bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 410: SPARC expression enhances cellular aggregation and resistance to gemcitabine in pancreatic cancer cells
Authors: Salvatierra Edgardo , Rivas Elvia , Guttlein Leandro , Robitschek Emily , Llera Andrea , Podhajcer Osvaldo
Published: August 2015

Pancreatic cancer (PaCa) is a very aggressive disease with a high mortality rate, characterized by a survival time of only few months after diagnosis. Pancreatic cancers also demonstrate great resistance to chemotherapeutic regimes, associated with an extensive desmoplastic reaction. The matricelular protein SPARC is involved in the interaction between the cell and the extracellular matrix, and its expression has been associated with worse prognosis in general, and in patients treated with gemcitabine. To evaluate its role in pancreatic cancer and chemotherapeutic resistance, we overexpressed SPARC in the SPARC-negative pancreatic cancer cells lines Miapaca2, Sw1990, and Panc1. The in vitro growth of two stable clones obtained from the different cell lines was compared with eGFP-transduced controls. No significant differences were observed in the 2D in vitro growth or the clonogenic capacity between pancreatic cancer cells expressing or not expressing SPARC. However, cell growth in 3D spheroid culture was markedly different. Indeed, SPARC-expressing cells exhibited more compact structures, with 45% reduction in spheroid size compared to control eGFP-expressing cells. This could partially be explained by the fact that the SPARC-expressing cells themselves exhibited more than 50% reduction in their size compared to the eGFP cells. While SPARC expression in pancreatic cancer cells did not affect Gemcitabine EC50 in 2D culture, SPARC-positive Panc1 and SW1990 cells exhibited six to ten times increased EC50 in 3D spheroids. Because the cell-cell interactions could potentially affect drug efficacy, and because increased resistance to gemcitabine has been associated with epithelial-mesenchymal transition (EMT), we analyzed the expression of EMT-associated genes (SNAI1 and OCLN), and genes also involved in cellular junctions (CDH1 and OCLN). SPARC-positive pancreatic cancer cells down regulated the expression of SNAI1 and CDH2, while CDH1 and OCLN were up-regulated Taken together, these data indicate that SPARC expression in PaCa cell lines diminishes cells size and modulates gemcitabine resistance in 3D models of cell culture, possibly through the modulation of epithelial genes related to cell-cell connections and EMT.

Citation Format: Edgardo Salvatierra, Elvia Rivas, Leandro Guttlein, Emily Robitschek, Andrea Llera, Osvaldo Podhajcer. SPARC expression enhances cellular aggregation and resistance to gemcitabine in pancreatic cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 410. doi:10.1158/1538-7445.AM2015-410
Abstract 4108: NKX6.1 suppresses cancer invasion and epithelial-mesenchymal transition in cervical cancer
Authors: Li Hsin-Jung , Yu Pei-Ning , Shih Yu-Lueng , Lin Ya-Wen
Published: August 2015

Cervical cancer is a common women malignancy worldwide. Recent reports emphasize inactivation of tumor suppressor genes are due to promoter hypermethylation in cervical cancer. Our previous study from methylation array has shown NK6 homeobox 1 (NKX6.1) is hypermethylated and downregulated in cervical cancer cell lines and clinical samples. Nkx6.1 contains a homeobox domain and functions as a transcription factor that regulates insulin-secreting β cell differentiation and neuronal fate determination. However, the biological function of NKX6.1 in cervical cancer remains elusive. Overexpression of NKX6.1 suppressed the transformation and invasive ability of HeLa and CaSki cells in vitro as well as decreased tumor formation and metastatic property in vivo. Conversely, knockdown of NKX6.1 strongly enhanced the transformation and invasiveness of SiHa cells. Epithelial-mesenchymal transition (EMT) has been shown to play a key role in the progression of uterine cervix carcinoma in situ (CIS) to invasive squamous cell carcinoma (SCC). NKX6.1 suppresses cancer invasiveness through coordinately increasing E-cadherin and decreasing Vimentin. Moreover, we demonstrated E-cadherin and vimentin were direct targets of NKX6.1 by luciferase reporter assay, electrophoretic mobility shift assay and chromatin immunoprecipitation quantitative polymerase chain reaction (Chip-qPCR) assay. Collectively, our data demonstrate NKX6.1 acts a tumor suppressor, and inhibits tumor invasion and metastasis by suppressing Epithelial-mesenchymal transition (EMT) in cervical cancer. These data imply that modulation of NKX6.1 expression may be a novel therapeutic option for treatment of cervical cancer.

Citation Format: Hsin-Jung Li, Pei-Ning Yu, Yu-Lueng Shih, Ya-Wen Lin. NKX6.1 suppresses cancer invasion and epithelial-mesenchymal transition in cervical cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4108. doi:10.1158/1538-7445.AM2015-4108
Abstract 4113: Characterization of a novel TIMP-2 deficient mouse model of experimental lung metastasis
Authors: Kumar Sarvesh
Published: August 2015

It is well known that the balance between matrix metalloproteinases (MMPs) and their endogenous inhibitors, the tissue inhibitors of metalloproteases (TIMPs) impacts tumor progression, thereby providing a novel avenue for cancer therapies. We have previously shown that forced TIMP-2 expression inhibits lung tumor growth independent of MMP inhibition via direct effects on tumor cells and modulation of the tumor microenvironment.

The current study was undertaken to examine how reduced TIMP-2 expression impacts lung tumor growth using a novel TIMP-2 deficient (T2D) mouse model in a clean C57BL6 background. This is first time we fully developed and characterized TIMP2 deficient mice, validated by genotyping, histologic staining, protein and mRNA expression. The deletion of TIMP2 gene down regulated the expression TIMP2 mRNA and protein as demonstrated by quantitative RT-PCR and immunoblot experiments, respectively. Further, we investigated the impact of TIMP2 deletion on Lewis lung carcinoma model of metastasis. Lewis lung carcinoma cells transfected with luciferase (LL/2-Luc-M38, Caliper) were intratracheally instilled in the lungs into group's have heterozygous (T2D+/−), homozygous (T2D−/−), and wild type (WT) littermates. Tumor progression was monitored by IVIS imaging after 2nd week of intratracheal instillation and continued till 4th week, mice were sacrificed and lungs were perfused and harvested for further analysis. The IVIS analysis showed higher tumor burden in TIMP2 deficient and heterozygous mice as compared to wild type littermate, suggested that TIMP2 deficiency aggravates tumor development in deficient mice. The effects of recombinant TIMP2 on tumor microenvironment and microarray experiments are currently in progress. The molecular mechanism underlying the observed activities will be investigated. Collectively, these preliminary findings suggest that TIMP2 may be a novel target for lung cancer therapy.

Citation Format: Sarvesh Kumar. Characterization of a novel TIMP-2 deficient mouse model of experimental lung metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4113. doi:10.1158/1538-7445.AM2015-4113
Abstract 4120: Tasquinimod inhibits local invasion and metastases in two preclinical models of renal cell carcinoma
Authors: Shen Li , Pierron Valerie , Bauchet Anne-Laure , Nakhle Jessica , Orillion Ashley , Adelaiye Remi , Ciamporcero Eric , Meyer-Losic Florence , Eriksson Helena , Lassau Nathalie , Leguerney Ingrid , Schmidlin Fabien , Olsson Anders , Pili Roberto
Published: August 2015

Renal clear cell carcinoma (RCC) is the most common type of kidney cancer in adults and represents 80% of cases. At the time of diagnosis, approximately 30% of patients have distant metastases. In addition, one third of patients who have surgical resection of the primary tumor eventually relapse and develop metastases. Effective agents that help to prevent the development of metastatic RCC are not available. Tasquinimod is a novel agent in clinical development for metastatic castration-resistant prostate cancer that has been shown to have immunomodulatory, anti-angiogenic, and anti-metastatic properties. In the current study, we tested whether tasquinimod effectively inhibits local invasion and spontaneous lung metastases in RCC. Two preclinical mouse models of RCC were used for this purpose: a xenograft nude model A498 and a recently established patient-derived xenograft model, RP-R-02 LM. In both models, tasquinimod had a modest effect on the growth of the primary tumors implanted subcutaneously. Interestingly, in the A498 RCC model, gene expression analysis in tumors revealed that tasquinimod increased E-cadherin and decreased vimentin expression, suggesting an effect on reversal of epithelial-mesenchymal transition. In addition, tasquinimod significantly decreased CXCR4 expression, suggesting a reduction in invasiveness and metastatic phenotype. To further test this hypothesis, we used the RP-R-02 LM model that is able to develop lung metastases. Tasquinimod induced a 66% reduction of lung metastases in comparison with the control group (p<0.01). Following tasquinimod treatment CD206+/Arginase 1+/high suppressive M2-polarized infiltrating macrophages were decreased in primary tumors, but even more so in the lung parenchyma and tumor nodules. In summary, our results demonstrate that tasquinimod has immunomodulatory and anti-metastatic properties in RCC models, providing a biological rationale for the clinical assessment of tasquinimod in RCC patients.

Citation Format: Li Shen, Valerie Pierron, Anne-Laure Bauchet, Jessica Nakhle, Ashley Orillion, Remi Adelaiye, Eric Ciamporcero, Florence Meyer-Losic, Helena Eriksson, Nathalie Lassau, Ingrid Leguerney, Fabien Schmidlin, Anders Olsson, Roberto Pili. Tasquinimod inhibits local invasion and metastases in two preclinical models of renal cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4120. doi:10.1158/1538-7445.AM2015-4120
Abstract 4126: CSF-1R inhibition blocks rhabdomyoscarcoma metastasis by polarizing macrophage differentiation
Authors: Evans Justin , Giles Amber , Reid Caitlin , Kaplan Rosandra
Published: August 2015

Colony Stimulating Factor 1 (CSF-1) is overexpressed by many tumors and predicts poor clinical outcome. CSF-1 expression results in the recruitment of CSF-1R (Fms)-expressing cells, which have been shown to suppress tumor immunity, thereby promoting cancer progression. PLX3397 is a novel, orally active small molecule inhibitor that selectively targets CSF-1R, KIT, and oncogenic FIT3. CSF-1R and KIT regulate the tumor, tumor microenvironment, and the pre-metastatic niche. However, it is unknown if targeting the myeloid CSF-1R expressing component of the tumor microenvironment regulates metastasis of pediatric tumors, such as rhabdomyosarcoma. Using the M3-9-M murine syngeneic orthotopic rhabdomyosarcoma model, we evaluated the effects of PLX3397 on primary tumor growth and metastatic progression. This highly metastatic rhabdomyosarcoma model has high infiltration of CSF-1R-expressing bone marrow-derived cells, which includes inflammatory monocytes, M2 macrophages, and myeloid-derived suppressor cells. Therefore, we hypothesized PLX3397 would have a direct effect on CSF-1R expressing M2 macrophages and inflammatory monocytes, resulting in decreased metastasis. Using both in vitro and in vivo models, we found that PLX3397 treatment decreased tumor-infiltrating M2 macrophages and skewed the differentiation of inflammatory monocytes toward M1 macrophages and development of immunocompetent mature dendritic cells. This resulted in impairment of spontaneous M3-9-M metastasis to secondary sites. In addition, we have found that the polarization of inflammatory monocytes reversed the T cell suppressive capability of tumor-derived myeloid cells. In conclusion, CSF-1R blockage with PLX3397 inhibited metastatic progression by decreasing the infiltration of M2 macrophages in the primary tumor and pre-metastatic niche. The polarization of inflammatory monocytes to dendritic cells restored the immune recognition and decreased metastasis. This data supports our rationale for testing PLX3397 in patients with high M2 infiltration and suggests value in this agent as an adjuvant setting for metastasis prevention in high-risk patients.

Note: This abstract was not presented at the meeting.

Citation Format: Justin Evans, Amber Giles, Caitlin Reid, Rosandra Kaplan. CSF-1R inhibition blocks rhabdomyoscarcoma metastasis by polarizing macrophage differentiation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4126. doi:10.1158/1538-7445.AM2015-4126
Abstract 4142: A role for apolipoprotein E in invasion in oral squamous cell carcinoma
Authors: Jayakar Sangeeta K. , Loudig Olivier D. , Brandwein-Gensler Margaret , Kim Ryung , Ow Thomas J. , Prystowsky Michael B. , Childs Geoffrey , Segall Jeffrey E. , Belbin Thomas J.
Published: August 2015

Oral squamous cell carcinomas (OSCC) have a poor patient prognosis, which is attributed to their invasive nature. Our goal was to identify a novel gene that is important in OSCC invasion. We utilized genome-wide expression data generated from OSCC patient tumor samples with varying degrees of invasiveness, as measured by the histological parameter worst pattern of invasion (WPOI). RNA extracted from tumors that were classified as either WPOI 5 or WPOI 3, two categories of tumors that have been previously shown to correlate with local recurrence and poor overall survival, were evaluated utilizing two microarray platforms. When both gene expression datasets were analyzed, we identified 104 genes that were overexpressed at least 1.5-fold in the WPOI type 5 tumors compared to the less invasive WPOI type 3 tumors. Fifty genes were chosen based on functional criteria to be assessed using in vitro invasion assays following treatment with siRNA pools. In our initial screen, 16 of these genes showed significant reductions in in vitro invasion when their expression was knocked down by siRNA treatment in the UMSCC1 cell line. The gene with the greatest effect on invasion from the screen was Apolipoprotein E (APOE).

In addition to in vitro invasion, we also observed that matrix degradation and mature invadopodia numbers were decreased with APOE knockdown in the UMSCC1 cell line, suggesting that APOE affects invasion in part through regulating invadopodial activity. We observed that APOE knockdown resulted in decreased phospho-cJun and phospho-ERK1/2 in the UMSCC1 and PCI-15B cell lines. Since cJun is a component of the AP1 transcription factor, we assessed AP1 promoter activity using a luciferase reporter assay. We found that AP1 promoter activity decreased with APOE knockdown in both cell lines. Our proposed model is that APOE knockdown suppresses the cells’ ability to invade via decreased activation of ERK1/2 and cJun, thus reducing AP1 transcriptional activity. Further studies are being done to determine which downstream targets of AP1 are being altered with APOE knockdown and if those targets are known to be implicated in invasion.

Citation Format: Sangeeta K. Jayakar, Olivier D. Loudig, Margaret Brandwein-Gensler, Ryung Kim, Thomas J. Ow, Michael B. Prystowsky, Geoffrey Childs, Jeffrey E. Segall, Thomas J. Belbin. A role for apolipoprotein E in invasion in oral squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4142. doi:10.1158/1538-7445.AM2015-4142
Abstract 4149: Epidermal growth factor-induced cyclooxygenase-2 enhances head and neck squamous cell carcinoma metastasis through fibronectin up-regulation
Authors: Hsu Jinn-Yuan , Chang Kwang-Yu , Chen Shang-Hung , Lee Chung-Ta , Chang Sheng-Tsung , Cheng Hung-Chi , Chang Wen-Chang , Chen Ben-Kuen
Published: August 2015

Epidermal growth factor receptor (EGFR) activation is a major cause of cell metastasis in many cancers, such as head and neck squamous cell carcinoma (HNSCC). However, whether the induction of cyclooxygenase-2 (COX-2) correlates with EGF-enhanced HNSCC metastasis remains unclear. Interestingly, we found that EGF induced COX-2 expression mainly in HNSCC. The tumor cell transformation induced by EGF was repressed with COX-2 knockdown, and this repression was reversed by simultaneously treating the cells with EGF and prostaglandin E2 (PGE2). The down-regulation of COX-2 expression or inhibition of COX-2 activity significantly blocked EGF-enhanced cell migration and invasion, but the addition of PGE2 compensated for this blockage in COX-2-knockdown cells. Interestingly, COX-2 depletion inhibited EGF-induced matrix metalloproteinase-1 (MMP-1), MMP3 and fibronectin expression and Rac1/cdc42 activation; this reduction in MMPs and the fibronectin/Rac1/cdc42 axis by the depletion of COX-2 was also rescued when the cells were treated with PGE2. Furthermore, the depletion of fibronectin impeded the COX-2-enhanced binding of HNSCC cells to endothelial cells and tumor cells metastatic seeding of the lungs. These results provide new insight that EGF-induced COX-2 enhances HNSCC metastasis via activation of the fibronectin signaling pathway. The inhibition of COX-2 expression and activation suggests a potential strategy for the treatment of EGFR-mediated HNSCC metastasis.

Citation Format: Jinn-Yuan Hsu, Kwang-Yu Chang, Shang-Hung Chen, Chung-Ta Lee, Sheng-Tsung Chang, Hung-Chi Cheng, Wen-Chang Chang, Ben-Kuen Chen. Epidermal growth factor-induced cyclooxygenase-2 enhances head and neck squamous cell carcinoma metastasis through fibronectin up-regulation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4149. doi:10.1158/1538-7445.AM2015-4149
Abstract 415: Differential expression of therapeutic targets across tumor micro-environments and at infiltrative margins in glioblastoma
Authors: McCrary Myles R. , Gutman David , Dunn William , Kaluzova Milota , Bouros Alexandros , Williams Merete , Zhang Xialong , Cooper Lee AD , Hadjipanayis Constantinos G. , Brat Daniel J.
Published: August 2015

Glioblastoma (GBM; WHO grade IV) is the most common primary brain tumor and is associated with a poor prognosis. The Cancer Genome Atlas (TCGA) project has revealed discrete molecular classes of GBM, raising the possibility of targeted therapies. Molecular heterogeneity across tumor microenvironments presents a challenge to targeted therapy, since effective therapies will be directed at targets near resection margins rather than those expressed in bulk resected tumor. To this end, we initiated studies to delineate spatial and temporal variations of potential druggable targets in GBM, focusing on potential protein targets at the tumor margin.

By utilizing 5-ALA (5-aminolevulinic acid, an oral compound that accumulates in malignant glioma cells and is metabolized to the fluorescent agent, protoporphyrin IX) fluorescence-guided surgery of primary and recurrent GBMs in a Phase II clinical trial, we were able to precisely define perinecrotic, bulk, and marginal regions within the glioma intraoperatively in order to sample tissues for further molecular characterization. Tissues harvested from these 3 tumor regions from 5 patients were analyzed using reverse phase protein array (RPPA) analysis with 218 antibodies (Array-Pro). Relative protein levels were determined and a heatmap was generated as a hierarchical cluster using Pearson Correlation and a center metric.

Forty proteins had statistically significant altered expression at the tumor margin compared with bulk and perinecrotic tumor. Among these, STAT1, STAT2, STAT3, PDGFRA, PDGFRB, EGFR, JAK2, MET, PIK3R1, and ERBB2 were upregulated at the margins and are potential druggable targets worthy of further investigation. Also upregulated at the tumor margins were NFKBIA, JUN, ZAP70, GAPDH, and CD28, whereas STAT5A, EFNB2, NTRK1, KDR, FGFR1, PTPRA, IFNGR1, and EFNB1 were downregulated at margins.

Our data demonstrate significant variation of druggable targets and signaling networks across tumor microenvironments in GBM. Marginal tumor differs from bulk and perinecrotic tumor by the relative overexpression of STAT proteins, growth factor receptors, and receptor kinases. Directing therapy at networks and targets present at the tumor margin of GBM may be an effective strategy.

Citation Format: Myles R. McCrary, David Gutman, William Dunn, Milota Kaluzova, Alexandros Bouros, Merete Williams, Xialong Zhang, Lee AD Cooper, Constantinos G. Hadjipanayis, Daniel J. Brat. Differential expression of therapeutic targets across tumor micro-environments and at infiltrative margins in glioblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 415. doi:10.1158/1538-7445.AM2015-415
Abstract 4151: Interleukin 17 promotes tumor progression in oral squamous cell carcinoma
Authors: Avadhani Avadhoot , Milne Trudy , Seymour Gregory , Rich Alison
Published: August 2015

Background: Interleukin 17 (IL17) is a proinflammatory cytokine with increased expression in some cancers. It has been demonstrated to exhibit both pro- and anti-tumor effects. Our group has previously demonstrated expression of IL17 from multiple cells in oral squamous cell carcinoma (OSCC) such that the aim of the current study was to examine the role of IL17 in OSCC progression.

Methods: The cytoplasmic SEFIR sequence of the transmembrane IL17 receptor (IL17R) was detected in formalin Fixed Paraffin Embedded (FFPE) OSCC tissues (n = 14) using immunohistochemistry. Soluble IL17R was detected in the cell culture supernatants of three OSCC cell lines (SCC4, SCC15 and SCC25) using a sandwich ELISA. Human recombinant IL17 over a range of concentrations (0, 10, 50, 100 ng/mL) was used to stimulate the three OSCC cell lines.Proliferation at 0, 24, 48, 72 hours was then assayed by cell titer blue and invasion at 48 hours using a QCM ECMatrix cell invasion assay. The data were analyzed using unpaired Student's t test and ANOVA as appropriate using GraphPad Prism 6 software.

Results:Cytoplasmic expression of transmembrane IL17R was detected in all FFPE OSCC tissues. Soluble IL17R was detected in the cell culture supernatants of all three OSCC cell lines and its concentration increased in a time dependent manner. SCC25 had significantly higher concentration of soluble IL17R at 72 hours than SCC4 and SCC15. The rate of proliferation of all three OSCC cell lines was not affected with the addition of recombinant IL17. However, IL17 promoted the in vitro invasion of SCC25 and SCC15 in a dose dependent manner. At the highest concentration of IL17 (100ng/mL), the SCC25 cells showed significant invasion compared to control cells (p<0.05), while the SCC15 cells exhibited significant invasion at both 50ng/mL and 100 ng/mL of IL17 (p<0.05). In contrast, IL17 failed to stimulate invasion in the SCC4 cell line.

Conclusions: This study is the first to demonstrate in vitro association between IL17 and possible invasion of OSCC. The exact mechanism by which IL17 enhances invasion and thereby tumor progression in vivo remains to be elucidated and could involve a number of different mechanisms including cell adhesion and extracellular matrix proteins.

Citation Format: Avadhoot Avadhani, Trudy Milne, Gregory Seymour, Alison Rich. Interleukin 17 promotes tumor progression in oral squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4151. doi:10.1158/1538-7445.AM2015-4151
Abstract 4155: Interstitial fluid flow-induced hepatocellular carcinoma cell invasion requires MEK/ERK signaling
Authors: Shah Arpit D. , Bouchard Michael , Shieh Adrian C.
Published: August 2015

Background: Worldwide hepatocellular carcinoma (HCC) is the third leading cancer-related cause of death. Within the United States, HCC maintains a rising incidence rate of 3% each year since 1992 with a 5 year survival rate of less than 15%. The American Cancer Society has estimated 33,190 new cases off HCC and 23,000 HCC related deaths in the year 2014 within the United States. Our work aims to determine the role of biomechanical forces such as interstitial fluid flow (IFF) in HCC cell invasion. We hypothesized that HCC cells utilize the hypothesized ‘autologous chemotaxis’ mechanism to invade via secretion of the chemokine CXCL12 and expression of its receptor CXCR4. Autologous chemotaxis occurs when a biologically significant chemokine gradient is established by IFF, driving downstream invasion. In an effort to elucidate signaling pathways involved in IFF-induced HCC invasion, we have identified a potential role for MEK/ERK signaling.

Materials and Methods: HCC cells or primary rat hepatocytes (PRH) were seeded into in a 3-D matrix composed of Matrigel and type I rat tail collagen, then exposed to static or interstitial flow conditions for 24 hours. The functional role of CXCR4 was tested using the CXCR4-antagonist AMD3100. MEK and ERK were inhibited using U0126 (25 μM) and FR180294 (10 μM), respectively. Phospho-MEK/ERK was assessed using western blot.

Results and Discussion: The 3D invasion assay demonstrated that the HCC cells, Huh7 and Hep3B, invaded significantly more in response to IFF (5.5 fold increase in invasion compared to their respective static condition), while PRHs showed no response. AMD3100 was used to block the CXCR4 receptor which resulted in reduced invasion in Huh7 and Hep3B cells. Furthermore when U0126 and FR180204 were incorporated into the 3D invasion assay a significant reduction in flow-induced invasion was observed in Huh7 cells. However, interstitial flow or CXCR4 inhibition did not affect pMEK/ERK, suggesting that MEK/ERK are not modulated directly by IFF and are not downstream of CXCR4.

Conclusion: Our results demonstrate that interstitial fluid flow stimulates HCC cell invasion. CXCR4/CXCL12 signaling and MEK/ERK signaling both are required for invasion, but appear to operate via separate mechanisms. Further investigation into these two separate mechanisms would provide better insight into flow-induced HCC invasion. This information could potentially enhance current cancer therapies and provide patients with better treatment options.

Citation Format: Arpit D. Shah, Michael Bouchard, Adrian C. Shieh. Interstitial fluid flow-induced hepatocellular carcinoma cell invasion requires MEK/ERK signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4155. doi:10.1158/1538-7445.AM2015-4155
Abstract 4173: Endoglin regulation of Smad2/3 function mediates beclin1 expression and endothelial autophagy during angiogenesis
Authors: Lee Nam Y. , Pan Christopher , Kumar Sanjay
Published: August 2015

Autophagy is the targeted degradation of proteins and organelles critical for homeostasis and cell survival. Transforming growth factor beta (TGF-beta) is known to differentially regulate autophagy under distinct physiological conditions by inducing inhibitory, stimulatory, or a combination of these opposing signaling pathways. Importantly, how TGF-beta signaling controls autophagic responses in the endothelial cell (EC) system during angiogenesis remains unknown. Here we identified endoglin, an EC-specific TGF-beta co-receptor essential for angiogenesis, as a key determinant of autophagy. We found that Smad2/3 are major transcriptional regulators of basal autophagy that specifically target beclin1 (BECN1) gene expression. Selectively blocking ALK5 activation or silencing endogenous Smad2/3 markedly increased BECN1 expression whereas altering ALK1/Smad1/5/8 signaling had no effect. Endoglin potentiates basal autophagy by suppressing ALK5/Smad2/3 activation. Notably, increased beclin1 levels upon Smad2/3 knockdown directly correlated with enhanced autophagy during angiogenesis. Taken together, these results establish endoglin as a critical mediator of autophagy and demonstrate a new cellular mechanism by which Smad2/3 inhibits angiogenesis.

Citation Format: Nam Y. Lee, Christopher Pan, Sanjay Kumar. Endoglin regulation of Smad2/3 function mediates beclin1 expression and endothelial autophagy during angiogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4173. doi:10.1158/1538-7445.AM2015-4173
Abstract 4190: A mouse model of pRb2/p130 in prostate cancer
Authors: Boffo Silvia , Klein-Szanto Andres J. , Rizzolio Flavio , Giordano Antonio
Published: August 2015

INTRODUCTION

Prostate Cancer (PCa) is the most commonly diagnosed cancer in men and it is responsible for the second highest number of cancer-related deaths in the United States. There is an urgent need for new treatments and better experimental animal models to study PCa development, progression, and metastasis.

Prostate cancer is a heterogeneous and multifocal disease that arises from several genetic and epigenetic aberrations. Among different altered pathways, the Retinoblastoma is inactivated in more than fifty percent of PCa. Despite a plentiful literature, the activity of pRb members (pRb, pRb2/p130 and p107) is not fully elucidated.

From a previously published mouse model, we know that the inactivation of all the pRb members induces aberrant proliferation and it is sufficient for tumor initiation.

Here, we describe a novel double ko (Rb, pRb2/p130) mouse model, which offers a new opportunity to dissect prostate tumor initiation and development.

MATERIALS AND METHODS

To study the effect of pRb2/p130 inactivation on prostate tumorigenesis and progression, we knocked out pRb and/or pRb2/p130 in the prostate tissue (Probasin-CRE). The genotypes of each gene were determined by PCR. To analyze the effects of pRb2/p130 loss, we collected prostate tissues at different time points (3, 6, 9, 12 and 18 months). A portion of each prostate sample was fixed in formalin and embedded in paraffin to perform immunohistochemistry analyses and another portion was flash-frozen for molecular studies.

The histological classification of each prostate samples was made on H&E-stained sections by A.K.S., a veterinary pathologist with extensive experience in murine prostate pathology.

RESULTS

As reported in literature, pRb family disruption in the prostate epithelium induces prostatic intraepithelial neoplasia. The prostate of triple ko mice showed extensive abnormal gland architecture beginning as early as 2 months of age with a focal hyperplasia. By 3 months of age, all the triple ko mice displayed characteristic traits of murine prostatic intraepithelial neoplasia (mPIN). Double ko (pRb and pRb2/p130) mice developed hyperplasia after 6 months and focally invasive well-differentiated adenocarcinomas by 12-18 months.

CONCLUSIONS

Our preliminary findings suggest that the inactivation of pRb and pRb2/p130 leads to the development of a prostate tumor phenotype and highlight the importance of the function of pRb2/p130 in the prostatic epithelium.

This model will be useful to clarify the molecular mechanism of pRb2/p130 function and represents a new opportunity for cancer therapy.

Citation Format: Silvia Boffo, Andres J. Klein-Szanto, Flavio Rizzolio, Antonio Giordano. A mouse model of pRb2/p130 in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4190. doi:10.1158/1538-7445.AM2015-4190
Abstract 420: The role of tumor microenvironment in therapy resistance and melanoma progression
Authors: Somasundaram Rajasekharan , Zhang Gao , Wagner Stephan N. , Fukunaga-Kalabis1 Mizuho , Herlyn Meenhard
Published: August 2015

Melanoma patients develop resistance to both chemo- and targeted-therapy drugs. Promising pre-clinical and clinical results with immune checkpoint inhibitors using antibodies directed against CTLA4 and PD1 have re-energized the field of immune-based therapies in melanoma. However, similar to chemo- or targeted-therapies only subsets of melanoma patients respond to immune checkpoint blockade. Studies from our laboratory have indicated that the tumor microenvironment (TME) might play a role in the emergence of therapy resistant tumor subpopulations. The TME actively recruits a number of immune and non-immune cells. A recent study describes immune cells as an important component of TME. About 33% of the infiltrating immune cells are of ‘B-cell’ lineage and yet, there is very little information on the role of these cells in melanoma cell biology. Our results suggest that B cells isolated from the tumor tissues of melanoma patients show higher inflammatory cytokine expressions (IGF-1, IL-1, PDGF and VEGF) when compared to circulating B cells. Presence of inflammatory cytokines in the TME results in the induction of heterogeneous melanoma subpopulation, down modulation of melanoma associated antigens (MAA), and thus, therapy resistance of melanoma. Here, we report a novel mechanism of acquired drug resistance in melanoma induced by tumor-associated B cells involving FGF-2/FGFR-3 and IGF-1 signaling. B-cell derived IGF-1 modulates the emergence of therapy resistant heterogeneous melanoma subpopulations. Neutralization of IGF-1 reverses the induction of heterogeneous melanoma subpopulations. We are currently evaluating the mechanism of down modulation of MAA in presence of inflammatory cytokines. Our studies confirm the important role of the TME in the induction of therapy resistant tumor subpopulations and offer a new combination treatment approach of targeting melanoma (Supported by AMRF and NIH grant CA114046).

Citation Format: Rajasekharan Somasundaram, Gao Zhang, Stephan N. Wagner, Mizuho Fukunaga-Kalabis1, Meenhard Herlyn. The role of tumor microenvironment in therapy resistance and melanoma progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 420. doi:10.1158/1538-7445.AM2015-420
Abstract 4201: Development of a syngeneic metastatic mouse model of malignant lymphoma
Authors: Matsumoto Takuro , Suetsugu Atsushi , Shibata Yuhei , Nakamura Nobuhiko , Aoki Hitomi , Kunisada Takahiro , Shimizu Masahito , Tsurumi Hisashi , Hoffman Robert M.
Published: August 2015

We have developed a mouse model of highly malignant lymphoma. The EL4 cell line was established from a lymphoma induced in a C57/BL6 mouse by 9,10-dimethyl-1,2-benzanthracene. Red fluorescent protein (RFP)-EL4 lymphoma cells were established. EL4-RFP cancer cells were injected in the tail vein of C57/BL6 GFP transgenic mice. EL4-RFP metastasis was observed in the lymph nodes of the superior mediastinum and in the liver 28 days later. The EL4-RFP tumors were visualized in the liver with the Olympus SZX7 microscope. Large EL4-RFP liver metastases in C57/BL6 GFP mice had GFP stromal cells derived from the host animal around the edge of EL4-RFP tumors. In addition, EL4-RFP lymphoma cells metastasis was formed in perigastric lymph node. EL4-RFP lymphoma cells circulated in the peripheral blood. Furthermore, EL4-RFP lymphoma cells were observed in bone marrow of C57/BL6 GFP transgenic mice. In summary, lymph node metastasis, liver metastasis and bone marrow metastasis were found approximately 4 weeks after transplantation. This malignant lymphoma model can be used to study early tumor development, metastasis, the role of stroma and discovery and evaluation of novel therapeuticus for this treatment-resistant disease.

Citation Format: Takuro Matsumoto, Atsushi Suetsugu, Yuhei Shibata, Nobuhiko Nakamura, Hitomi Aoki, Takahiro Kunisada, Masahito Shimizu, Hisashi Tsurumi, Robert M. Hoffman. Development of a syngeneic metastatic mouse model of malignant lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4201. doi:10.1158/1538-7445.AM2015-4201
Abstract 4216: Targeting NANOG: genes, proteins and response to viral RNAi in preclinical models
Authors: Jessup J. Milburn , Mattoo Abid R. , Korokhov Nikolay
Published: August 2015

Purpose: Since the NANOG family controls stemness and neoplastic progression in human colorectal carcinoma (CRC), our postulate is that targeting the dominant NANOG and NANOGP8 members with viral vector delivered shRNA will inhibit CRC progression in preclinical models and that replicating viral vectors are more active than non-replicating vectors.

Methods: Cells were human CRC lines (Clone A, CX-1 and LS174T) and mouse fibroblasts (3T3). shRNA specific to NANOG (shNG-1), NANOGP8 (shNp8-1) or a control (shNEG) were created in lentivirus (LV) and chimeric adenovirus 5/3 (Ad5/3). CRC and 3T3 were cultured in monolayer with serum-containing medium or in suspension in ultra low attachment plates without serum. Nanostring 770 gene cancer panel was analyzed in 72 hr cultures with NMF clustering and GSEA analysis. Annexin V expression and caspase-3, 8 or 9 activation in suspension culture assessed Apoptosis. LV or Ad5/3 shRNAs were added to spheroids in 3-D suspension cultures to test therapy in vitro. Intratumoral injection of LV or Ad5/3 shRNA into 5-10 mm sub cutis nodules in groups of 5-10 NOD/SCID mice measured activity in vivo.

Results: LV shNp8-1 in both Clone A and CX-1 caused significant inhibition of TGF-beta, EGFR, NRF2, E2F3 and the Cyclin D1 pathways by NMF and GSEA analysis in monolayer culture. LV shNp8-1 inhibited growth of LS174T, Clone A and CX-1 by 50% in 3 days in 3-D suspension cultures without further decrease. Apoptosis was due to Caspase 9 activation. In contrast, Ad5/3-shNp8-1 decreased spheroid growth by up to 94% at 9 -14 days while Ad5/3 decreases spheroid growth by 80%. Ad5/3-shNp8-1 but nit Ad5/3 was active in co-cultures with 3T3 to a 2:1 CRC:3T3 ratio. Transduction level was ∼50% in LV treated cells but ∼90% in Ad5/3 treated cells. Intratumoral injection of LVshNG-1 or LVshNp8-1, decreased tumor size by 50% compared to LVshNEG or untreated CX-1 tumors for 11 days after which tumor growth rebounded to match controls. Ad5/3-shNp8-1 injection into 5-9 mm nodules inhibited CX-1 and LS14T growth by ∼50% until euthanized.

Conclusions: Targeting NANOGP8 more than NANOG inhibits proliferation and activates apoptosis through multiple pathways. Nonreplicating LV delivered shRNA inhibited tumor growth in in vitro and in vivo preclinical models but replicating oncolytic Ad5/3 infects more cells and promotes shRNA efficacy. Replicating vectors targeting NANOG are important for further development.

Citation Format: J. Milburn Jessup, Abid R. Mattoo, Nikolay Korokhov. Targeting NANOG: genes, proteins and response to viral RNAi in preclinical models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4216. doi:10.1158/1538-7445.AM2015-4216
Abstract 4217: Targeting head and neck cancer-initiating cells with combinatorial therapeutics through pharmacological inhibition of ROS scavenger and conventional chemotherapy
Authors: Chang Ching-Wen , Lo Jeng-Fan
Published: August 2015

Background: Cancer-initiating cells (CICs) contribute to tumor initiation, progression and therapy resistance through critical physiology mechanisms. Redox homeostasis plays an important role in the regulation of cancer stemness. We recently have reported that cancer cells containing differential level of ROS display distinct physiological properties of stemness, proliferative activity, chemoresistance and tumorigenicity. Therefore, to uncover the regulatory physiological mechanisms by which to sustain the low lever of ROS in CICs warrants an important study for future therapy development.

Results: In this study, we demonstrated that lower ROS levels in head and neck CICs (HN-CIC) enriched population are associated with increased expression of ROS scavenger such as catalase and SOD2. Pharmacological and genetic inhibition of SOD2 or catalase caused the increased expression of differentiation marker, cytokeratin 18, but the decreased expression of CIC marker in HN-CICs. In addition, the sphere formation abilities of HN-CICs under genetic inhibition of SOD2 or catalase were also significantly abolished. Notably, combinatorial treatment with ROS scavenger inhibitors and conventional anticancer drugs synergistically reduced the stemness properties and tumorigenicity both in vitro and in vivo. Using the Oncomine database, we found associations between SOD2 and catalase expression and increasing head and neck cancer recurrence, tumor invasion and resistance to Gemcitabine treatment.

Conclusion: Together, these data demonstrate the importance of antioxidant defense to maintain the low ROS level, cell viability and chemosensitivity of HN-CICs. Pharmacological inhibition of ROS scavenger (SOD2 and catalase) might be a future alternative HNSCC treatment in combination with conventional chemotherapy

Citation Format: Ching-Wen Chang, Jeng-Fan Lo. Targeting head and neck cancer-initiating cells with combinatorial therapeutics through pharmacological inhibition of ROS scavenger and conventional chemotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4217. doi:10.1158/1538-7445.AM2015-4217
Abstract 4218: Phenotypic selection screening reveals cancer stem cell therapeutic targets
Authors: Hurt Elaine M. , Flynn Matt , Thomas Suneetha , van Vlerken-Ysla Lilian , Sandercock Alan , Rust Steven , Ralph Minter , Hollingsworth Robert
Published: August 2015

Cancer stem cells (CSCs) represent an important subpopulation of tumor cells that are untouched and perhaps even expanded after chemotherapy and radiation. This population has been shown in murine models of cancer to be solely responsible for the regrowth of tumors following chemotherapy, underscoring the importance of therapeutically targeting this population. Therefore, we sought to find novel targets against CSCs using a phenotypic selection screen. Using a designed ankyrin repeat (DARpin) library, we panned cells disaggregated from tumor spheres from three patient-derived xenograft models of late stage pancreatic cancer to enrich for DARPins that bind CSCs. Once released from the cells, the DARPins were further analyzed for binding to both normal, pancreatic cancer total tumor cells and pancreatic CSCs in order to choose targets that showed either pancreatic cancer-specific or pancreatic CSC-specific binding. These DARPins were then tested in a sphere formation assay, an EZH2-based high content imaging screen, and a standard proliferation assay. Those DARPins showing good cancer-specific binding profiles were also tested for internalization and killing using a saporin-conjugated secondary antibody. Using this approach, we identified 30 DARPins of interest for either direct inhibitory antibody approaches or for potential antibody drug conjugate or nanoparticle delivery approaches. Identification of the proteins bound by each of the DARPins was done using either immunopreciptation followed by liquid chromatography tandem-mass spectrometry or through Retrogenix's cell expression microarray technology. Identified targets included known CSC antigens such as integrin alpha6 (CD49f) as well as other potentially novel targets. These results highlight the possibilities of finding therapeutic targets for CSCs and opens up an exciting possibility for novel therapies.

Citation Format: Elaine M. Hurt, Matt Flynn, Suneetha Thomas, Lilian van Vlerken-Ysla, Alan Sandercock, Steven Rust, Minter Ralph, Robert Hollingsworth. Phenotypic selection screening reveals cancer stem cell therapeutic targets. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4218. doi:10.1158/1538-7445.AM2015-4218
Abstract 422: The role of CCL2 in the breast cancer tumor microenvironment and metastasis
Authors: Lavender Nicole , Sai Jiqing , Yang Jinming , Novitskiy Sergey V. , Richmond Ann
Published: August 2015

Background: Although many effective breast cancer treatments exist, metastasis remains a major problem. Increasing evidence suggests that leukocyte-associated chemokines may play an important role in tumor growth and metastasis. For instance, recent data shows that reduction of CCL2 diminishes the recruitment of inflammatory monocytes to the tumor microenvironment (TME) and inhibits metastatic seeding to lung and bone marrow. Therefore, our goal is to examine the impact of CCL2 on the TME and pre-metastatic niche (i.e., lung).

Methods: We are investigating how delivery of this chemokine affects breast tumor growth and metastasis by treating mice possessing highly metastatic and poorly metastatic tumors with CCL2, a CCL2 receptor (CCR2) antagonist, or vehicle for two weeks. Treatment will start 7 days after tumor implantation. On days 7, 14 or 21, tumors, lungs, and regional lymph nodes will be harvested and tumor size, number of metastases, and leukocyte infiltrate in both the tumor and the metastatic niche will be analyzed by FACS. This will allow us to fully characterize how intranasal delivery of CCL2 will affect the presence of mesenchymal stem cells (CD44+, CD73+, CD90+, CD146+/NOTCH3+, CD19-, CD45-), subsets of T cells, and myeloid cells in the TME or pre-metastatic niches. We will evaluate the intracellular cytokine and enzymatic profile (IL-4, IL-13, IL-17, ARG1 or IFNγ, IL-12, iNOS2) of Brefeldin-treated leukocytes to reveal whether they are pro- or anti-tumorigenic, respectively.

Results: Our preliminary studies demonstrate that CCL2 enhances the release of hydrogen peroxide from neutrophils, a mechanism utilized by these cells to kill tumors cells. Furthermore, the addition of CCL2 to co-cultures of neutrophils and tumor cells increases the ability of neutrophils to kill tumor cells.

Conclusions & Future Directions: Our data shows that delivery of CCL2 in vitro enhances the activity of neutrophils and their ability to kill tumor cells. Ongoing studies are examining the effects of in vivo and ex-vivo delivery of CCL2 or a CCR2 antagonist on primary tumor growth and metastasis. We will also investigate how administration of CCL2 or a receptor antagonist will affect the presence or absence of leukocytes in the TME. These findings will determine whether the addition of CCL2 during treatment of the primary tumor will provide a mechanism for reducing the potential of future breast cancer metastases.

Citation Format: Nicole Lavender, Jiqing Sai, Jinming Yang, Sergey V. Novitskiy, Ann Richmond. The role of CCL2 in the breast cancer tumor microenvironment and metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 422. doi:10.1158/1538-7445.AM2015-422