bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2334: TWIST1, a B-RAF effector, promotes melanoma cell invasion
Authors: Weiss Michele B. , Aplin Andrew E.
Published: June 2012

Metastatic melanoma is the deadliest form of skin cancer and a paradigm for highly aggressive, chemotherapy-resistant tumors. Therefore, determining the factors impacting melanoma advancement toward metastasis is an important and clinically-relevant task. The TWIST1 protein is up-regulated in melanoma and its expression correlates strongly with poor clinical prognosis. We tested the role of high TWIST1 expression in traits associated with the progression of melanoma, as well as sought to delineate the mechanism underlying TWIST1 up-regulation. Our data shows that TWIST1 protein levels are higher in melanoma cells, especially invasive lines, compared to normal melanocytes. To evaluate the biological relevance of TWIST1 expression, we generated invasive melanoma cell lines depleted of TWIST1 by shRNA and non-invasive melanoma cell lines which constitutively overexpress TWIST1. First, we utilized these cell lines in standard Matrigel invasion assays through boyden chambers. Depletion of TWIST1 in several invasive melanoma cell lines significantly reduces Matrigel invasion by approximately 40-50%. The inverse phenotype is apparent when TWIST1 is overexpressed in low TWIST1-expressing, non-invasive melanoma cell lines. Additionally, we tested the effect of altered Twist expression in 3D collagen spheroid outgrowth assays, which mimic both tumor architecture and the in vivo collagen-rich dermal layer. We have found significant reduction of spheroid outgrowth when invasive cell lines are depleted for TWIST1 as well as increases in outgrowth when non-invasive cells overexpress TWIST1. Alterations to spheroid outgrowth were not as a result of apoptotic changes or proliferative rate as assessed by live/dead and EdU staining. Importantly, the mechanisms regulating high TWIST1 expression in melanoma are unclear. A comparison between wild-type and mutant B-RAF melanoma cell lines demonstrates that TWIST1 is more highly expressed in mutant B-RAF cells. We have found that disruption of B-RAF/MEK signaling, through siRNA/shRNA knockdown or pharmacological inhibition, strongly represses TWIST1 at the mRNA and protein level. An area of active investigation is the determination of the transcription factor responsible for TWIST1 regulation downstream of mutant B-RAF in melanoma. The data generated from these studies will allow for a greater understanding of the role and regulation of TWIST1 during the progression of melanoma towards metastasis. In addition, it may highlight TWIST1 as an attractive molecule/pathway for novel targeted therapies for melanoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2334. doi:10.1158/1538-7445.AM2011-2334
Abstract 2339: Overexpression of PKCα in T47D breast cancer cells induces migration via p120-catenin transcriptional downregulation
Authors: Perez White Bethany E. , Zhao Huiping , Tonetti Debra A.
Published: June 2012

Several factors have been identified that influence epithelial-mesenchymal transition (EMT) in breast cancer cells. We previously reported that overexpression of protein kinase C α (PKCα) in breast cancer predicts tamoxifen resistant secondary tumors (Tonetti, 2003). In T47D breast cancer cells, stable overexpression of PKCα (T47D/PKCα) leads to hormone-independent growth as well as tamoxifen resistance (Chisamore, 2001). In our current study we report that PKCα overexpression increases the metastatic potential of breast cancer cells through induction of a nonclasssical EMT. T47D/PKCα cells are morphologically distinct from T47D/neo cells and do not retain the cuboidal structure of epithelial breast cancer cells. Overexpression of PKCα resulted in significantly induced migratory capabilities in the Boyden chamber assay and PKC activation by phorbol 12-myristate 13 acetate further enhanced T47D/PKCα cell migration. Pharmacological inhibition with the classical PKC inhibitor Go6976 significantly reduced the migratory capacity of T47D/PKCα cells. Transient siRNA-mediated knockdown of PKCα (120 h) significantly reduced both basal and NIH3T3 fibroblast conditioned media-induced T47D/PKCα cell migration. Levels of adherens junction proteins E-cadherin, α-E-catenin, β-catenin and p120-catenin were significantly downregulated in T47D/PKCα cells compared to T47D/neo cells as determined by western blot. Levels of E-cadherin protein were restored by more than 50% after 168 h of PKCα knockdown in T47D/PKCα cells. While SYBR green RT-qPCR revealed that E-cadherin transcripts were significantly higher in T47D/PKCα cells compared to T47D/neo control cells, there was no change in expression of β-catenin or α-E-catenin transcripts. However, p120-catenin transcripts were significantly lower in T47D/PKCα cells compared to T47D/neo cells. Treatment with the proteasomal inhibitor MG132 induces accumulation of E-cadherin but not p120-catenin. Taken together, these data suggest that PKCα may be responsible for EMT in breast cancer cells through upstream signaling that leads to transcriptional inhibition of p120-catenin and subsequent degradation of E-cadherin protein. Further, PKCα expression may not only be predictive of tamoxifen-resistance and hormone-independent growth but also of increased potential for metastasis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2339. doi:10.1158/1538-7445.AM2011-2339
Abstract 2359: Regulation of cytoskeleton organization by sphingosine in a mouse cell model of ovarian cancer progression
Authors: Creekmore Amy L. , Schmelz Eva M. , Roberts P. C.
Published: June 2012

Ovarian cancer is the fifth leading cause of cancer deaths among women mainly because of late detection of the disease due the lack of symptoms and reliable biomarkers. Approximately 90% of ovarian cancers are derived from the surface epithelium of the ovary. To further investigate the events that lead to progression of epithelial ovarian cancer, we previously developed a cell model of ovarian cancer. Mouse ovarian surface epithelial (MOSE) cells that have undergone spontaneous transformation in cell culture were categorized into distinct morphological and phenotypical stages based on passage history: early passage cells (MOSE-E) represent a pre-neoplastic, non-tumorigenic phenotype whereas late passage cells (MOSE-L) exhibit a highly aggressive malignant phenotype both in vitro and in vivo. In depth studies of genetic changes during MOSE cell progression in our labs have identified the cytoskeleton and its regulatory genes as critical early progression events. These changes in gene expression led to an increasingly dysregulated cytoskeleton which may affect the intracellular localization of signaling intermediates, and, thereby, important downstream signaling events involved in cancer progression. Sphingosine (SO), a bioactive metabolite of complex sphingolipids, has been shown to have chemopreventive activity against cancer, including cancer of the colon, breast and ovarian. In the present study, we have investigated the hypothesis that SO prevents or reverses the observed aberrant cytoskeleton organization, and thereby affects the cells metastatic potential. In MOSE-L cells, SO treatment did not significantly influence the gene expression or protein levels of several cytoskeleton components or regulatory proteins. However, immunostaining of SO-treated MOSE-L cells revealed a reversal of the aberrant cytoskeleton organization and mislocalization of regulatory proteins, many of which are associated with cell migration. This corresponds to observations that SO reduces cell motility in MOSE cells. Our data indicates that cytoskeleton re-organization induced by exogenous SO treatment may be involved in the regulation of signaling pathways that reduce proliferation, migration and invasion of the MOSE cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2359. doi:10.1158/1538-7445.AM2011-2359
Abstract 236: DKK-3, a secreted Wnt antagonist, suppresses tumorigenic potential and lung metastasis of osteosarcoma
Authors: Guo Yi , Rubin Elyssa , Pourmorady Jonathan , Christ Alexander , Rooney Kevin , Eskander Ramez , Sakai Toshinori , Bhatia Nitin , Zi Xiaolin , Hoang Bang H
Published: June 2012

Osteosarcoma (OS) is the most common primary malignancy of bone. Current therapeutic protocols focus on intensive chemotherapy and radical surgical resection. However, 5-year survival rate remains low when patients present with pulmonary metastasis, suggesting a need for more effective targeted therapies. In this study, we examined the in vivo anti-tumor effects of Dickkopf-3 (Dkk-3), a secreted Wnt antagonist, in a mouse model of osteosarcoma. Osteosarcoma 143B cells over-expressing Dkk-3(143B/Dkk-3) was established by stable gene transfection. To examine the effects of Dkk-3 on tumorigenesis and lung metastasis of osteosarcoma, nude mice were injected subcutaneously or orthotopically in the proximal tibia with 143B/Dkk-3-transfected vs. control cells to establish local tumor and lung metastasis. Dkk-3 dramatically inhibited local tumor growth in engrafted mice. In the orthotopic intratibial injection model, 143B/Dkk-3 cells formed significantly fewer lung nodules than control cells. In order to determine the potential molecular mechanism underlying the anti-tumor effect of DKK3 on osteosarcoma, we examined the reversal of EMT (Epithelial Mesenchymal Transition) and the expression of c-Met and MMPs (Matrix metalloproteinase) by real time PCR and western blot. Dkk-3 significantly reversed the epithelial-mesenchymal transition of osteosarcoma cells by inducing expression of epithelial markers (E-cadherin, keratin 8, and keratin18) and repressing the expression of mesenchymal markers (N-cadherin, Vimentin, and fibronectin). Dkk-3 also inhibited the expression and proteolytic activity of metalloproteinases and down-regulated oncogenic c-Met expression. Our study suggested that Dkk-3 effectively inhibits both local tumor growth and lung metastasis by suppressing multiple oncogenic pathways. Therefore, over-expressing DKK3 may represent a novel therapeutic strategy for osteosarcoma patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 236. doi:10.1158/1538-7445.AM2011-236
Abstract 2367: RhoA GTPase but not cortactin is required for tumor cell migration of human epidermoid carcinoma cells in vivo
Authors: Robertson Amy E. , Leong Hon S. , Lewis John D.
Published: June 2012

Metastasis contributes to over 90% of cancer mortalities. Metastatic disease is a dynamic, multi-step process that requires invasion and migration of cancer cells from the primary tumour site and leads to colonization at a distant location. Migration events mediated by the family of Rho GTPases are facilitated by acto-myosin contraction at the cell rear. In vitro studies have demonstrated that knockdown of RhoA causes insufficient actomyosin contraction and decreased cell detachment. Migration of cancer cells in vivo does not only require detachment from the primary tumor, but also relies on invasion of the cell into the surrounding stromal tissue. Formation of functional invadopodia drives invasion and a key component of invadopodia is cortactin. The objective of this study is to combine inhibitory short hairpin RNA (shRNA) technology with an in vivo metastasis model to identify mechanisms of cancer cell migration that have not previously been studied in an animal model. I hypothesize that inhibition of mediators required for cellular rear detachment and invadopodia formation, such as RhoA and cortactin respectively, will prevent migration of human fibrosarcoma (HT1080) and human epidermoid carcinoma (Hep3) cell lines in vivo.

Inhibition of RhoA and cortactin was performed by lentiviral infection of Hep3 and HT1080 cell lines with individual pLOK.1-puro vectors containing a 21-mir sequence to target and decrease mRNA expression. The migratory ability of both cell lines following RNA inhibition was analyzed in vitro using a scratch wound assay and in vivo using a migration and metastasis model in the shell-less chicken embryo, whereby transduced HT1080 and Hep3 cells are injected intravenously and subsequently arrest in the chorioallantoic membrane (CAM). We used a microscope mounted incubator and spinning disc confocal microscopy to visualize cells in real time and quantify their migration parameters.

Results obtained from in vitro migration assays indicate that inhibition of cortactin does not affect the migration of Hep3 and HT1080 cell lines across a rigid substratum. Diminished levels of cortactin also had no significant effect on the migration of these cells in vivo. Importantly, inhibition of RhoA caused a significant decrease in cell migration in vitro for both cell lines and in vivo for Hep3 cells (p<0.05). This data suggests that RhoA, a key mediator of cellular rear detachment, is required for in vivo migration of human epidermoid carcinoma cells while cortactin, implicated in invadopodia structures, is not required.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2367. doi:10.1158/1538-7445.AM2011-2367
Abstract 2369: Isolation of circulating tumor cells by size exclusion using lithography fabricated precision microfilters
Authors: Adams Daniel , Makarova Olga , Zhu Peixuan , Li Shuhong , Amstutz Platte , Tang Cha-Mei
Published: June 2012

Background: We present a novel precision microfilter that achieves rapid and highly efficient isolation of circulating tumor cells (CTCs) from peripheral blood. Isolation of CTCs by size exclusion is a widely researched technique with the advantage of capturing cells without reliance on cell surface expression markers. For many years CTC filtration technology has relied on track-etch microfilters with randomly located pores and low-porosity. More recently, precision microfilters with high porosity have been tested for CTC isolation, but they are not widely available. We have developed a new method of fabricating precision, high porosity microfilters that are capable of high volume manufacturing. These microfilters are strong, clear, non-fluorescent, and have high CTC capture efficiencies.

Methods: We describe an assay to capture and enumerate prefixed and live CTCs. MCF-7 human breast adenocarcinoma cells (ATCC) were cultured in DMEM until confluent. Cells were stained in a fixative/staining solution containing paraformaldehyde, acridine orange, and DAPI dilacetate. After incubation, a cell sample was placed on a microscope slide and cells counted to obtain the exact input. Cells were then washed into 7.5 mL whole human blood with 7.5 mL PBS and placed into a syringe. An 8 µm pore precision microfilter was placed into Swinnex filter holder, and the sample drawn by negative pressure through the filter at ∼10 mL/min. The microfilter was mounted onto a microscope slide and counted using a fluorescence microscope under FITC and DAPI settings. Cells were defined by their shape and coloration under FITC fluorescence and nuclear signature under DAPI fluorescence. The procedure was then repeated using a staining solution without fixative to capture live MCF-7 cells. Both live and fixed MCF-7 cell isolation by microfiltration were performed in triplicate.

Results: Precision microfilters were able to capture MCF-7 cells spiked into 7.5 mL human blood with a recovery rate of 98±3% for fixed cells, and 90±2% for live cells. The filtration was performed in 2 minutes, and cell counts were obtained in 10 minutes. By contrast, track-etched microfilters captured only 73% of fixed cells and cell counts required 20 minutes.

Conclusions: Creatv's precision microfilters are shown to recover MCF-7 cells more efficiently than track-etch filters. The pore uniformity and low fluorescent background allowed for faster and more accurate counts of spiked CTC in human peripheral blood compared to track-etch microfilters.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2369. doi:10.1158/1538-7445.AM2011-2369
Abstract 2382: A critical role of tumor-specific hedgehog signaling for pancreatic cancer metastasis
Authors: Sheng Tao , He Jing , Zhang Xiaoli , Rychahou Piotr , Yang Ling , Xie Guorui , Wang Huamin , Evers Nark , Xie Jingwu
Published: June 2012

The hedgehog (Hh) pathway plays a vital role in embryonic development and tissue differentiation, and aberrant activation of this pathway is found in a variety of human cancer, including pancreatic cancer. Despite the significance of Hh signaling for cancer development, it is not clear whether Hh signaling in the tumor compartment is required for tumor metastasis of pancreatic cancer. Here we show that elevated expression of hedgehog target genes is highly associated with poor outcomes of pancreatic cancer patients, but not always with elevated expression of hedgehog molecules (SHH and IHH) or the known downstream signaling molecules. Target genes of Hh signaling were expressed in the tumor as well as in the stroma. We found that elevated Hh target gene expression is associated with increased expression of snail1 or loss of E-cadherin, implying that Hh signaling may be associated with the pancreatic cancer metastasis potential. To investigate how Hh signaling may affect cancer metastasis, we established an orthotopic mouse model of pancreatic cancer. We showed in this model that Hh signaling is elevated in lymph node and liver metastasis. The role of Hh signaling for pancreatic cancer metastasis was demonstrated in an orthotopic mouse model of pancreatic cancer, which involves activated hedgehog signaling. We found that knocking down Gli2 expression in the cancer cells, which was associated with reduced Hh target gene expression, prevents tumor metastasis to liver. Based on these data, we conclude that tumor specific Hh signaling activation is required for pancreatic cancer metastasis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2382. doi:10.1158/1538-7445.AM2011-2382
Abstract 2400: Tumor suppressor EAF2 cooperates with p53 in control of prostate tumorigenesis and vascularity
Authors: Pascal Laura E. , Ai Junkui , Graham Lara E. , Xu Jianhua , Rigatti Lora , Pilch Jan , Zhou Wang
Published: June 2012

Introduction: Inactivation of EAF2 has been shown previously to lead to tumorigenesis in multiple organs; however the mechanism of EAF2 tumor suppression remains unclear. Demonstrating functional interactions between p53 and EAF2 would link the newly discovered tumor suppressor EAF2 to the well-established p53 pathway, providing new insights into the mechanisms of p53 and EAF2 action. We previously have shown that EAF2 can co-localize and co-immunoprecipitate with p53 in transfected cells. In a TSP-1 promoter-driven luciferase reporter assay, p53 transfection suppressed the TSP-1 promoter activity and EAF2 co-transfection blocked the p53 suppression of TSP-1 promoter. However, EAF2 transfection alone had little or no effect on the TSP-1 promoter. In a murine model, EAF2 knockout caused TSP-1 down-regulation, supporting an in vivo role for EAF2 in regulation of TSP-1 expression.

Methods: Mouse knockout, transient transfection, co-localization, co-immunoprecipitation, and luciferase-based promoter assay were employed in this study.

Results: Ongoing animal studies show that loss of EAF2 and p53 displayed much more dramatic phenotypes than loss of EAF2 or p53 individually and resulted in more severe splenic extramedullary hematopoiesis, and further increased vessel density in the prostate and liver. Furthermore, double knockout of EAF2 and p53, but not knockout of EAF2 or p53 alone, resulted in prostate tumorigenesis.

Conclusion: The above observations together suggest that EAF2 and p53 cooperate in prostate tumorigenesis and increased vascularity.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2400. doi:10.1158/1538-7445.AM2011-2400
Abstract 2415: TGF-ß signaling inactivation inhibits the formation of hepatocellular carcinomas induced by loss of p53
Authors: Morris Shelli M. , Baek Ji Yeon , Koszarek Amanda , Kanngurn Samornmas , Knoblaugh Sue E. , Grady William M.
Published: June 2012

Background: Hepatocellular carcinoma (HCC) is one of the most deadly forms of cancer and results from the accumulation of mutated or altered tumor suppressor genes and/or oncogenes. The tumor suppressor and oncogenes commonly affected include growth factors, receptors and downstream signaling components. Inactivation of the TGF-ß signaling pathway and mutation of TP53, the gene for p53, are common in HCCs suggesting they may cooperate in HCC formation. Thus, we assessed whether inactivation of TGF-ß signaling, by deletion of the TGF-ß receptor type II, TGFBR2, affects the formation of HCCs arising secondary to loss of p53.

Aim: To determine if loss of TGFBR2 and p53 cooperate in vivo to affect HCC formation.

Methods and Results: Albumin-cre (Alb-Cre) transgenic mice were crossed with mice homozygous for floxed Tgfbr2 (Tgfbr2flx/flx) and/or floxed Trp53 (Trp53flx/flx) to generate mice lacking TGFBR2 and/or p53 in the liver. Deletion of Tgfbr2 alone did not induce liver tumors, while inactivation of both Tgfbr2 and Trp53 resulted in a subset of mice (19%) that developed liver tumors by 65 weeks of age. Surprisingly, deletion of p53 alone, in the context of intact TGF-ß receptors, resulted in an increased number of mice developing tumors (38%), as well as increased morbidity (42 weeks of age). Interestingly, in both genotypes, Alb-Cre;Trp53flx/flx;Tgfbr2flx/flx and Alb-Cre;Trp53flx/flx;Tgfbr2wt/wt, the mice develop both hepatocellular carcinomas (HCC) and cholangiocarcinomas (CC), suggesting these tumors originate from a common liver stem cell population. Quantitative RT-PCR analysis of tumor and non-tumor tissue demonstrated a subset of Alb-Cre;Trp53flx/flx;Tgfbr2wt/wt (p53 null) tumors express significantly increased levels of alpha-fetoprotein mRNA, a clinical marker for HCC. Additionally, tumors from the Alb-Cre;Trp53flx/flx;Tgfbr2wt/wt displayed increased TGFβ-1 protein levels as compared to tumors from Trp53 and Tgfbr2 null mice. Furthermore, increased phosphorylated ERK1/2 expression was also present in the tumors from the p53 null mice and correlated with a slight up-regulation in beta-catenin expression, another common molecular event observed in human HCC.

Conclusions: The Alb-Cre;Trp53flx/flx;Tgfbr2flx/flx mouse model recapitulates many molecular features of human HCC and demonstrates that TGF-ß signaling is oncogenic in the setting of loss of p53 in the liver.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2415. doi:10.1158/1538-7445.AM2011-2415
Abstract 2421: Extracellular DNA in pancreatic cancer metastasis
Authors: Wen Fushi , Shen Alex , Choi Andrew , Shi Jiaqi
Published: June 2012

Extracellular nucleic acids have received increasing attention for their potential application in the rapid, accurate, and non-invasive early detection of cancer and other diseases, but less attention is given to their potential roles in cancer biology. We initiated a study to examine the role of extracellular DNA in pancreatic cancer metastasis. Pancreatic cancer is one of the most lethal cancers with a prognosis of just six months to live, and a five-year survival rate less than 5%. This is mainly due to the fact that at the time of diagnosis, 80-90% of patients have metastatic cancer already. To better understand the metastasis mechanism of this cancer, we tested the hypothesis that pancreatic cancer cells produce extracellular DNA to promote their metastasis and cancer cell adhesion to a new micro-environment.

We first examined the presence of extracellular DNA associated with pancreatic cancer cells by DAPI and CYTOX Green stains and by immunofluorescence assay (IFA) with the DNA-specific antibody. A large amount of extracellular DNA was observed on the surfaces and in the vicinity of the MiaPaCa-2 and BxPC3 pancreatic cancer cells but not the normal pancreas HPDE cells cultured on coverslips and in the 3D matrigel. A similarly larger amount of cell-free DNA was also found associated with the cancer cell line when the DNA from the cell culture media was extracted and analyzed. We subsequently stained tissue slides from human pancreatic cancer tissues, normal pancreas tissues, and the pancreatic cancer tissue microarrays using DNA-specific dyes and DNA -specific antibody in IHC and IFA. These experiments further confirmed the presence of extracellular DNA associated with the pancreatic cancer cells.

To understand the role of extracellular DNA in cancer metastasis, we studied the effect of treatment with DNase I on cell migration, invasion, proliferation, and colonization in vitro. We measured cell adhesion to plates coated with adhesion proteins. We also investigated de-attachment of cells from coverslips by proteinase K in the presence or absence of DNase I. These functional studies showed DNase I inhibited cell migration, invasion, and adhesion without affecting cell viability. In the 3D matrigel, DNase I completely changed the pancreatic cancer cell growth pattern.

We conclude that extracellular DNA associated with cancer cells play a novel role in cell migration, invasion, and cell adhesion in vitro. To our knowledge, this is the first report on the presence of a large amount of extracellular DNA on the surface and in the surrounding of pancreatic cancer cells and on the potential novel role of extracellular DNA in pancreatic cancer metastasis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2421. doi:10.1158/1538-7445.AM2011-2421
Abstract 2423: B7-H3 contributes to increased metastatic potential of melanoma cells by interfering with the expression of metastasis-related genes
Authors: Nygren Marit K. , Tekle Christina , Chen Yih-Wen , Kristian Alexandr , Nesland Jahn Marthin , Mælandsmo Gunhild Mari , Fodstad Øystein
Published: June 2012

Metastatic malignant melanoma is a disease with a long time survival rate of only 5%. Since there is no effective treatment for advanced melanoma, detection of molecular markers involved in tumor progression and drug resistance can hopefully provide new insight in therapeutically aspects of this disease. We have been studying B7-H3 – a glycosylated transmembrane protein which has been shown to play a role in cancer progression through its immunoregulatory properties.

B7-H3 is highly expressed in melanoma cell lines as well as in clinical melanoma specimens, and we have previously demonstrated a role of the protein in chemoresistance. Thus, in this study, we wanted to investigate the possible function of B7-H3 in melanoma metastasis development.

We found that short hairpin RNA silencing of B7-H3 reduced the migratory potential as well as the matrigel-invasiveness of the malignant melanoma cells in vitro. In an in vivo experimental metastasis model, silencing of B7-H3 increased the symptom-free survival of nude mice and rats significantly, this in parallel with a lower fraction mice having microscopically detectable metastasis compared to control mice. The brain was the predominant organ with metastases and a clear difference between the nude mice injected with B7-H3 expressing cells and B7-H3 knock down cells was observed, with 10/14 and 6/17 mice having brain tumors, respectively. Furthermore, we found the expression of several metastasis-associated proteins, such as MMP-2, TIMP1 and TIMP2 to be regulated by B7-H3. In addition, the secretion of IL-8 was markedly reduced in the B7-H3 knock down cells as well as the phosphorylation of Stat3. Interestingly, activated Stat3 is known to play a role in brain metastasis.

Taken together, our findings suggest that B7-H3 might be a potential therapeutic target for anti-metastasis therapy in human melanoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2423. doi:10.1158/1538-7445.AM2011-2423
Abstract 2424: Interferon consensus sequence binding protein promotes epithelial to mesenchymal transition and cell motility in human osteosarcoma cells
Authors: Park Byung-Kiu , Sung Jee Young , Na Yoon Sook , Kim Yong Nyun
Published: June 2012

Interferon consensus sequence binding protein (ICSBP) is a regulatory factor induced by interferon gamma (IFN-γ) and a member of interferon regulatory family (IRF). ICSBP is mainly expressed in hematopoietic cell and regulates immune response and differentiation but its role in non-hematopoietic cells is largely unknown. Here we investigated a role of ICSBP in human osteosarcoma U2OS cell line. Basal level of ICSBP expression was low but was induced by interferon-γ in U2OS cells. In addition, induced ICSBP promoted U2OS cell proliferation. To further elucidate ICSBP functions in osteosarcoma cells, we generated the U2OS stable cell line that overexpresses ICSBP. ICSBP overexpressing cells grew faster compared with mock cells and showed morphological changes. ICSBP upregulated TGF-βRI expression resulting in activation of TGF-β signaling pathways. In addition, ICSBP expression altered cell morphology and increased cell adhesion and motility. Furthermore ICSBP increased vimentin expression but decreased ZO-1 and E-cadherin expression, which is EMT related phenomena. These motility and EMT related phenomena were inhibited by TGF-βRI inhibitor. Taken together, these findings indicate that ICSBP promotes EMT and cell motility via TGF-β pathways in U2OS cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2424. doi:10.1158/1538-7445.AM2011-2424
Abstract 2430: Aromatase expression inhibits anoikis and promotes bone metastasis of ERα positive breast cancer cells
Authors: Liu Zhao , De Mukhopadhyay Keya , Yu Lan , Sun Lu-Zhe
Published: June 2012

Aromatase is the rate-limiting enzyme that catalyzes the final step in estrogen biosynthesis. In postmenopausal women the ovary ceases to make estrogen. Although it has been shown that estrogen is produced locally in breast cancer (BCa) tissue of postmenopausal women for the proliferation and survival of ERα (+) BCa cells, there is little information on the role and the regulation of aromatase in breast cancer cells for their survival when they intravasate into the circulation where estrogen level is very low.

Human breast cancer CAMA-1 cell along with aromatase-transfected MCF-7 (MCF-7/Aro) and ZR-75-1 (ZR-75-1/Aro/CL10) cells were cultured in suspension to mimic circulating tumor cells. Interestingly, all the three ERα(+) BCa cell lines expressed more aromatase in suspension culture compare to the adhesion culture. Moreover both endogenous and transfected aromatase were upregulated in suspension culture. Additionally, a higher expression of ERα was also observed in suspension culture, which could increase the sensitivity of the breast caner cells to intracrine estrogen. We found that anoikis was shown to be inhibited by the supplementation of testosterone, which is converted to estradiol by aromatase. Conversely, addition of the aromatase inhibitor letrozole blocked the testosterone-induced anoikis inhibition. The increase of pro-apoptotic protein BAX and BAD were observed in suspension culture, while the treatment of estrogen inhibited the expression of BAX and BAD in both adhesion culture and suspension culture. More interestingly, mice with intracardiacally inoculated ZR-75-1/Aro cells presented distant bone metastasis in the mandible and tibiae/femora after 2-weeks of inoculation without estrogen supplement, while the control ZR-75-1 cells were incapable of forming metastasis without estrogen. Our findings provide an important foundation for future investigation on how hormone-dependent BCa cells survive in circulation during the process of metastasis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2430. doi:10.1158/1538-7445.AM2011-2430
Abstract 2450: Resistance of CD44+ hepatocellular carcinoma cancer stem cellsto immune effector responses
Authors: Wan Shanshan , Vatan Linda , Simeone Diane M. , Kryczek Ilona , Zou Weiping , Welling Theodore H.
Published: June 2012

Hepatocellular carcinoma (HCC) is a common cause of cancer-related deaths worldwide. There is little therapy for advanced disease and recurrence rates can be as high as 50% following resection. Evidence in several tumor types suggests that cancer stem cells (CSCs) contribute to tumorigenicity, therapeutic resistance and recurrence, however the HCC CSC phenotype remains to be completely defined. HCC recurrence is influenced by the degree of immune response in the tumor microenvironment. Therefore, we aimed to characterize HCC-CSCs phenotype and investigate the interaction between CSCs and immune cell subsets.

To identify a dominant CSC population in HCC, we examined several putative CSC markers, including CD44, CD90, CD133 and EpCAM on primary human resected HCCs (n=6), HCC xenografts (n=2), and HCC cell lines (n=3) using flow cytometry. Cells from all sources consistently showed discrete populations of CD44+ cells (2-40%), while other common CSC markers showed significant heterogeneity. CD44+ cells isolated from HCC xenografts or HepG2 cells by fluorescence-activated cell sorting (FACS) demonstrated enhanced tumorigenic potential with as little as 100 cells implanted into NOD-SCID/IL-2Rgnull mice compared to no tumor from 100 CD44− cells. CD44+ cells were also able to produce heterogenic tumor mass in vivo. Moreover, CD44+ cells demonstrated increased sphere forming capacity in serum free media (2 fold, p<0.05), and increased resistance to the chemotherapeutic agents cisplatin and doxorubicin in vitro. Additionally, CD44+ HCC cells were resistant to effector cytokines such as IFNγ and TNFα and effector responses during co-culture with anti-CD3/anti-CD28 activated T cells from healthy donor PBMCs. CD44+ HCC cells had 2-fold increased expression of B7-H1, a co-inhibitory molecule for T cells, when compared to non-CSCs during exposure to activated T cells or effector cytokine IFNγ, suggesting a possible mechanism of resistance to immune effectors in the HCC microenvironment.

In conclusion, CD44 discriminates for the HCC CSC as evidenced by CD44+ subset demonstrating enhanced tumorigenicity, sphere forming capacity and resistance to chemotherapeutic agents. CD44+ CSC are also resistant to activated T cells and effector cytokines suggesting that microenvironmental factors related to immune response may be important for HCC recurrence or progression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2450. doi:10.1158/1538-7445.AM2011-2450
Abstract 2462: Effects of KRASG12D and P53 oncogenes on pancreatic cellular plasticity
Authors: Chen Jeff , Contino Gianmarco , Gurumurthy Sushma , Bardeesy Nabeel
Published: June 2012

Pancreatic Ductal Adenocarcinoma (PDAC) is fourth most common cause of cancer death in the United States. Improved understanding of the early stages of this cancer could enable interventions at a time where therapies will be most effective. The cells that give rise to PDAC have not been fully defined, although experimental studies have revealed considerable plasticity for different pancreatic cell types to interconvert their cellular identities. Notably, recent studies have shown that a subpopulation of pancreatic centroacinar cells_marked by the expression of Aldehyde dehydrogenase (ALDH) -has progenitor-like properties with the potential to give rise to both exocrine and endocrine lineages. Here, we used published protocols (Rovira et al., Proc Natl Acad Sci U S A. 2010 Jan 5;107(1):75-800) to isolate ALDH+ centroacinar cells and examined the effect of the common gene mutations found in PDAC on the clonogenic growth of the cells. We found that activating KRAS mutations and p53 inactivation both significantly enhanced the growth of ALDH+ cells as spheroids. These cells represent a potentially powerful tool to elucidate mechanism of transdifferentiation in the pancreas and may help to more fully define how key oncogenic mutations drive PDAC initiation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2462. doi:10.1158/1538-7445.AM2011-2462
Abstract 2466: RON receptor tyrosine kinase as a targeting moiety for pancreatic cancer stem-like cells by antibody directed immunoliposomes for enhanced cytotoxicity
Authors: Padhye Snehal , Wang Ming-Hai
Published: June 2012

Pancreatic adenocarcinoma (PAC) is a malignant disease characterized by metastasis and chemo-resistance. Accumulated evidence has suggested that PAC malignancies arise due to functional presence of cancer stem cells that have high potential for self renewal, differentiation, and resistance to cytotoxic agents. Currently, therapeutics targeting PAC stem cells are not available. The study in this project describes a novel strategy and potential therapeutics targeting PAC stem cells for enhanced cytotoxicity. PAC stem like cells (PSLC) were obtained from human L3.6pl pancreatic cancer line under stem cell culture conditions. These cells show a typical sphere-like morphology in suspension and re-differentiate into epithelial appearance when subjected to serum inclusive adherent conditions. PSLC express transcription factor Bmi-1, a unique self-renewal marker, Aldh-1a, a metabolic enzyme expressed in stem-like cells, and cell-surface markers including CD24, CD44, and ESA as evidenced by Western blot and flow cytometry analyses. PSLC also displayed a phenotype characterized as epithelial to mesenchymal transition. Analysis of a panel of receptor tyrosine kinases has revealed that expression of EGFR, VEGFR, and MET was significantly reduced in PSLC. In contrast, RON receptor tyrosine kinase expression was sustained, which provides the molecular basis for antibody-directed approach for targeting PSLC. To achieve this goal, Zt/c9 monoclonal antibody was post-inserted onto doxorubicin liposomes to form anti-RON immunoliposomes (Zt/c9-Dox-IL). In vitro analysis indicated that; a) Zt/c9 was highly effective in inducing receptor internalization, which facilitates intracellular delivery of chemoagents; b) Zt/c9-Dox-IL was effective in killing Panc 0327 and L3.6pl PAC cells. An average of 7 fold decrease in IC50 value was obtained compared to control Dox-IL inserted with mouse IgG (mIgG-Dox-IL); and c) Zt/c9-Dox-IL displayed increased cytotoxicity against PSLC with the IC50 at 8 µM compared to 69 µM for mIgG-Dox-IL. In conclusion, PSLC enriched from L3.6pl cells display a phenotype similar to cancer stem cells with properties of increased self renewal and EMT. RON expression was maintained in PSLC, which was used as the drug delivery moiety for enhanced cytotoxicity. Increased PSLC cytotoxicity by Zt/c9-Dox-IL suggests that antibody-directed RON targeted delivery of cytotoxic agents is a rational approach for targeting PSLC to achieve enhanced cytotoxicity.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2466. doi:10.1158/1538-7445.AM2011-2466
Abstract 2468: ATM signaling to mTORC1 in response to DNA damage independent of AMPK activation
Authors: Alexander Angela , Kim Jinhee , Walker Cheryl L.
Published: June 2012

When DNA becomes damaged either as a result of endogenous processes or exposure to radiation or chemicals, the DNA damage response is rapidly engaged. DNA damage response pathways have been well-characterized at the molecular level, and are known to involve a large network of proteins that trigger cell cycle checkpoints to allow time for DNA repair or trigger apoptosis via p53-dependent mechanisms if the damage is too severe to be repaired. However in addition to these classic damage responses, our previous work on ROS-induced pathways indicated that an additional aspect of the cellular damage response to many types of damage or stress could be induction of autophagy via repression of mTORC1.

We found previously that ROS could activate ATM in the cytoplasm, independently of the DNA damage produced by ROS, and subsequently activate the LKB1-AMPK energy-sensing pathway to activate TSC2 and repress mTOR, therefore inducing autophagy. We next asked whether a similar pathway could be induced in response to DNA damage, since there have been a few reports of AMPK activation by DNA damage in some cell lines, and ATM has been shown to activate AMPK via both LKB1-dependent and LKB1-independent mechanisms.

In a panel of cancer cell lines, including MCF7 breast carcinoma cells and SKOV3 ovarian carcinoma and other cells such as MEFs and HEK293, we profiled the signaling events in response to classic double-strand break agents such as etoposide, ionizing radiation or radiomimetic neocarzinostatin, as well as the intrastrand crosslinking agent cisplatin. In a cell-line and damage-specific manner, mTORC1 could be repressed, but unlike the previously identified ROS-induced signaling pathway, AMPK was not activated. In addition, in contrast to the rapid ROS-induced signaling pathway, DNA damage induced mTORC1 suppression occurred over a period of hours (6-24 hours), suggesting that additional mechanisms may be involved in signaling to mTORC1. Using Tsc2-deficient MEFs, we demonstrated that mTORC1 suppression requires TSC2, a negative regulator of mTORC1. This dichotomous response between ROS-induced mTORC1 suppression via AMPK activation of TSC2, and DNA damage, which does not require AMPK, indicates that mTORC1 regulation by DNA damage is complex and context-dependent, which may have implications for understanding mechanisms of action of DNA damaging cancer therapeutics.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2468. doi:10.1158/1538-7445.AM2011-2468
Abstract 2477: RBM3 prevents radiation-induced mitotic catastrophe by promoting anaphase
Authors: Moran Elizabeth , Ramalingam Satish , Subramaniam Dharmalingam , Moriasi Cate , Jensen Roy A. , Anant Shrikant
Published: June 2012

Deregulation of tumor suppressor and oncogenes is a key event in tumorigenesis and enhanced radioresistance. Post-transcriptional control of mRNA stability and translational control are important mechanisms by which these genes may be deregulated. We have previously demonstrated that RBM3, a glycine-rich RNA binding protein, is upregulated in cancers. Overexpression of RBM3 also transformed normal cells and led to tumor formation in vivo, suggesting that the protein is a protooncogene. Conversely, knockdown of RBM3 in cancer cells reduced tumor growth, in part due to cells undergoing mitotic catastrophe. Mitotic catastrophe is a common form of radiation-induced cell death. We are currently seeking to determine whether ectopic overexpression of RBM3 confers radioresistance. Our preliminary findings show increased survival of RBM3-overexpressing cells and a decrease in radiation-induced G2/M arrest. Additionally, RBM3 overexpression led to decreased expression level and activity of cellular machinery promoting apoptosis. Conversely, proteins related to cellular survival were upregulated in RBM3-overexpressing cells. The CDC20-APC complex protects cells from mitotic catastrophe by allowing the cells to transition successfully through anaphase. Inhibition of CDC20 therefore results in mitotic catastrophe. We recently observed that RBM3 overexpression resulted in CDC20 upregulation and increased the degradation of proteins regulated by the APC-CDC20 complex. Additionally, CDC20 activator Aurora Kinase B, but not Aurora Kinase A, was upregulated while CDC20 inhibitors Bub1b and Bub3 were downregulated. Preliminary findings would therefore suggest that RBM3 protects cells from mitotic catastrophe and does so in part by enhancement of anaphase. These findings imply that RBM3 confers radioresistance in part through upregulation of CDC20 and its enhancers and downregulation of its inhibitors, thereby preventing mitotic catastrophe.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2477. doi:10.1158/1538-7445.AM2011-2477