bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 496: Adaptive response to chronic oxidative stress involves epithelial-mesenchymal transition in MCF-7 breast cancer cells
Authors: Mahalingaiah Prathap Kumar S. , Ponnusamy Logeswari , Singh Kamaleshwar P.
Published: May 2015

Though the increased oxidative stress by endogenous and exogenous factors has been hypothesized as a potential mechanism for breast cancer growth and progression, but there is no direct mechanistic evidence for adaptation to ROS-induced toxicity by the breast cancer cells. Therefore the objective of this study was to evaluate effects of chronic oxidative stress on the growth, survival and tumorigenicity of MCF-7 breast cancer cells. MCF-7 cells were exposed to hydrogen peroxide (H202) induced oxidative stress for both acute (48 hrs) and chronic (3 months) period. Cell growth and viability were evaluated by cell count and MTT assay and were confirmed by cell cycle analysis. Expression of genes related to cell cycle, cell survival, and metastasis were measured by quantitative real-time PCR. Effect of oxidative stress on tumorigenic phenotype was determined in-vitro by soft agar assay. Results of cell count, MTT and cell cycle analysis revealed increased growth, and survival of MCF-7 cells that was chronically exposed to H2O2. This was further confirmed at molecular level by increased expression of cell cycle and cell survival genes, whereas down-regulation of apoptotic genes. Chronic oxidative stress also increased tumorigenic phenotype and metastatic potential of MCF-7 cells as revealed by increase in soft agar grown colonies and expression of metastasis related genes. Additionally, fibroblast-like appearance with down regulation of epithelial markers, whereas up-regulation of mesenchymal markers in cells with chronic exposure to oxidative stress further suggest that adaptation to chronic oxidative stress by MCF-7 cells involves epithelial to mesenchymal transition (EMT). Dysregulation of epigenetic regulatory genes expression also suggest potential role of epigenetic mechanism in increased survival and tumorigencity of breast cancer cells.The findings of this study for the first time provided direct evidence for involvement EMT and epigenetic changes in adaptation of MCF-7 breast cancer cells to chronic oxidative stress. Results of this study are also highly significant in understanding the mechanism for acquired resistance to chemotherapeutic drugs that are known to produce ROS in breast cancer cells.

Citation Format: Prathap Kumar S. Mahalingaiah, Logeswari Ponnusamy, Kamaleshwar P. Singh. Adaptive response to chronic oxidative stress involves epithelial-mesenchymal transition in MCF-7 breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 496. doi:10.1158/1538-7445.AM2014-496
Abstract 4967: ESRP1 regulated alternative splicing of CD44mRNA enhances lung colonization of metastatic cancer cell
Authors: Tsuchihashi Kenji , Nagano Osamu , Yae Toshifumi , Ishimoto Takatsugu , Motohara Takeshi , Yoshikawa Momoko , Yoshida Go J , Wada Takeyuki , Masuko Takashi , Mogushi Kaoru , Tanaka Hiroshi , Osawa Tsuyoshi , Kanki Yasuharu , Minami Takashi , Aburatani Hiroyuki , Ohmura Mitsuyo , Kubo Akiko , Suematsu Makoto , Takahashi Kazuhisa , Baba Eishi , Akashi Koichi , Saya Hideyuki
Published: May 2015

Cancer metastasis is established by the seeding and successful colonization of stem-like cancer cells which show tumor-initiating ability at distant organs. CD44 is a cell surface marker protein which is highly expressed in stem-like cells of several human cancers including breast cancer. CD44 exists in various isoforms produced through alternative splicing of mRNA. Increased expression of CD44 variant isoform (CD44v) has been shown to correlate with metastatic potential and aggressive clinical behavior of various human cancers. We recently showed that CD44v contributes to reactive oxygen species (ROS)-defense in cancer cells by enhancing cystine uptake by the cystine transporter xCT. However, the functional relevance of CD44v and xCT in the metastatic spread of stem-like cancer cells has remained elusive.

In the present study, we show that metastatic breast cancer 4T1 cells consist of CD44v+ and CD44v- cells. The orthotopic transplantation of CD44v+ population but not that of CD44v-, in mice resulted in efficient lung metastasis. We found that CD44v expressing metastatic cancer cells highly express xCT and thereby maintain high reduced glutathione (GSH) level. A specific xCT inhibitor sulfasalazine suppressed lung metastatsis of CD44v+ cells. Alternative splicing of CD44mRNA in such metastatic cancer cells is regulated by Epithelial Splicing Related Protein1 (ESRP1) and short hairpin RNA (shRNA) targeting ESRP1 switched isoform from CD44v to CD44 standard (CD44s), leading to the suppression of lung metastasis. Furthermore, we found that ESRP1 expression is regulated epigenetically through histone modification by performing chromatin immunoprecipitation (ChIP) sequencing analysis of the ESRP1 locus in CD44v+ and CD44v- cells.

These findings establish a novel role of ESRP1-regulated alternative splicing program in the regulation of antioxidant status of stem-like cancer cells which is crucial for the metastatic ability.

Citation Format: Kenji Tsuchihashi, Osamu Nagano, Toshifumi Yae, Takatsugu Ishimoto, Takeshi Motohara, Momoko Yoshikawa, Go J Yoshida, Takeyuki Wada, Takashi Masuko, Kaoru Mogushi, Hiroshi Tanaka, Tsuyoshi Osawa, Yasuharu Kanki, Takashi Minami, Hiroyuki Aburatani, Mitsuyo Ohmura, Akiko Kubo, Makoto Suematsu, Kazuhisa Takahashi, Eishi Baba, Koichi Akashi, Hideyuki Saya. ESRP1 regulated alternative splicing of CD44mRNA enhances lung colonization of metastatic cancer cell. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4967. doi:10.1158/1538-7445.AM2014-4967
Abstract 4969: Hypo adenosine-to-inosine editing of microRNA-455 promotes melanoma metastasis
Authors: Shoshan Einav , Mobley Aaron , Braeuer Russell , Kamiya Takafumi , Huang Li , Vasquez Mayra , Jeong Lee H. , kim Sun Jim , Calin George , Sood Anil , Markel Gal , Fidler Isaiah , Bar-Eli Menashe
Published: May 2015

We have previously reported that activation of CREB in metastatic melanoma cells resulted in reduced expression of adenosine deaminase acting on RNA 1 (ADAR1), an RNA editing enzyme. We hypothesized that disruption of A-to-I editing of microRNAs alters expression of genes regulating melanoma progression. By sequencing miRs in primary vs. metastatic melanoma cells, we show that the overall microRNA editing frequencies were reduced in metastatic melanoma cells. Specifically, we found A-to-I editing in two sites of miR-455 in non-metastatic melanoma cells with accumulation of unedited miR-455 in metastatic melanoma cells. Using two animal models, we demonstrated that the unedited miR-455 promoted melanoma growth and metastasis, while the edited form suppressed these features. Unedited miR-455 promotes melanoma progression via suppression of the tumor suppressor cytoplasmic polyadenilation element binding protein (CPEB1). Thus, miR-455 A-to-I editing alters the selection of its target genes and directs its function to inhibit melanoma growth and metastasis. Taken together, our results provide a previously undescribed mechanism (microRNA editing) contributing to melanoma progression.

Citation Format: Einav Shoshan, Aaron Mobley, Russell Braeuer, Takafumi Kamiya, Li Huang, Mayra Vasquez, Lee Ho Jeong, Sun Jim kim, George Calin, Anil Sood, Gal Markel, Isaiah Fidler, Menashe Bar-Eli. Hypo adenosine-to-inosine editing of microRNA-455 promotes melanoma metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4969. doi:10.1158/1538-7445.AM2014-4969
Abstract 4972: In vivo whole genome shRNA screen reveals novel targets to block cancer metastasis
Authors: Stoletov Konstantin , Bond David , Leong Hon Sing , Woolner Emma , Raha Srijan , Robertson Amy , Wong Francis , Zijlstra Andries , Lewis John D.
Published: May 2015

The formation of invasive, rapidly growing metastatic lesions is a critical step in cancer metastasis, the cause of more than 90% of cancer deaths. Development of novel therapeutic approaches that block the invasion step of metastasis is one of the highest priorities for clinical cancer research. For this reason we completed the first genome-wide in vivo shRNA screen for genes that directly contribute to invasive metastatic lesion formation. Using state of the art intravital imaging, we identified over fifty novel regulators of invasive metastatic colony formation in vivo. Interactome analysis links these genes to key cellular processes including: transcriptional regulation of gene expression, mRNA processing and cytoskeletal remodeling. The target list was then prioritized based on clinical gene expression profiles that negatively correlated with key cancer endpoints including metastasis, cancer-specific and overall survival. Pharmacological and shRNA-mediated knockdown of the high priority targets in human cancer cell lines such as prostate cancer and melanoma specifically blocked cancer cell migration and invasion in vitro and in vivo. Moreover, shRNA-mediated knockdown of these genes blocked human cancer cell metastasis in the avian embryo and mouse preclinical models of metastasis. Finally, immunohistochemical analysis on clinical prostate cancer and melanoma tissue samples showed strong correlation with disease progression and metastasis. In summary, we have identified numerous novel genes that that are functionally involved in cancer invasion and metastasis that may serve as predictive markers for disease aggressiveness and represent exciting new pharmacological targets to block cancer invasion and metastasis.

Citation Format: Konstantin Stoletov, David Bond, Hon Sing Leong, Emma Woolner, Srijan Raha, Amy Robertson, Francis Wong, Andries Zijlstra, John D. Lewis. In vivo whole genome shRNA screen reveals novel targets to block cancer metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4972. doi:10.1158/1538-7445.AM2014-4972
Abstract 4978: Targeting of phosphatidylserine by monoclonal antibodies enhances activity of immune checkpoint inhibitors in tumors
Authors: Gong Jian , Nguyen Van , Yin Shen , Archer Rich , Hutchins Jeff , Freimark Bruce
Published: May 2015

Phosphatidylserine (PS) is a phospholipid normally residing in the inner leaflet of the plasma membrane and becomes exposed on tumor vascular endothelial cells (ECs) and tumor cells. PS exposure becomesenhanced in response to chemotherapy, irradiation, and oxidative stresses in the tumor microenvironment. PS exposure in tumors promotes an immunosuppressive microenvironment which includes the recruitment of myeloid derived suppressor cells (MDSCs), immature dendritic cells, and M2-like macrophages as well as the production of anti-inflammatory cytokines. Binding of PS targeting antibodies on tumor endothelial cells, tumor cells and their secreted microparticles triggers an Fc-FcR mediated pro-inflammatory cellular and cytokine response that reverses the immunosuppressive PS meditated checkpoint, thereby enhancing anti-tumor immunity. A chimeric PS-targeting antibody, bavituximab, is being used in combination with chemotherapy to treat patients with solid tumors in multiple late-stage clinical trials. Using multiple syngeneic tumors in immune competent mice, we demonstrate PS targeting antibodies enhance the anti-tumor activity of anti-CTLA-4 and anti-PD-1 antibodies. Tumor growth inhibition correlates with infiltration of immune cells in tumors and induction of adaptive immunity. The combination of these mechanisms promotes strong, localized, anti-tumor responses without the side-effects of systemic immune activation.

Citation Format: Jian Gong, Van Nguyen, Shen Yin, Rich Archer, Jeff Hutchins, Bruce Freimark. Targeting of phosphatidylserine by monoclonal antibodies enhances activity of immune checkpoint inhibitors in tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4978. doi:10.1158/1538-7445.AM2014-4978
Abstract 498: Blunting the radical scavenging property of mitochondria-targeted, triphenylphosphonium-linked antioxidants failed to mitigate their anti-proliferative and antitumor effects
Authors: Cheng Gang , Zielonka Jacek , Joseph Joy , Kalyanaraman Balaraman
Published: May 2015

Reactive oxygen species (ROS), including superoxide and hydrogen peroxide, are implicated in cancer cell proliferation. Therefore, strategies to mitigate ROS levels were proposed as a viable mechanism to inhibit cancer cell proliferation. As mitochondria are believed to be a major contributor to intracellular ROS production, mitochondria-targeted antioxidants (MTAs) (e.g., Mito-Q, Mito-CP, Mito-Vitamin-E or Mito-chromanol) were successfully used to stop proliferation and kill cancer cells in vitro and attenuate tumor growth in vivo. MTAs also inhibited mitochondrial oxygen consumption and ATP production. In this study, we tested whether the antioxidant moiety and related antioxidant function of MTAs or other mitochondrial mechanism is responsible for inhibiting tumor cell proliferation and resulting cytotoxicity. To this end, we compared the anti-proliferative and antitumor effects of Mito-CP, a mitochondrially-targeted nitroxide exhibiting superoxide dismutase (SOD)-like activity, and a synthetic analog of Mito-CP (Mito-CP acetamide), lacking the nitroxide moiety and SOD-like activity. Both Mito-CP and Mito-CP acetamide were equally potent in different cancer cell types (breast, pancreatic, colon, skin, bladder and lung). Additionally, both compounds inhibited mitochondrial function and decreased intracellular ATP levels to the same extent. We conclude that MTAs such as Mito-CP, Mito-Vitamin-E exert their antiproliferative effects in cancer cells due to mitochondrial localization and targeting cellular bioenergetics and not due to their antioxidative activity. Results from this study strongly question the present dogma on mitochondrial ROS involvement in cancer cell proliferation.

Citation Format: Gang Cheng, Jacek Zielonka, Joy Joseph, Balaraman Kalyanaraman. Blunting the radical scavenging property of mitochondria-targeted, triphenylphosphonium-linked antioxidants failed to mitigate their anti-proliferative and antitumor effects. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 498. doi:10.1158/1538-7445.AM2014-498
Abstract 4994: E2F1 deficiency causes increased migration through upregulation of MYLK
Authors: Meng Peng , Ghosh Rita
Published: May 2015

Malignant melanoma is an extremely aggressive disease with high metastatic potential. Several studies have shown that the transcription factor E2F1 is overexpressed in melanoma. E2F1 belongs to the family of E2F transcription factors; is more abundant in melanoma cells than in normal melanocytes, which highlights its relevance to melanoma biology. E2F1 participates in a variety of biological processes such as proliferation, apoptosis, development, differentiation, DNA damage and repair, oxidative metabolism, and self-renewal by virtue of its transcriptional activator functions. However, its role in many of these biological processes in melanoma is not yet fully addressed. In this study, we show that E2F1 deficiency increased migration and invasion of melanoma cells, which was associated with increased MYLK at both RNA and protein level. MYLK is a calcium-calmodulin (Ca2+//CaM)-dependent myosin light chain kinase, which phosphorylates Ser-19 of myosin II after activation. This process is implicated in the regulation of smooth muscle contraction. In non-smooth muscle, MYLK is known to be involved in many inflammatory diseases through regulation of tight junction and intestinal epithelial barrier function. MYLK is also involved with proliferation and migration in cancers of the breast, prostate and colon, but its expression and role in melanoma cells is unknown. ML-7, the specific inhibitor for MYLK abrogated the increased migration and invasion due to E2F1 deficiency. Transient knockdown of MYLK also significantly abrogated the increased migration and invasion from E2F1 deficiency. Our results strongly suggest that E2F1 deficiency enhanced migration and invasion through regulating MYLK expression. We also found increased pERK in E2F1 knockdown cells, which suggest that ERK activation may be involved in the up-regulation of MYLK. All of our data taken together suggests that E2F1-mediated inhibition of melanoma cell migration occurs through a complex cross-talk between E2F1-ERK-MYLK and is not through a simple linear relationship between signaling molecules. Supported by R21 CA125719 (RG).

Citation Format: Peng Meng, Rita Ghosh. E2F1 deficiency causes increased migration through upregulation of MYLK. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4994. doi:10.1158/1538-7445.AM2014-4994
Abstract 4995: Role of MICAL1 in NEDD9 invasive signaling
Authors: Holmes Casey O. , Brock Amanda M. , Sobash Philip T. , Tiedeken Jessica A. , Rosenzweig Steven A.
Published: May 2015

Metastasis is the final step leading to patient death in most solid tumors, including head and neck squamous cell carcinoma (HNSCC). For metastasis to proceed, tumor cells must become mobile and invasive, losing their communal behavior. A goal of our work is to delineate the downstream effectors of growth factor signaling pathways that contribute to invasion. Using a phosphotyrosine proteomics screen we previously identified neural precursor cell expressed, developmentally down-regulated 9 (NEDD9; CasL) as a mediator of VEGF and IGF-1 signaling to invasion in HNSCC cells. NEDD9 tyrosine phosphorylation led to matrix metalloproteinase9 (MMP9) expression and secretion, invadopodia formation and enhanced invasion. To determine the molecular details of NEDD9 protein:protein interactions leading to invasion, we have generated a series of NEDD9 substrate domain tyrosine (Y to F, YXXP) and SH3 domain mutants. Mutation of all 13 YXXP motifs to FXXP blocked NEDD9 tyrosine phosphorylation, MMP9 expression and invadopodia formation. Of note, we found that NEDD9 co-immunoprecipitated with Molecule Interacting with CasL 1 (MICAL1). To further define this interaction and its downstream impact, stable NEDD9 or MICAL1 silenced HNSCC cell lines expressing select shRNAs were generated, with each exhibiting reduced migration and invasion. MICAL shRNA transfected cell lines also exhibited decreased MMP9 secretion, consistent with our findings that lack of MICAL1 decreases cell invasion and with findings of other labs that MICALs interact with Rab proteins altering intracellular trafficking. Further, we have verified that MICAL1 and NEDD9 form complexes in cells based on coimmunoprecipitation analyses. Better understanding of this pathway will undoubtedly lead to the identification of novel therapeutics for the prevention of invasion and metastasis. Supported by NIH R01CA134845 (SAR), P30 CA138313 and NIH/NCRR UL1 TR000062/TL1 TR000061 from the South Carolina Clinical & Translational Research Institute (COH).

Citation Format: Casey O. Holmes, Amanda M. Brock, Philip T. Sobash, Jessica A. Tiedeken, Steven A. Rosenzweig. Role of MICAL1 in NEDD9 invasive signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4995. doi:10.1158/1538-7445.AM2014-4995
Abstract 500: Heat shock protein gp96 regulates LRP6-Wnt-Survivin pathway and myeloma progression
Authors: Hua Yunpeng , White-Gilbertson Shai , Kellner Joshua , Rachidi Saleh , Li Zihai , Liu Bei
Published: May 2015

Multiple myeloma (MM) is an incurable plasma cell neoplasm whose pathogenesis is closely linked to dysregulated unfolded protein response (UPR) in the endoplasmic reticulum (ER). Constitutive activation of UPR in mice causes myeloma, as demonstrated by transgenic expression of a master UPR transcription factor XBP1s. However, the underlying mechanism remains unknown. Heat shock protein gp96 (grp94) is an ER paralogue of the cytosolic HSP90 and its expression can be induced by the accumulation of misfolded proteins. As one of the most abundant proteins in the ER lumen, gp96 is a key downstream chaperone in the ER to mediate UPR. However, the roles of individual ER HSPs in plasma cell and myeloma have not been reported. In this study, we demonstrated that the persistence of plasma cells as well as the development of myeloma in XBP1s-transgenic mice is critically dependent on gp96 in vivo. Furthermore, we demonstrated that gp96 silencing in human MM cells led to significant reduction of surface expression of Wnt coreceptor LRP6 and the downstream canonical Wnt signaling. gp96 knockdown causes severe compromise in MM cell growth which can be significantly rescued by GSK3β inhibitors. In the absence of gp96, MM cells undergo mitotic catastrophe and apoptosis which correlated with decreased expression of survivin, a downstream molecule of Wnt in controlling cell survival. This is the first study to uncover the critical roles of gp96 in the initiation and progression of multiple myeloma, suggesting that blockade of gp96 is a novel therapeutic strategy against multiple myeloma.

Citation Format: Yunpeng Hua, Shai White-Gilbertson, Joshua Kellner, Saleh Rachidi, Zihai Li, Bei Liu. Heat shock protein gp96 regulates LRP6-Wnt-Survivin pathway and myeloma progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 500. doi:10.1158/1538-7445.AM2014-500
Abstract 5004: Iqgap2 is downregulated in colorectal cancer (crc) and involved in cellular migration
Authors: Gröschl Benedikt , Bettstetter Marcus , Widmann Tamara , Hofstädter Ferdinand , Dietmaier Wolfgang
Published: May 2015

IQGAP2 is a member of three IQGAP scaffolding proteins and involved in cytoskeletal reorganization and adhesion of cells. It has been described as a putative tumor suppressor affecting tumor development and progression. Our aim in this study was to analyze the role of IQGAP2 in colorectal cancer (CRC).

By means of microarray analyses we previously found IQGAP2 expression to be downregulated in CRC and suppressed by Maspin. Here we measured IQGAP2 expression by RT-qPCR in 17 MSI-H (high microsatellite instability) and 19 MSS (microsatellite stable) CRCs as well as in three MSI and three MSS CRC cell lines, respectively. Furthermore, we analyzed the methylation status of the IQGAP2 promoter and studied the subcellular localization of the IQGAP2 protein. Using an in vitro system for overexpressing and silencing IQGAP2 we investigated the effect of IQGAP2 on cellular migration applying Real-Time Cell Analyzer (RTCA) technology.

Stably overexpressing Maspin transfected CRC cell line SW480 showed IQGAP2 loss on mRNA and protein level. IQGAP2 expression was reduced in 86% (30/36) of tumors compared to normal tissue (p<0.001) and lower in MSS tumors compared to MSI tumors (p=0.039). Consistently, IQGAP2 mRNA levels were also downregulated in CRC cell lines. None of the cell lines, tumor or normal tissue samples analyzed showed IQGAP2 promoter methylation. Immunofluorescence analysis of CRC cells revealed IQGAP2 distribution in plasma membranes and more intensely in areas of cell-cell contacts. Migration assays showed increased cellular migration after IQGAP2 knockdown and impaired migration after overexpression of IQGAP2.

We provide evidence that IQGAP2 expression is regulated by Maspin and may have an important effect on cellular migration in colorectal cancer.

Citation Format: Benedikt Gröschl, Marcus Bettstetter, Tamara Widmann, Ferdinand Hofstädter, Wolfgang Dietmaier. Iqgap2 is downregulated in colorectal cancer (crc) and involved in cellular migration. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5004. doi:10.1158/1538-7445.AM2014-5004
Abstract 5006: A 3-dimensional cell invasion assay compatible with high content screening
Authors: Fronczak Jennifer A. , Gajeski Matthew S. , Beckman Erica L. , Vollmer Laura , Vogt Andreas , Krishnan Gopal
Published: May 2015

The transition from non-invasive phenotype to invasive phenotype of tumor cells marks the switch from a benign tumor that can be successfully treated via surgery to a more malignant form of cancer. Understanding the mechanisms underlying this hallmark event, which enables tumor cells to invade through extracellular matrix, is critical for discovering pathways and new targets to develop anti-metastatic strategies. Previously, we developed a cell based assay for the study of cell migration in 96-well plates. A self-dissolving biocompatible gel (BCG) is utilized to form uniformly sized, cell-free detection zones in collagen I coated 96-well plates. When cells are seeded into these wells, they pattern in an annular monolayer surrounding the BCG. Once the BCG dissolves, an overlay of collagen I is applied to the assay wells, following which cells can invade in 3-dimensions into the detection zone previously occupied by the BCG. This “Oris™ Pro” cell invasion assay presents a straightforward, accessible and quantifiable cell method to study cell invasion, and can be used for example to evaluate candidate drugs targeting tumor invasion. Microscopic visual assessment of cell invasion in the presence of inhibitors is made by use of cell stains at multiple time-points. In this study we demonstrate the use of this assay platform for robust and reproducible analysis of HT-1080 and MDA- MB-231 cell invasion in 3-D. The presentation will discuss (i) factors that influence cell invasion, including density of collagen overlay and time; (ii) microscopic monitoring of cell invasion in the presence of potential inhibitors throughout the duration of the experiment; and (iii) the qualitative and quantitative readouts that can be obtained using this 3-dimensional cell invasion assay.

Citation Format: Jennifer A. Fronczak, Matthew S. Gajeski, Erica L. Beckman, Laura Vollmer, Andreas Vogt, Gopal Krishnan. A 3-dimensional cell invasion assay compatible with high content screening. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5006. doi:10.1158/1538-7445.AM2014-5006
Abstract 5007: RSK isoform regulation of migration and invasion in glioblastoma
Authors: Prechtl Amanda , Sulzmaier Florian , Jiang Pengfei , Kesari Santosh , Ramos Joe
Published: May 2015

Glioblastoma (GBM) is associated with high mortality in patients due in large part to the highly migratory and invasive nature of glioma cells. The 90kDa ribosomal S6 Kinases (RSKs) are a family of serine/theronine kinases that are an integral component of the extracellular signal regulated kinase (ERK)/ mitogen activated protein kinase (MAPK) pathway, which is highly active in many types of cancer. The RSK family contains four human isoforms (RSK1-4) that regulate a variety of cellular processes including cell proliferation, survival, and motility, however a role for individual RSK isoforms in GBM cell invasion has not clearly been defined. Here, we analyze the roles of RSK1-4 in glioblastoma cell invasion. Using transwell assays and a novel three-dimensional tumor spheroid assay, we found that the highly specific RSK inhibitor, BI-D1870, decreases glioblastoma cell motility and invasion. To identify which RSK isoform was associated with this decrease in invasion, we knocked down each RSK isoform using lentiviral mediated shRNA transduction and found that different RSK isoforms could decrease invasion depending on the cell line, without significantly affecting proliferation. Additionally, RSK2 knockdown in primary patient-derived glioblastoma cells decreased cell migration, and Kaplan-Meier analysis revealed that patients overexpressing RSK2 had a poorer prognosis compared to those expressing normal levels of RSK2. Our studies support a differential role for individual RSKs in glioblastoma invasion, suggesting that targeting RSKs may provide an effective means for the treatment of glioblastoma.

Citation Format: Amanda Prechtl, Florian Sulzmaier, Pengfei Jiang, Santosh Kesari, Joe Ramos. RSK isoform regulation of migration and invasion in glioblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5007. doi:10.1158/1538-7445.AM2014-5007
Abstract 5018: Combination immunotherapy with PD-L1 blockade and Poly I:C in a murine breast cancer model
Authors: Soliman Hatem , Nelson Ashley R.
Published: May 2015

Background: Programmed death ligand 1 (PDL1) is commonly expressed on the surface of many tumor cells, including breast cancer. The activity of tumor infiltrating lymphocytes (TIL) is inhibited by PDL1. Blocking PDL1 with monoclonal antibodies can prevent this immune suppression and enhance anti-tumor activity. Polyinosinic-polycytidylic acid (Poly I:C) is a Toll like receptor 3 (TLR3) agonist which was shown to synergize with anti-PDL1 antibodies in a B16 melanoma model (Celis et. al, Blood 2010). We sought to investigate if synergy could be demonstrated in a murine breast cancer model.

Methods: Five week old female BALB/c mice were injected with 3x106 4T1 cells in a mammary fat pad and divided into 4 groups with ten mice/group (PBS control, 200µg anti-PDL1 antibody (10F.9G2 Bioxcell) D1,5 IP, 50µg Poly I:C (Invivogen) D1,6,11,16 IV, anti-PDL1+Poly I:C). Treatment started when tumors were palpable and measured twice weekly. The mice were sacrificed 20 days later with tumors, spleens, and lymph nodes harvested. Tumors were measured twice weekly. All experiments were carried out under approved IACUC #4277R per institutional guidelines. Statistical analysis performed using Prism Graphpad.

Results: Mean tumor measurements (mm2) at day 20 for control=207.5, anti-PDL1=188.5, Poly I:C=182.6, anti-PDL1+Poly I:C=149.4. Two way ANOVA was statistically significant at p<.0001. Mean tumor weight (gm) at day 20 of anti-PDL1+Poly I:C=.99 vs control=1.23 p=.002 using t test.

Conclusions: The combination of anti-PDL1 and Poly I:C TLR3 agonist demonstrates improved anti-tumor activity against established 4T1 orthotopic murine breast tumors compared to control or either agent alone. Characterization of TIL infiltrates in tumor specimens and immune activation in harvested lymphocytes is ongoing and will be presented. This data provides preliminary evidence supporting continued investigation of the combination of PDL1 and Poly I:C immunotherapy for breast cancer.

Citation Format: Hatem Soliman, Ashley R. Nelson. Combination immunotherapy with PD-L1 blockade and Poly I:C in a murine breast cancer model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5018. doi:10.1158/1538-7445.AM2014-5018
Abstract 5025: Dissecting the dynamics of anti-PD1 immunotherapy in preclinical tumor models
Authors: Sriram Venkataraman , Bigler Michael E. , Cherwinski Holly , Murphy Erin , McClanahan Terrill K. , Phillips Joseph H.
Published: May 2015

MK-3475 is an antagonistic humanized IgG4 anti-PD1 antibody currently in late stage clinical development for the treatment of cancer. MK-3475 exerts its anti-tumor effect through blocking the interaction of the immune inhibitory molecule PD-1 with its ligands. MK-3475 does not bind to mouse PD-1. Therefore, we generated a surrogate antibody, mDX400 for use in preclinical rodent models. In multiple syngeneic tumor models, treatment with mDX400 murinized anti-mouse antibody results in complete and durable tumor regression. We have used mDX400 as a mouse equivalent of MK-3475 to aid in our understanding of the cellular and molecular dynamics of anti-PD1 immunotherapy.

In the current study, we examined peripheral and intratumoral lymphoid compartments pre- and post-mDX400 treatment. The data indicated that:

1. Anti-tumor efficacy was primarily driven by the level of PD-1 receptor blockade in tumor, but not blood.

2. An altered molecular signature, including significant upregulation of genes involved in monocyte, B- and T-cell activation and differentiation, was observed post-treatment in the tumors, but not peripheral blood or draining lymph nodes, of responding animals.

3. Tumor-specific immune memory was evident, as assessed by ultrahigh-throughput DNA sequencing of TCR CDR3 region, following successful treatment with mDX400.

These data provide insights into the molecular dynamics following anti-PD1 immunotherapy and will guide hypothesis generation for biomarkers to inform our clinical strategy. In addition, the cellular and molecular pathways that predominate in preclinical tumor models insensitive to anti-PD1 immunotherapy are being evaluated. These types of studies may inform us on potential therapies that can be combined with MK-3475 to expand the therapeutic benefit of this biologic.

Citation Format: Venkataraman Sriram, Michael E. Bigler, Holly Cherwinski, Erin Murphy, Terrill K. McClanahan, Joseph H. Phillips. Dissecting the dynamics of anti-PD1 immunotherapy in preclinical tumor models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5025. doi:10.1158/1538-7445.AM2014-5025
Abstract 5029: Comparative combination cancer immunotherapy with vaccination, checkpoint inhibition and TNFRSF stimulation
Authors: Schreiber Taylor H. , Schilling Neal , Deyev Vadim , Podack Eckhard R.
Published: May 2015

Therapeutic tumor immunity requires the presence and appropriate activation of tumor antigen specific CD8+ T cells and migration of activated tumor-antigen specific CD8+ T cells into a tumor microenvironment where immunosuppressive barriers have been eliminated. Antibody mediated blockade of CTLA-4 and PD-1 is a clinically effective strategy to dampen tumor mediated immunosuppression in a minority of patients with advanced cancer, and this approach may be potentiated through vaccination to prime additional CTL clones and through direct stimulation of T cell costimulatory molecules of the TNF superfamily. Here we provide a systematic comparison of anti-tumor vaccination with either heat shock protein gp96-Ig or traditional peptide/adjuvant vaccines given alone or in combination with CTLA-4 or PD-1 blockade and direct T cell costimulation via OX40, 4-1BB and TNFRSF25. Through the tracking of tumor-antigen specific CD4+ and CD8+ T cell responses, these studies demonstrate that both TNFRSF4 and TNFRSF25 independently and additively costimulate vaccine induced CD8+ T cell proliferation following both primary and secondary antigen challenge. In contrast, the activity of TNFRSF4 and TNFRSF25 were observed to be divergent in the costimulation of CD4+ T cell immunity. Interestingly, antigen-specific cellular and humoral responses were uncoupled upon secondary immunization, which was dramatically effected by the presence of OX40, 4-1BB or TNFRSF25 costimulation. When combined with checkpoint inhibition in therapeutic murine orthotopic tumor models, synergistic anti-tumor activity was observed with various triple combinations with vaccine, checkpoint inhibition and T cell costimulation. These studies highlight the complementary activity of vaccination, checkpoint inhibition and direct T cell costimulation, which may guide the application of combination immunotherapy for effective anti-tumor immunity.

Citation Format: Taylor H. Schreiber, Neal Schilling, Vadim Deyev, Eckhard R. Podack. Comparative combination cancer immunotherapy with vaccination, checkpoint inhibition and TNFRSF stimulation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5029. doi:10.1158/1538-7445.AM2014-5029
Abstract 5034: The antitumor immune response generated by radiation therapy may be limited by tumor cell adaptive resistance and can be circumvented by PD-L1 blockade
Authors: Dovedi Simon J. , Lipowska-Bhalla Graznya , Cheadle Eleanor , Poon Edmund , Morrow Michelle , Stewart Ross , Wilkinson Robert , Honeychurch Jamie , Illidge Timothy
Published: May 2015

Radiation therapy (RT) is administered to around 50% of all cancer patients making it one of the most important cancer treatments. In addition to the direct cytoreductive effect of RT there is increasing evidence that radiation-induces immunogenic tumor cell death. Despite the immunogenicity of RT-induced tumor cell death, RT delivered to tumours in the clinic rarely generates therapeutic systemic anti-cancer immune responses or ‘abscopal effects’.

Here we show that tumor infiltrating CD8+ cytotoxic T lymphocytes (CTL) have increased expression of PD-1 following RT in vivo. Moreover, our data demonstrate that treatment of established syngeneic tumors with RT leads to upregulation of tumor cell expression of PD-L1 in vivo but not when cells are irradiated in vitro. Using depleting antibodies we determined that the depletion of CD8+ T cells but not CD4+ T cells or NK cells could abrogate this RT-induced increase in tumor cell expression of PD-L1 in vivo. Furthermore, silencing of IFNγR1 using ShRNA confirmed that this process was dependent on CD8+ T cell production of IFNγ suggesting an adaptive upregulation of PD-L1 following RT occurs in response to CTL activation. This novel finding suggests that the immunogenicity of RT may be limited via the PD-L1/PD-1 signalling axis and may contribute to treatment failure.

We next sought to determine whether blockade of the PD-1/PD-L1 signalling axis could enhance the therapeutic response to RT. Our study demonstrates that administration of either an anti-PD-1 or anti-PD-L1 mAb in combination with RT leads to substantially improved survival when compared to either monotherapy alone with approximately 60% of treated mice undergoing a complete response. In addition, our data reveal that combination therapy generates long-term immunological memory in mice that have undergone complete response protecting against tumor rechallenge.

This is the first report to our knowledge that demonstrates adaptive resistance through the upregulation of tumour cell PD-L1 expression in response to an RT-induced CTL response. This study demonstrates the potential for enhancing the efficacy of conventional RT through blockade of the PD-1/PD-L1 axis and warrants clinical evaluation.

Citation Format: Simon J. Dovedi, Graznya Lipowska-Bhalla, Eleanor Cheadle, Edmund Poon, Michelle Morrow, Ross Stewart, Robert Wilkinson, Jamie Honeychurch, Timothy Illidge. The antitumor immune response generated by radiation therapy may be limited by tumor cell adaptive resistance and can be circumvented by PD-L1 blockade. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5034. doi:10.1158/1538-7445.AM2014-5034
Abstract 5036: Blockade of the CD39 immunoregulatory pathway by monoclonal antibodies
Authors: Bastid Jeremy , Dejou Cécile , Giustiniani Jérôme , Cochaud Stéphanie , Alberici Gilles , Bensussan Armand , Eliaou Jean-François , Bonnefoy Nathalie
Published: May 2015

The CD39/ CD73/ adenosine pathway is an important regulator of effector immune cell response. We previously demonstrated that, in human cancer specimen, CD39 is expressed by infiltrating regulatory T cells, tumor cells and the tumor associated stroma. CD39 enzymatic activity decreases peritumoral ATP, a potent tumor cell toxicity and immunogenic inducer, and generates immunosuppressive adenosine that binds adenosine receptors and inhibits CD4, CD8 T cell and NK cell responses. We and other demonstrated that CD39-mediated decrease of extracellular ATP and increase of adenosine promote tumor progression and immune escape as well as resistance to chemotherapy-induced immune response. We therefore generated several CD39 blocking monoclonal antibodies and present here the latest developments of these antibodies. We provide evidence that CD39 blocking antibodies restore the proliferation of CD4 and CD8 T cells inhibited by melanoma cells expressing CD39 and increase the generation of CD8 cytotoxic T cells against CD39+ melanoma cells. We also demonstrated that CD39+ lymphoma cells are less sensitive to NK cell cytotoxic activity than CD39- lymphoma cells, and that CD39-blocking monoclonal antibodies increase NK-mediated lysis of CD39+ lymphoma cells but not CD39- lymphoma cells. In conclusion, CD39 blocking antibodies may represent a novel immunotherapy strategy for inhibiting regulatory T cells and tumor cell-mediated immunosuppression. The results presented here support the ongoing development of CD39 blocking monoclonal antibodies as potential anticancer drugs to restore antitumor immune response.

Citation Format: Jeremy Bastid, Cécile Dejou, Jérôme Giustiniani, Stéphanie Cochaud, Gilles Alberici, Armand Bensussan, Jean-François Eliaou, Nathalie Bonnefoy. Blockade of the CD39 immunoregulatory pathway by monoclonal antibodies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5036. doi:10.1158/1538-7445.AM2014-5036
Abstract 5038: Telomere length linking social contexts and cancer risk in Mexican Americans
Authors: Han Lixia , Zhao Hua , Strom Sara , Daniel Carrie R. , Chang David , Zhang Hui , Ye Yuanqing , Gu Jian , Chow Wong-Ho , Wu Xifeng
Published: May 2015

Social contexts have a strong impact on chronic disease risk, including cancer, but how these influences “get under the skin” remains unclear. We hypothesize that telomere length may serve as the molecular link between social contexts and cancer risk. Using quantitative real-time PCR, we measured relative telomere length (TL) on baseline whole blood DNA samples collected from 6,574 participants of the ongoing Mano-a-Mano Mexican American Cohort study. We found that TL decreased linearly with increasing age (P-trend<0.001). In multivariable-adjusted Cox proportional-hazard regression models, longer TL was directly associated with a 2.66-fold increased risk of all-cancer incidence (95% CI= 1.40-5.03). TL was positively associated with several acculturation-related variables. For example, participants with a higher acculturative score with greater stays in the US had a longer TL among participants older than the median age of 38 (P=0.05 and 0.04, respectively). Participants born in the US and with at least a high school education were also more likely to have a longer TL. Among the modifiable risk factors, TL was inversely associated with sedentary or sitting time among women (P=0.01) and among young participants (P=0.01). In other chronic diseases, we found that longer TL was associated with diabetes and gallbladder disease in older participants (P=0.04 and 0.01, respectively). Our results suggest that TL may mediate the relationship between acculturation and related risk factors such as physical inactivity, education, and cancer risk among Mexican Americans. This is our first step in a continuum of research focused on understanding how social and behavioral disparities influence biological pathways in cancer development.

Citation Format: Lixia Han, Hua Zhao, Sara Strom, Carrie R. Daniel, David Chang, Hui Zhang, Yuanqing Ye, Jian Gu, Wong-Ho Chow, Xifeng Wu. Telomere length linking social contexts and cancer risk in Mexican Americans. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5038. doi:10.1158/1538-7445.AM2014-5038
Abstract 5042: Using a statewide collaborative approach to improve tobacco cessation referral rates for cancer patients
Authors: Severson Jane , Baca Hilary , Blayney Douglas , Brown Karen , Galiyas Grayce , Minasian Mara , Schmidt Joan , Schulze Anna , Warren Graham
Published: May 2015

Purpose:

Though tobacco use by cancer patients is associated with poor therapeutic outcomes, most oncologists do not regularly provide tobacco cessation support for cancer patients. The purpose of this study was to determine if dedicated statewide services could substantially enhance delivery of evidence based cessation support to broad cancer patient populations across many oncology clinics.

Methods:

The Michigan Oncology Quality Consortium (MOQC) collaborated with the Michigan Department of Community Health and the Michigan Cancer Consortium to identify and address tobacco cessation support that could be improved for cancer patients. A statewide Tobacco Cessation Demonstration Project was developed to reduce patient barriers to access by increasing education, identifying all cancer patients who use tobacco, referring them to the Michigan Tobacco Quitline, and providing funding for free Quitline telephone counseling services and nicotine replacement therapy for cancer patients. Reported are results in 19 clinical practices.

Results:

Implementation of this project resulted in tobacco assessments in more than 98% of patients and increased tobacco cessation referral rates for participating practices from 15% in May 2013 to over 60% in July and September of 2013 (p < .001). Total statewide referrals to the Quitline increased by between 32% and 60% over the first 6 months, with an increasing proportion of patients (between 31% and 50%) being cancer patients referred by MOQC participating sites.

FebMarAprMayJunJulAugSepOctTotal Michigan # Referrals140153153202233245238220210MOQC # Referralsn/an/an/a628812397104102MOQC Referrals % of Total Michigann/an/an/a31%38%50%41%47%49%

Conclusions:

Reducing patient barriers to access to tobacco cessation services and supplying clinic teams with scientific evidence, standard workflows, and lean tools improved the referral rate to a tobacco cessation program for oncology patients. Critical factors for success included strong physician leadership, adoption of an auto referral process, and front line staff accountability for identifying all patients who use tobacco and assessing their tobacco cessation status at subsequent clinic visits.

Citation Format: Jane Severson, Hilary Baca, Douglas Blayney, Karen Brown, Grayce Galiyas, Mara Minasian, Joan Schmidt, Anna Schulze, Graham Warren. Using a statewide collaborative approach to improve tobacco cessation referral rates for cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5042. doi:10.1158/1538-7445.AM2014-5042