS) has been found to be overexpressed in a wide range of epithelial tumors, including ovarian cancer. Expression of FAS, the key enzyme in de novo synthesis of long-chain fatty acids, is normally low but increases in cancer. The increase of FAS levels regulates cell proliferation, apoptosis and migration in tumors. In tumor cells, the inhibition of FAS elicits cell cycle arrest and apoptosis, so it is considered a potential drug target for cancer therapy. A2780 and SKOV3 ovarian cancer cells were grown in vitro and exposed to culture media containing 10 μM of HO-3867 for a period of 12 or 24 hours. FAS protein level analysis and examination of proliferation and migration genes was performed by Western blotting. RT-PCR was used to examine mRNA levels. FAS activity levels were measured by spectrophotometry. FAS, Cyclin D1 and VEGF expression levels were analyzed in vivo using both Western blotting, RT-PCR and immunohistochemistry. We found that HO-3867 significantly inhibits the FAS activity levels in A2780 and SKOV3 ovarian cancer cells. FAS mRNA levels were significantly decreased in HO-3867-treated cells. Based upon these results, we investigated how HO-3867 inhibits FAS expression and activity in ovarian cancer cells. We found that FAS-regulating genes such as SREBP1 & 2 and pHER1 were clearly inhibited by HO-3867 exposure. Collectively, our results indicate that HO-3867 inhibition of FAS leads to inhibition of the cell migration genes FAK, ERK1/2, c-Myc, upregulation of the proliferation-regulating genes p21, p27 and downregulation of cyclin D1, resulting in inhibition of ovarian cancer cell proliferation, migration and tumor growth.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5289.
Abstract 529: Matrix metalloproteinase expression and secretion in papillary thyroid carcinoma
Authors: Kummer Nicolas T. , Bedwell Josh , Nowiki Theodore S. , Schantz Stimson , Shin Edward , Schaefer Steven , Tiwari Raj K. , Geliebter Jan
Published: February 2011

In the last decade, the incidence of thyroid cancer has risen ∼84% and mortality ∼34%. The ACS estimates ∼ 37,200 new cases of thyroid cancer, with 1,630 deaths in 2009. Papillary thyroid carcinoma (PTC) is the most common thyroid and endocrine malignancy, accounting for ∼80% of all thyroid cancer. It typically occurs from a gain-of-function mutation in the RET, RAS or BRAF genes which make up a linear signaling cascade for ERK activation. Recurrent or persistent disease prevails in up to 40% of thyroidectomy cases with a poor prognosis when aggressive cancer is evident. Thus, further research into factors contributing to aggressive disease is merited. Matrix metalloproteinases (MMPs) represent a family of secreted proteases with diverse functions implicated in the pathogeneses of aggressive tumors. Their functions include degrading the extracellular matrix (ECM) to facilitate cell migration, cell signaling through ECM cleavage products that can affect apoptosis and cell growth, and they can act directly on growth factor precursors and receptors. MMP upregulation has been demonstrated in many types of cancers yet its pattern of expression is poorly understood in PTC. Analysis of MMPs and tissue inhibitors of metalloproteinases (TIMPs) in the human PTC cell line, BCPAP, and the normal thyroid cell line, NTHY-ori by protein array and real-time RT-PCR revealed significantly elevated levels of MMPs and decreased levels of TIMP-4. Real time RT-PCR demonstrated the following fold increases in expression in BCPAP compared to NTHY-ori: 16. 9 (SE=0.79) for MMP-1, 1.80 (SE=0.41) for MMP-3, 1.40 (SE=0.08) for MMP-9, 7.12 (SE=2.14) for MMP-10, 12.60 (SE=6.90) for MMP-13 and increased levels of TIMP-4 in NTHY-ori compared to BCPAP of 2.38 (SE=1.04). Protein array data from serum free conditioned media of the cell lines reflected a similar pattern of secreted MMP and TIM4 levels. Fold increases in the optical concentration (determined by spot densitometry) of secreted proteins were detected in BCPAP compared to NTHY-ori of 29.48 (SE=1.92) for MMP-1, 16.80 (SE=1.07) for MMP-3, 1.66 (SE=0.07) for MMP-9, 3.66 (SE=0.55) for MMP-10, 1.59 (SE=0.11) for MMP-13 and increased levels of TIMP-4 in NTHY-ori compared to BCPAP of 1.27 (SE=0.06). For the first time, these results demonstrate increased production and secretion of a group of MMPs and down regulation of TIMP-4 which have been characterized in tumor progression in a PTC cell line. Further investigation into the role these agents play in the tumorgenesis of aggressive PTC may yield insight into future treatment and screening alternatives.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 529.
Abstract 5290: Motility of B16 mouse melanoma cells is driven by phospho-MARCKS
Authors: Rotenberg Susan A. , Chen Xiangyu
Published: February 2011

Phosphorylation of Myristoylated Alanine-rich C-Kinase Substrate (MARCKS) by protein kinase Cα (PKCα) is known to trigger its release from the plasma membrane/cytoskeleton into the cytoplasm, thereby promoting actin reorganization during migration. This study shows that cytoplasmic phosphoMARCKS directly promotes motile behavior of melanoma cells. Aggressively motile B16 F10 mouse melanoma cells express high levels of phosphoMARCKS, whereas weakly motile F1 cells express negligible levels. When treated with okadaic acid (OA) (a protein phosphatase inhibitor), F1 cells exhibit a dramatic increase in phosphoMARCKS coincident with a 5-fold increase in motility. This motility is lost if OA-treated cells are also treated with a shRNA reagent targeted to MARCKS. Pre-treatment of cells with calphostin C (a PKC inhibitor) prevents OA-mediated elevation of phosphoMARCKS. These findings imply that PKC is highly active in F1 cells but that its phosphorylation of MARCKS is efficiently reversed by protein phosphatases. The mechanistic significance of phosphoMARCKS to motility is established with a pseudo-phosphorylated mutant of GFP-MARCKS in which Asp residues replaced Ser residues known to be phosphorylated by PKCα. This mutant localizes to the cytoplasm and engenders three-fold higher motility in F1 cells. However, when the mutant is fused to a nuclear localization signal, motility is substantially decreased. Expression of an unmyristoylated, phosphorylation-resistant MARCKS mutant that localizes to the cytoplasm, blocks motility by 40-50% of both OA-stimulated F1 cells and intrinsically motile F10 cells. These findings demonstrate the importance of phosphoMARCKS to the metastatic potential of melanoma cells, and reveal a previously undocumented cytoplasmic role for this phospho-protein.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5290.
Abstract 5291: Exploring mechanisms underlying invasion in HLRCC: Assessing the contribution of HIF and ROS
Authors: Sourbier Carole , Kim Yeong-Sang , Lee Sunmin , Trepel Jane , Neckers Len , Linehan W. Marston
Published: February 2011

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is a highly metastatic disease characterized by a disruption of the Krebs cycle due to a germline mutation of the fumarate hydratase gene (FH). We have recently shown that HLRCC kidney cancer cells are in a pseudo-hypoxic state due to normoxic stabilization of both HIF1 and HIF2 and that the expression of HIF1 can be decreased using ROS scavengers such as pyruvate or N-acetylcysteine (NAC). Our objective was to study the involvement on invasion of aberrant HIF1 and HIF2 expression in HLRCC cells. When either HIF1 or HIF2 was knocked down, invasion was dramatically inhibited in vitro. Invasion of HLRCC cells was also inhibited by decreasing the ROS level with NAC. To study further the mechanisms by which HIF1 and HIF2 mediate invasion in HLRCC, we evaluated several markers of glycolysis and ROS formation. Our results show that when HIF1, but not HIF2, is knocked down, Glut1 mRNA level, glucose uptake, and lactate secretion are decreased. Even though N-acetylcysteine treatment decreased HIF1 and HIF2 expression, it did not affect glucose uptake or lactate secretion by HLRCC cells. Moreover transient knock-down of HIF1 or HIF2 by siRNAs did not affect the ROS level in vitro. To study further the mechanism of action of both HIF1 and HIF2, microarrays and in vivo experiments with stably transfected cell lines with shRNA for HIF1, HIF2 and ARNT are in progress. These results suggest that HIF1-mediated invasion may partially involve glycolysis. The mechanisms by which HIF2 and ROS mediate invasion are still under study. Our data support the hypothesis that HIF1, HIF2 and ROS are important for invasion. If one of them is inhibited, invasiveness of the HLRCC cells is compromised.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5291.
Abstract 5292: Role of voltage-gated Na+ channels in the metastatic potential of colon cancer cells
Authors: House Carrie , Vaske Charles , Schwartz Arnold , Obias Vincent , Sarvazyan Narine , Hales Tim , Stuart Joshua , Lee Norman
Published: February 2011

Ion channels are major signaling proteins spanning the plasma membrane where they enable the diffusion of specific ions into and out of a variety of cells. Voltage-gated Na+ channels (VGSCs) are responsible for the depolarization phase of neuronal action potentials and their activity is required for neurite extension and neurotransmitter release. Aberrant expression of VGSCs has recently been implicated in cancer. Overexpression of VGSCs appears to be associated with increased motility and invasive potential in vitro, whereas treatment with pharmacological agents that block Na+ currents, significantly reduce invasion. We have recently identified functional VGSCs in SW480, SW620, and HT29 colon cancer cells and examined their role in invasion. Electrophysiological techniques were employed to confirm functional expression of the SCN5A isoform in colon cancer cells. The more invasive SW620 cell line has a higher current density and increased protein expression compared to the less invasive SW480 and HT29 cell lines. Furthermore, we used RNA interference and VGSC blockers (tetrodotoxin and lidocaine) to inhibit channel expression and activity, respectively, and demonstrated a loss of invasive potential. Conversely, treatment with veratridine, a neurotoxin that prevents complete inactivation of VGSCs, increased Na+ influx and led to higher invasive potential. In addition to colon cancer cell lines, the expression of Nav1.5, the protein encoded by SCN5A, was examined in colon cancer patient samples by immunohistochemistry. Expression of this channel was restricted to the lumen of colon cancer specimens when compared to their normal matched controls. The contribution of this channel to metastatic behavior has not been previously demonstrated in colon cancer. To facilitate a further understanding of the mechanism by which this channel induces invasive potential, we have analyzed the involvement of SCN5A in activation of the MAPK pathway, which is deregulated in many cancers. Coincidentally, this pathway is also activated after depolarization in neurons during neurogenesis. Lastly, we show that SCN5A participates in the regulation of a colon cancer transcriptional network important for invasion. Taken together, our data suggest that VGSCs contribute to the metastatic potential of colon cancer cells through activation of the MAPK pathway and transcriptional regulation of genes important for the invasion process.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5292.
Abstract 5293: CCN3 increases motility of human chondrosarcoma via PI3K, AKT and NF-κB pathways
Authors: Chen Ruei-Ching
Published: February 2011

Nephroblastoma overexpressed (Nov; CCN3), from the CCN gene family, which is involved in many cellular activities such as growth and differentiation. Most of the CCN family can regulate the cell motility, adhesion and division. Here, we found that CCN3 increased the migration and expression of matrix metalloproteinase (MMP)-13 in human chondrosarcoma cells (JJ012 cells). Activations of focal adhesion kinase (FAK), phosphatidylinositol 3-kinase (PI3K), Akt and NF-κB pathways after CCN3 treatment was demonstrated, and CCN3-induced expression of MMP-13 and migration activity was inhibited by the specific inhibitor of PI3K, Akt and NF-κB cascades. Transfection of cells with FAK, p85, Akt, IKKα and IKKβ mutant also reduced CCN3-mediated cancer migration. Taken together, our results suggest that CCN3 acts through FAK/PI3K/Akt, which in turn activates NF-κB, resulting in the activation of MMP13 andcontributing to the migration of human chondrosarcoma cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5293.
Abstract 5298: Human enhancer of filamentation 1 induction by Prostaglandin E2 enhances the cell motility of colorectal cancer cells
Authors: Xia Dianren , Menter David G. , DuBois Raymond N.
Published: February 2011

Prostaglandin (PG) E2 promotes cell motility during colorectal carcinogenesis. The mechanism by which PGE2 signaling regulates cell motility is not completely understood. Here, we demonstrated that PGE2 treatment induces HEF1 expression that links with cell motility in colorectal cancer cells. PGE2 rapidly stimulated HEF1 expression in colorectal cancer cells. Both PGE2 treatment and HEF1 overexpression activated Rac 1 and resulted in similar effects on cell spreading. Moreover, knockdown of HEF1 using shRNA suppressed PGE2-driven cell spreading. PKA activator forskolin induced HEF1 expression and cell spreading in a similar manner as PGE2. Inhibition of PKA activity by H-89 attenuated HEF1 expression induced by PGE2. Furthermore, PKA knockdown by siRNA blocked the HEF1 expression and cell spreading induced by PGE2. These data suggest that PGE2 induces HEF1 expression to promote cell motility through a PKA-involved pathway.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5298.
Abstract 5299: Separate functions of caspase-3 in cell migration and apoptosis
Authors: Libby Erika , Eble Shannon , Adelson Martin , Mordechai Eli , Trama Jason
Published: February 2011

The cysteine protease function of caspase-3 in the execution phase of apoptosis is well known. However, even though subverting apoptosis is beneficial for tumor development and survival, some tumors maintain high expression levels of caspase-3 in positive correlation with malignancy. Here we show that caspase-3 expression is positively correlated with tumor cell migration and invasion. Furthermore, the enhanced migration and invasion is protease-independent and is specifically conferred by the inactive precursor form of caspase-3. Ectopic expression of caspase-3 in the MCF7 cell line, which is caspase-3 deficient and showed low migration ability, enhanced wound healing and chemotaxis. In contrast, shRNA reducing the expression of caspase-3 in the 5637 cell line, which expressed a high level of caspase-3 and high migration ability, hampered wound healing, chemotaxis, and matrigel invasion. Use of a caspase cysteine protease inhibitor or site directed mutagenesis of caspase-3 either rendering the ectopically expressed precursor form non-processable or inactivating the cysteine protease site did not diminish the caspase-3-dependent increase of cell migration. In addition, only reconstitution of the MCF7 cell line with the wild-type or non-processable precursor form of caspase-3 enhanced cell migration. Ectopic expression of the p17 or p12 fragment alone, the cysteine protease active p17 and p12 fragments together, or the cysteine protease inactive p17 fragment and p12 fragment together did not enhance cell migration. Fluorescence microscopy showed the enrichment of the non-processable mutant of caspase-3 in the leading edge of cells under experimental conditions of wound healing. In summary, the precursor form of caspase-3 induces cell migration by a protease-independent mechanism. This indentifies a new role for caspase-3 in tumor metastasis and represents a novel target for cancer therapeutics. Since the current evidence indicates that the role of caspase-3 in apoptosis and the role in migration are functionally separate, it is conceivable to design a molecule that disrupts the metastasis-inducing function of caspsase-3 while preserving the apoptosis-inducing function. We are currently investigating proteins that associate with caspase-3 under experimental conditions of caspase-3-dependent migration.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5299.
Abstract 5302: Overcoming cancer immune evasion in lung carcinoma using EGFR-TKI (Gefitinib)
Authors: Satomi Ryosuke , Naoki Katsuhiko , Yaguchi Tomonori , Yasuda Hiroyuki , Nakayama Sohei , Yoda Satoshi , Terai Hideki , Ikemura Shinnosuke , Sato Takashi , Soejima Kenzo , Ishizaka Akitoshi , Kawakami Yutaka
Published: February 2011

Clinical studies for immunotherapy targeting various types of carcinoma, including lung carcinoma, are currently underway, yet the clinical effectiveness of these therapies is very limited. Recent findings demonstrate that cancer immune evasion is a major contributing factor to this problem. Cancer cells produce a variety of immunosuppressive molecules that inhibit the host cell's immune response, leading to conditions such as cachexia that decrease patients’ quality of life. EGFR mutations in lung carcinoma are known to lead to processes characteristic of malignancies, such as cell cycle progression, metastasis, and angiogenesis. Based on these observations, we tested the hypothesis that EGFR activation leads to immune evasion by cancer cells via the production of various immunosuppressive molecules.

In this study, 6 lung adenocarcinoma cell lines were used, namely A549, RELF-LC-OK, PC-9, HCC-827, NCI-H1650, and NCI-H1975. We demonstrate that these cells secrete immunosuppressive cytokines such as IL-6, TGF-beta and VEGF, which inhibit the maturation of dendritic cells and prevent them to activate T cells. Then, we show that Gefitinib administration at sub-lethal concentration inhibits the production of immunosuppressive cytokines in EGFR-TKI sensitive PC-9 and HCC-827 cells those with EGFR mutations as well as in EGFR-TKI resistant NCI-H1650 cells that have an EGFR mutation but lack the PTEN gene. While, cytokine production is not inhibited by Gefitinib in A549 and RELF-LC-OK cells with wild type EGFR genes and NCI-H1975 cells that have both EGFR-TKI sensitive and resisitant mutations. Additionally, EGFR-TKI administration in PC-9, HCC827, and NCI-H1650 cells restores dendritic cell function, which was initially reduced by the supernatant of tumor cells.

These results show that lung adenocaricinoma harboring EGFR mutations evade immune response through secreteting immunosuppressive agents and that the immune response is restored by Gefitinib administration. Thus, the combination of Gefitinib and immunotherapy may have synergistic effect and will be a promising treatment of lung cancer patients harboring EGFR mutation, including certain population of EGFR-TKI resistant cases.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5302.
Abstract 5308: Targeting the Cox2 pathway prostaglandin EP receptors to regulate NK cell functions
Authors: Holt Dawn M. , Ma Xinrong , Kundu Namita , Fulton Amy M.
Published: February 2011

Breast malignancies often have high levels of Cox-2. The Cox-2 product prostaglandin E2 (PGE2) contributes to the high metastatic capacity of breast tumors. Our published data indicates that inhibiting either PGE2 production or PGE2-mediated signaling through the PGE2 receptor (EP4) reduces metastasis by a mechanism that requires Natural Killer (NK) cells. In order to better understand the mechanism by which inhibiting PGE2 production or signaling reduces metastatic disease, we examined the role of PGE2 in regulating protective host effector cells. It is known that NK cell function is compromised by PGE2, but very little is known about the mechanism by which PGE2 affects NK effector activity. NK effector function is regulated by complex interactions of recognition receptors expressed on NK cells and cognate ligands expressed on target cells. Both stimulatory and inhibitory signals are delivered through these receptor-ligand interactions. We have previously shown that both Cox inhibitors and antagonists of the EP4 receptor modulate the expression of NK cell stimulatory (H60) and inhibitory (MHC class I) ligands on the tumor target cell in favor of NK recognition and killing. We now report the direct effects of PGE2 on the NK cell. Murine splenic NK cells express all four PGE2 receptors (EP1-4). We examined the role of EP receptors in three NK cell functions; cytokine release, cytotoxicity, and migration. Activating all four NK-EP receptors with PGE2 leads to inhibited IFNγ secretion, induced TNFα production, less migration of NK cells and reduced ability to lyse tumor target cells. Using agonists and antagonists of individual EP receptors, we showed that only certain EP receptors contribute to loss of function. Next, we showed that all NK-EP receptor expression is downregulated during progressive tumor growth, which produced an environment that induced a less mature NK phenotype. EP2 and EP4 are linked to protein kinase A and elevations in intracellular cAMP. NK cells from tumor bearing mice respond poorly to stimulation with 10µM PGE2 and intracellular cAMP levels were increased by only 1.45-1.78 fold. In contrast, cAMP was induced by 6.08-10.58-fold in NK cells from normal mice. Thus, the tumor milieu results in changes in both NK-EP expression levels and EP receptor functions. NK mediated cytotoxicity, cytokine release, and migration were also compromised in tumor bearing mice. These results taken together support a mechanism whereby inhibiting PGE2 production or preventing signaling through some, but not all EP receptors may prevent suppression of NK functions that are critical to control breast cancer metastasis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5308.
Abstract 531: RNA interference-mediated downregulation of uPARAP decreases invasion and migration property in glioma cells through reorganization of the actin cytoskeleton
Authors: Takahashi Satoshi , Toda Masahiro , Sasaki Hikaru , Kawase Takeshi
Published: February 2011

OBJECTIVE: The aim of this study was to evaluate the role of membrane protein urokinase-type plasminogen activator receptor associated protein (uPARAP), which is one of the member of serine-protease cascade, urokinase plasminogen activator system, in the motile and invasive activities of glioma cells.

METHODS: In the present study, we first evaluate expression of uPARAP in various normal tissues as well as glioma patients’ samples and glioma cell lines using quantative PCR and Western blotting. To evaluate the function of uPARAP, we silence the uPARAP gene using two independent siRNAs designed for uPARAP, and evaluate its function on motile and invasive activities of glioma cells by Boyden chamber migration and invasion assays. We also evaluate the function of uPARAP on rearrangement of actin cytoskeleton by staining glioma cells with Alexa Fluor 488 conjugated Phalloidin.

RESULTS: uPARAP protein was expressed four out of four (100%) glioma samples regardless of its World Health Organization grade but did not express in normal brain control. Introduction of small interfering RNA targeted uPARAP into two different glioma cell lines; KNS42 and KNS81, resulted in down-reguration of uPARAP expression, and significantly suppressed glioma cells’ migration and invasion properties in vitro. Rearrangement of actin cytoskeleton was observed at Alexa Fluor 488 conjugated Phalloidin staining. In uPARAP knocked-down glioma cells, polymeric actin became organized in stress fibers and the lamellipodia disappeared.

CONCLUSION: On the basis of our findings, we conclude that RNA interference-mediated down-regulation of uPARAP decreases invasion and migration property in glioma cells. The inhibition of invasion and migration property was mediated via reorganization of the actin cytoskeleton. Downregulation of uPARAP may be a novel anti-invasion therapeutic strategy for malignant gliomas.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 531.
Abstract 5311: Myeloid-derived suppressor cells and immune cell responsiveness in tumor-bearing mice
Authors: Mundy Bethany L. , Lesinski Gregory , Benninger Kristen , Khan Mahmood , Kuppusamy Periannan , Guenterberg Kristan , Kondadasula Sri Vidya , Chaudhury Abhik Ray , Kreiner Melanie , Young Gregory , Guttridge Denis C. , Carson William E.
Published: February 2011

Conditions that inhibit IFN-induced signal transduction and gene transcription can dampen the host immune system and its ability to recognize and eliminate tumors leading to impaired immunoediting. We hypothesized that in the setting of cancer, myeloid-derived suppressor cells (MDSC) could impair the responsiveness of immune effector cells to interferons. We used the murine adenocarcinoma cell line C26 which induces MDSC generation when implanted in C2DF1 mice. C26-bearing mice had significantly elevated levels of GR1+CD11b+ MDSC (p<0.0001) and plasma IL-6 as compared to controls (p<0.0001). The ability of IFNa or IFNy to stimulate phosphorylation of the STAT1 transcription factor (P-STAT1, a direct measure of IFN action) was measured by intracellular flow cytometry. Induction of P-STAT1 following a 15 minute stimulation of splenocytes with IFNa (102-104 U/mL) was significantly reduced in C26-bearing mice (Mean P-STAT1 = 18.8; Range = 4.0 - 33.4) as compared to control mice (Mean P-STAT1 = 37.9; Range = 27.0 - 50.0; p<0.0001). A similar decrease in P-STAT1 in splenocytes of C26-bearing mice was observed following IFNy stimulation (1 ng/mL; 15 min; p=0.002). This decrease in IFN responsiveness was also observed in cells derived from inguinal lymph nodes following IFN-stimulation. The level of IFNa induced P-STAT1 was inversely correlated with the plasma IL-6 levels (Pearson correlation = −0.635; p=0.019). However, direct exogenous treatment of mice with IL-6 did not decrease splenic IFN response. In vitro co-culture experiments revealed that MDSC inhibited IFN-responsiveness of splenocytes from normal mice. Reduction of MDSC with either gemcitabine or anti-GR1 antibody treatment restored IFN responsiveness in splenocytes from C26-bearing mice as compared to tumor-bearing mice treated with vehicle or isotype control antibody. Spleens from C26-bearing animals displayed elevated levels of iNOS protein and nitric oxide (NO). The elevation in NO in C26-bearing mice was associated with increased levels of nitration on STAT1. In vitro treatment of splenocytes with a nitric oxide donor led to a decreased STAT1 IFN response. Taken together, these data suggest a novel link between MDSC and a decreased molecular response of the immune effectors responsible for promoting functional anti-tumor responses.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5311.
Abstract 5317: HLA-E and HLA-G tumor expression is of prognostic value for clinical outcome of early breast cancer patients, but exclusively in classical HLA class I tumor-negative patients
Authors: de Kruijf Esther M. , Sajet Anita , van Nes Johanna Gh , Natanov Russ , Putter Hein , Smit Vincent Thbm , Liefers Gerrit Jan , van den Elsen Peter J. , van de Velde Cornelis Jh , Kuppen Peter Jk
Published: February 2011

Background Non-classical human leukocyte antigens (HLA), HLA-E and HLA-G, are known to affect clinical outcome in various tumor types. We examined the clinical impact of HLA-E and HLA-G expression in early breast cancer patients, and related the results to tumor expression of classical HLA class I molecules, as together these cell surface molecules may determine natural killer (NK) cell responses.

Material and Methods Our study population (n=677) consisted of all early breast cancer patients primarily treated with surgery in our center between 1985 and 1995. Tissue micro array (TMA) sections of formalin-fixed paraffin-embedded tumors were immunohistochemically stained for HLA-E and HLA-G. For evaluation of HLA-E and HLA-G expression and the combined variable, HLA-EG, a binary score was used. Expression of classical HLA class I expression was previously determined.

Results HLA-E, HLA-G and HLA-EG were expressed in breast tumors in 50%, 60% and 23% of patients respectively. Remarkably, only in patients with loss of classical HLA class I tumor expression, expression of HLA-E (p=0.027), HLA-G (p=0.035) and HLA-EG (p=0.001) resulted in a worse relapse free period. An interaction was found between classical and non-classical HLA class I expression (p=0.002), suggestive for a biological connection.

Conclusions We have demonstrated that expression of HLA-E and HLA-G are important factors in the prediction of clinical outcome of breast cancer patients, but exclusively in patients with classical HLA class I negative tumors. HLA-E and HLA-G expression may specifically prevent NK-cell recognition by the host in this subset of tumors. These results provide further evidence that breast cancer is highly immunogenic, but also capable of evading tumor eradication by the host immune system in which both T cells and NK cells play a role.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5317.
Abstract 5319: The role of Notch and Delta-like 1 in antitumor immunity
Authors: Dikov Mikhail M. , Huang Yuhui , Yang Li , Lin Lupin , Carbone David P.
Published: February 2011

Defects in T cell function are evident in tumor bearing hosts. One potential mechanism for these hematopoietic defects is disruption of Notch signaling which is essential for T- versus B-lineage commitment, and it is clear that Delta-like 1 (DLL1) and Delta-like 4 (DLL4) are critical in this process 1-4. Here we show that tumor-associated defects in T cell development are characterized by attenuated levels of DLL1 and DLL4 in the bone marrow (BM) microenvironment, and elevated circulating vascular endothelial growth factor (VEGF) is one causative factor. We also show that elective therapeutic stimulation of Notch signaling in animals using multivalent form of DLL1 significantly reduces tumor growth, rescues T cell development and enhances T cell-mediated immune responses and that over-expression of DLL1 alone in BM precursors also inhibits tumor growth and improves anti-tumor immunity. Thus, stimulation of DLL1-Notch signaling represents a new strategy to overcome tumor immune dysfunction. As neutralizing DLL4 has been proposed as an approach to inhibit tumor growth by regulating tumor angiogenesis, our data suggest that combining stimulation or blockade of specific context-dependent Notch signaling may optimally inhibit tumor growth via multiple distinct mechanisms.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5319.
Abstract 5323: Distinct roles of IL-17A in inflammation-induced tumor development and tumor immunosurveillance
Authors: Kato Takuma , Hiroyoshi Nishikawa , Linan Wang , Jun Mitusi , Yuka Maeda , Hiorshi Shiku , Hiorshi Shiku
Published: February 2011

While chronic inflammation is considered to be a critical component of tumorigenesis, the immune system can also prevent tumor development and/or growth. IL-17A and IL-17A secreting T cells, termed Th17 mediate inflammation via activation of a wide range of downstream inflammatory mediators that contribute to chronic inflammatory diseases. It also has been suggested that Th17 can be a constituent in tumor immunosurveillance since tumor-specific Th17 has been shown to eradicate established tumor. In the present study, we addressed the effects of inflammation/immunity driven by IL-17A on a primary tumor development model using a chemical carcinogen 3′-methylcholanthrene (MCA). We found that IL-17A deficient (IL-17A−/−) mice were protected from the primary tumor development by MCA. Paradoxically, the growth of primary tumors developed in IL-17A−/− mice was faster than that of tumors in WT (C57BL/6) mice. Although tumor cell lines derived from WT and IL-17A−/− mice grew equally in immunocompromised (T, B cell deficient C. B-17 scid) mice, those from IL-17−/− mice were easily rejected in C57BL/6 mice as compared to those from WT mice. Consistent with this, tumor growth of B16 melanoma, a non-immunogenic transplantable tumor cell line, was enhanced in IL-17A−/− mice. The apparently contrasting effects of IL-17A on tumor growth and development may reflect difference in cells producing IL-17A, namely cells mediate innate or acquired immunity. We transferred CD4+ T cells from WT mice into IL-17A−/−. While the transfer with CD4+ T cells from WT mice did not affect the primary tumor development in IL-17A−/− mice, the growth of primary tumors in IL-17A−/− mice transferred with WT CD4+ T cells was significantly suppressed. These results indicate that IL-17A promotes tumorigenesis possibly by enhancing inflammation and IL-17A produced by CD4+ T cells contributes to the immune surveillance to the emerging tumors as well. Overall, these results not only demonstrate that a single cytokine, IL-17A, plays contrasting role in tumor development and growth, but also indicate that immune inflammation-driven tumorigenesis and tumor immunosurveillance coexist in the same host.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5323.
Abstract 5326: CCL2 is critical for immunoresistance in association with cancer EMT
Authors: Kudo-Saito Chie , Kawakami Yutaka
Published: February 2011

Purpose: Epitherial-to-mesenchymal transition (EMT) is a key step toward cancer metastasis, which is still a critical issue in cancer therapy. We previously reported that Snail-induced EMT accelerates cancer metastasis through multiple immunosuppressions using murine and human melanoma cells. However, the molecular mechanism is not completely understood. In this study, we confirmed the Snail-induced immunosuppressions using various human cancer cell lines, and further elucidated the mechanisms by identifying the specific mediators.

Results: We first established human pancreatic cancer cell line Panc-1 cells showing typical EMT features such as reduced adhesion and enhanced invasion after transduction with snail gene, which is a transcription factor governing EMT. When human PBMCs were cocultured with the Snail transfectants, CD11c+ cell differentiation was significantly inhibited, and immunosuppressive CD4+FOXP3+ Treg cells were increased as similarly observed using Snail+ melanoma, suggesting the general features of the Snail-induced immunosuppressive mechanisms. We next conducted GeneChip analysis to identify other possible mechanisms by comparing gene profiles between the Snail transfectant and the parental cells. CCL2 was significantly upregulated in various Snail+ tumor cells after transduction with snail or treatment with TGFβ, a potent EMT inducer. CCL2 not only enhanced tumor invasion but also caused immunosuppressions through induction of Lipocalin 2 (LCN2), which was reported to induce apoptosis in various cells. CCL2 also increased LCN2 receptor Megalin and immunosuppressive molecules IDO and PD-L1 in DCs; in turn, LCN2 increased CCR2 in DCs. Utilizing the cooperative mechanism induced by CCL2 and LCN2, Snail+ cancer cells severely hampered APC function through induction of apoptosis and PD-L1/IDO expressions followed by Treg generation and then CTL attenuation. CCL2 blockade utilizing the specific siRNA or antibody significantly inhibited both tumor invasion and those immunosuppressions in vitro. Intratumoral injection with these anti-CCL2 reagents significantly inhibited tumor growth and metastasis by enhancing anti-tumor response systemically in vivo.

Conclusion: These results demonstrate that CCL2 plays a vital role in the Snail-induced immunosuppressions. CCL2 blockade in tumor microenvironment may be an attractive strategy to overcome metastasis and immunoresistance of Snail+ cancers by restoring immunocompetence in cancer patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5326.