bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5223: The role of bone-marrow derived cells in the intestinal tumor microenvironment of apcmin/+ mouse on tumor response to inhibitors of thymidylate synthase
Authors: Acharya Grishma , Davis Celestia , Tisdale Nikeya , Zhang Yu , Pena Maria Marjorette O
Published: August 2012

The vast majority of chemotherapeutic agents have been developed to specifically target actively dividing neoplastic cells. However, tumors are not exclusively composed of a heterogeneous population of neoplastic cells. They also include a heterogeneous collection of host derived infiltrating non-neoplastic cells that together with the extracellular matrix and secreted molecules compose the tumor stroma or microenvironment. Multiple interactions between stromal and tumor cells play a critical role in tumor growth and progression. Therefore, tumor response to chemotherapy and recurrence post-therapy must also be influenced by these interactions. For many decades, inhibitors of the enzyme thymidylate synthase (TS), such as fluoropyrimidines, have been used in the clinical management of various cancers; however, their clinical efficacy continues to be limited by their toxicity to normal cells and acquired resistance by tumor cells. Therapeutic efficacy is often measured by its impact on tumor burden, however, the impact of TS inhibitors on tumor stromal cells and their role on tumor response to therapy is largely unknown. The goal of this project is to determine the role of bone marrow derived tumor stromal cells (BMDCs) in mediating tumor response to systemic therapy with TS inhibitors. We hypothesize that, in addition to targeting tumor cells, TS inhibitors also directly target stromal cells and that the sensitivity of stromal cells to the therapy determines tumor response. Using the ApcMin/+ mouse as a model of intestinal tumorigenesis, we identified and characterized BMDCs in the intestinal tumor stroma. These included Macrophages, Fibroblasts, Pan T cells, Helper T cells, Leukocytes, Mast cells, T-regulatory cells (T-reg) and Myeloid derived suppressor cells (MDSCs), among others. We generated chimeric mice wherein the chemosensitivity of intestinal tumor cells to TS inhibitors was distinct from that of the BMDCs by transplanting eGFP positive bone marrow cells that were genetically modified to express a drug resistant variant of TS. We examined the effect of 5-FU on MDSCs, mast cells, and T-regs, that have previously been shown to promote intestinal tumor development in ApcMin/+ mice. The results showed that MDSCs were specifically sensitive to 5-FU due to its apoptotic effect on these cells. Current studies are aimed at identifying the combination of stromal cells that mediate tumor response to TS inhibitors in order to identify cellular targets for combined therapy that would enhance reduction of tumor burden or delay tumor recurrence.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5223. doi:1538-7445.AM2012-5223
Abstract 5225: Correlation between αvβ3 integrin expression, paclitaxel resistance and RGD-bearing conjugate efficacy
Authors: Eldar-Boock Anat , Sanchis Joaquin , Lupu Ruth , Vicent Maria J. , Satchi-Fainaro Ronit
Published: August 2012

Overcoming drug resistance, emerging either on recurring cancer or metastasis, is a major target of cancer treatments. The combination of anti-angiogenic therapy with cytotoxic therapy offers a promising therapeutic approach. Paclitaxel (PTX) is a widely-used potent cytotoxic drug, which also exhibits anti-angiogenic activity at low doses. The use of the hydrophobic PTX to its full potential is limited by severe side effects, caused by the drug and its solubilizing agents. Integrins play a key role in cell matrix interactions. The highly restricted expression of integrin αvβ3, overexpressed on tumor endothelial and some epithelial cells, during tumor growth, invasion, and metastasis present an interesting molecular target for metastatic breast tumors. In addition, PTX acquired resistance correlated with integrin αvβ3 overexpression on breast cancer cells. We designed and synthesized a novel polyglutamic acid (PGA)-PTX-E-[c(RGDfK)2] nano-scaled conjugate. Polymer conjugation converted PTX to a water-soluble macromolecule, which passively targeted the tumor tissue exploiting the enhanced permeability and retention (EPR) effect, while extravasating via the leaky tumor neovasculature. The E-[c(RGDfK)2] enhanced the effects previously seen for PGA-PTX alone, utilizing the additional active targeting to the αvβ3 integrin. PGA is enzymatically-degradable by cathepsin B, leading to PTX release. PGA-PTX-E-[c(RGDfK)2] displayed a potent anti-angiogenic therapy, determined by several, well-established, in vitro assays. Mice bearing orthotopic mammary tumors demonstrated preferential tumor accumulation of the RGD-bearing conjugate, leading to enhanced antitumor efficacy and a marked decreased in toxicity compared with free PTX. The correlation between αvβ3 integrin expression on tumor cells and acquired PTX-resistance was determined by in vitro manipulation of αvβ3 overexpressing-cells and by examining subsets of primary versus metastatic or recurring tumors of PTX-treated patients as well as free and conjugated-PTX-treated tumor-bearing mice. Taken together, our conjugate alters the pharmacokinetics of free PTX. Inclusion of an active targeting moiety to integrin expressing-cells, have the potential to manipulate and overcome acquired drug resistance, which will hopefully warrant it as a novel targeted, anti-angiogenic and anticancer therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5225. doi:1538-7445.AM2012-5225
Abstract 5230: Improving the specificity of drugs which target the actin cytoskeleton for cancer therapy
Authors: Sequeira Vanessa B. , Stehn Justine R. , Haass Nikolas , Bonello Teresa , Guven Kim , Desouza Melissa , Schevzov Galina , Kottyan Gregg , McCluskey Adam , Bischof Leanne , Palmer Stephen , Hardeman Edna C. , Allanson Munif , Reeve Vivienne , Weninger Wolfgang , Cripe Timothy P. , Gunning Peter
Published: August 2012

The actin cytoskeleton is fundamental in the regulation of cellular processes involved in tumourigenesis and is therefore a highly desirable chemotherapeutic target. However, the non-specific nature of actin-targeting drugs makes them ineffective due to their toxic action on the heart. We have designed a class of drugs that target the second core component of actin filaments, tropomyosin. Tm5NM1, a cytoskeletal tropomyosin isoform, is upregulated in a variety of tumour cells and patient samples, and is known to promote tumour cell growth. Our lead drug, TR100, targets Tm5NM1 and potentially other isoforms, reducing cell proliferation in vitro in a panel of melanoma and neuroblastoma cell lines and decreases the rate of tumour growth in vivo in melanoma and neuroblastoma mouse models without affecting heart integrity. The difficulty in improving the specificity of these anti-tropomyosin compounds for Tm5NM1 was the lack of a suitable cell-based system to test the drugs. NIH3T3 stable cells overexpressing fluorophore-tagged Tm5NM1 or the muscle-specific isoform αfastTm now allows us to determine the impact of our drugs on functionally distinct actin populations. Since tropomyosin is a structural protein with no known measurable catalytic activity it has previously been challenging to design a high throughput assay to quantitate specific changes in actin filament architecture. High content imaging provides a system to image actin filament integrity which can be quantified using an algorithm designed in collaboration with the CSIRO. Using this algorithm we can calculate the impact of the anti-tropomyosin compounds on actin filaments in co-cultured stable cells. Development of these assays will contribute to the design of more specific drugs which target defined tropomyosin containing actin populations highly expressed in cancer without showing toxicity in muscle, thereby improving the therapeutic index of anti-tropomyosin drugs for the clinical treatment of a wide array of cancers.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5230. doi:1538-7445.AM2012-5230
Abstract 5234: Microenvironmental signals dictate disseminated tumor cells (DTCs) fate through regulation of TGFβII and p38α
Authors: Bragado Paloma , Estrada Yeriel , Sosa Maria Soledad , Schewe Denis M. , Capobianco Carla , Moore Kateri , Farina Hernan G. , Aguirre-Ghiso Julio
Published: August 2012

Metastases develop from disseminated tumor cells (DTCs) years or even decades after primary tumor treatment. This is thought to be due to the ability of residual DTCs to remain dormant, but the mechanisms controlling this state are poorly understood. We have explored whether the target organ microenvironment where DTCs lodge can control their fate and the timing of DTCs dormancy in a HNSCC model. When injected in nude mice, HEp3-GFP cells form tumors with 100% efficiency and spontaneously disseminate to lungs (72% incidence) and bone marrow (BM) (28% incidence). Tracking of GFP-tagged DTCs showed that after ∼2 weeks the quiescent DTCs in lungs start to grow to form overt metastasis in 72% of mice. In contrast, those in the BM remain at constant numbers suggesting a dormant phenotype. Furthermore, DTCs isolated from lungs and expanded in culture (Lu-HEp3) display a high ERK/p38 signaling ratio and are tumorigenic when inoculated in vivo. Meanwhile in vitro expanded BM-HEp3-derived cell lines remain dormant when inoculated in vivo and are characterized by a low ERK/p38 activity ratio (predictive of quiescence for breast fibrosarcoma, squamous carcinoma and colon carcinoma cells) and by increased expression of the key dormancy transcription factors (TFs) Sharp1 and p53. In addition, treatment of THEp3 cells with BM conditioned media inhibits in vivo proliferation, activates TGFβ2 and p38α, and induces the expression of Sharp1 and p53. Moreover, inhibition of TGFβ signaling using a TGFβRI inhibitor, blocks p38 activation and downregulates Sharp1 and p53 in BM-HEp3 cells, suggesting that BM-HEp3 cells dormancy is functionally linked to TGFβ signaling. Importantly, TGFβ2, p38α or Sharp-1 knock down interrupted BM-HEp3 cell dormancy, while overexpression of p38α inhibits lu-HEp3 cells in vivo growth. In addition to that, systemic inhibition of p38α/β with SB203580 or TGFβRI/II with LY364947, dramatically accelerated lung metastasis as well as DTCs, micro- and macro-metastasis burden in growth restricted sites such as, spleen, liver or BM. We propose that reprogramming of DTCs into dormancy in growth restrictive microenvironments might induce a gene network activated by stress signaling mediated at least by TGFβ2 and p38α/β signaling.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5234. doi:1538-7445.AM2012-5234
Abstract 5235: Uncovering tumor-host molecular and cellular interactions involved in tumor dormancy using microRNAs
Authors: Tiram Galia , Segal Ehud , Sherberk Roni , Bachar Gal , Ofek Paula , Udagawa Taturo , Edry Liat , Shomron Noam , Satchi-Fainaro Ronit
Published: August 2012

There is an increasing interest in elucidating the mechanisms of the earlier stages in tumor progression, the point at which a dormant tumor acquires the ability to grow and metastasize. Although the tumor dormancy phenomenon has important implications for early detection and treatment of cancer, its biology and genetic characteristics are poorly understood. microRNAs (miRs), small non-coding RNA molecules which regulate gene expression at the post transcriptional level, are emerging as key regulators in physiological and pathological processes, including tumorigenesis. We hypothesized that specific miRs may also play a key role in the regulation of tumor dormancy and the angiogenic switch. We therefore established a pair of cell lines that generate dormant avascular and fast-growing angiogenic osteosarcomas in SCID mice (Saos-2-D and Saos-2-E respectively). Although these cells share similar growth rate in vitro, when inoculated into mice, Saos-2-E cells establish vascularized and palpable tumors within one month, while Saos-2-D cells remain avascular and non-palpable for a year. Following our hypothesis, miR array of Saos-2-D and Saos-2-E cells, was performed. Several miRs that were differentially-expressed in the dormant versus fast-growing-tumor-forming cells were selected for further evaluation. Out of those miRs, miR200c and miR93 (representing either a mild or a substantial difference in the expression profile respectively) reverted cells forming fast-growing tumors to a dormant non-angiogenic phenotype, both in vitro and in vivo. Preliminary in vitro analysis of human brain, breast and prostate cancer cell lines revealed a similar phenotypic effect following overexpression of those miRs. We therefore propose that miR200c and miR93 play a role in the switch from dormancy. Further analysis on the target genes of those miRs is required in order to fully elucidate their role in tumor dormancy. Uncovering new molecular targets involved in the tumor dormancy phenomenon could provide important tools for novel dormancy-directed tumor therapy strategies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5235. doi:1538-7445.AM2012-5235
Abstract 5238: Impaired angiogenesis as a hallmark of prolonged tumor dormancy in a mouse model of human glioblastoma
Authors: Ferber Shiran , Segal Ehud , Almog Nava , Satchi-Fainaro Ronit
Published: August 2012

Small sized, microscopic, non-invasive, avascular and therefore asymptomatic tumors can remain in their dormant stage for considerable period of time depending on numerous processes. One crucial mechanism underlying the transformation from a dormant phenotype to a fast-growing phenotype is the ability of the tumor cells to induce angiogenesis, a phenomenon termed as the “angiogenic switch”. Suspected dormant tumor-generating clone, derived from aggressive tumor-forming U-87 MG human glioblastoma cell line, was isolated using single-cell clone and identified by gene expression signature of dormant tumors (Almog et al., Cancer Research 2009). In order to evaluate the phenotypic differences between cell lines that generate dormant avascular tumors or fast-growing angiogenic tumors, we established a pair of mCherry-labeled human glioblastoma cell lines, U-87-D (Dormant) derived from the parental U-87-F (Fast-growing). While the two cell lines share similar growth rate in vitro, we found profound differences in tumor growth pattern when injected into mice. U-87-F established palpable and vascularized tumors only few days following inoculation, whereas U-87-D-generated tumors remained at a small size for more than 100 days. We further characterized both cell lines using migration, invasion and capillary-like tube formation assays in vitro. Major differences in invasiveness via a monolayer of human umbilical vein endothelial cells (HUVEC) were found. Furthermore, considerably increased number of tube-like structures formed from HUVEC were observed in the presence of U-87-F conditioned media (CM), compared with those formed in the presence of U-87-D CM. Similarly, HUVEC migration towards U-87-F CM was significantly higher compared with that towards U-87-D CM. Next, we utilized non-invasive intravital imaging to evaluate tumor progression, and non-invasive endo-microscopy imaging, as well as microbubbles contrast-enhanced ultrasound imaging, to track the blood flow within the tumor and blood vessels morphology at the tumor microenvironment. We concluded that the dormant and fast-growing tumors displayed distinct differences in their angiogenic potential leading to highly diverse tumor progression profiles when injected into mice.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5238. doi:1538-7445.AM2012-5238
Abstract 5241: Development of a single Listeria monocytogene-based vaccine simultaneously targeting two antigens
Authors: Wallecha Anu , Malinina Inga , Ramos Kimberly , Singh Reshma
Published: August 2012

Previously published studies reported that recombinant Listeria monocytogenes (Lm-LLO)-based vaccines expressing either tumor associated antigens (TAA) or angiogenesis associated antigens fused to an immunogenic fragment of listeriolysin O (LLO) have demonstrated efficacy in mouse models with several types of tumors such as lung, breast, prostate or melanoma. Therefore, we hypothesized that an Lm-LLO vaccine capable of delivering two disparate antigens with two different therapeutic mechanisms would likely have a synergistic effect on decreasing tumor growth by 1) targeting blood vessels, which may enhance effector T cell infiltration into the tumor and 2) improving the therapeutic efficacy of the vaccine. In addition, a dual vaccine construct creates a flexible platform for future use. The Lm-LLO-cHER2 vaccine was developed using a chimeric HER2/neu (cHER2) construct, and was found to regress tumors, elicit a strong T cell immune response and break immune tolerance towards the HER2/neu self-antigen in experimental animals. The Lm-LLO-VEGR2 vaccine has been shown to eradicate established breast tumors, reduce microvascular density, protect against tumor rechallange and metastases, and induce epitope spreading to various regions of the TAA such as HER2/neu. A dual Lm-LLO vaccine was developed that expresses and secretes both the VEGFR2 and cHER2 antigens as LLO-based proteins and is based on a highly attenuated Listeria monocytogenes backbone Lm Δ dal dat actA, which is cleared 48 hours post-injection in interferon gamma knock-out mice. Initial characterization of the dual vaccine indicates that the two antigens cHER2 and VEGFR2 are stably expressed and secreted after two in vivo mouse passages. Currently, we are evaluating the anti-tumor effects and antigen specific immune responses generated by the dual vaccine in mouse models. If successful, this dual vaccine may offer a new immunotherapy for the treatment of cancer in humans.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5241. doi:1538-7445.AM2012-5241
Abstract 5242: Pancreatic cancer cell type specific oxygen dependent growth and drug sensitivity
Authors: Gouw Arvin M. , Hsieh Annie L. , Le Anne , Maitra Anirban , Dang Chi V.
Published: August 2012

Most cell are cultured at 37°C, in 4% CO2 and 20% O2. Studies have shown that changing the temperature of the cell culture will lead to either cold shock or heat shock. Others have also shown that level of CO2 is crucial for pH balance in cell growth. Although cultured cells are exposed to 20% O2, oxygen tension in vivo is significantly lower in normal tissues ranging from 5 to 8% O2. Furthermore cancer tissues tend to be more hypoxic because of the poorly developed neo-vasculature. Hence, we sought to determine if pancreas cancer cells will grow and/or respond to drugs differently under 20% vs. 6% oxygen tension. Methods: Six pancreatic cancer cell lines were cultured in DMEM media with 10% FBS and 5% Penicillin in 4% CO2, 37°C with variable O2 conditions: 20% and 6%. Cells are plated onto 6-well plates. Triplicates of cells were trypsinized daily for 4 days for cell counts which was done by using hematocytometer. To test drug sensitivity, we used a known Lactate Dehydrogenase A (LDHA) inhibitor, FX11. LDHA inhibition has been shown to suppress tumor growth both in vitro and in vivo. Some cells were treated with an LDHA inhibitor, FX11 in varying concentrations: 5mM, 10mM and 20mM, while the controls were exposed to DMSO. Results: 1. Pancreas cancer cell lines E3, P215 and A6L grow better in 6% than in 20% oxygen level. 2. Pancreas cancer cell line A6L shows higher sensitivity to FX11 in 6% compared to 20% oxygen. 3. Pancreas cell line P215 shows lower sensitivity to FX11 in 6% compared to in 20% oxygen. Conclusion: The artificial nature of standard culture condition does not account for the significantly lower oxygen tension in vivo. Because cancer cell lines are established from tumors which tend to be hypoxic, measurements of cell growth rates and respond to therapeutic agents at 20% O2 may not reflect responses at 6% O2 and specifically not to in vivo conditions.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5242. doi:1538-7445.AM2012-5242
Abstract 5243: Reductions in hypoxia in the tumor microenvironment after Listeria monocytogenes-LLO based immunotherapy
Authors: Singh Reshma , Wallecha Anu
Published: August 2012

As a consequence of increasing growth and outpacing blood supply, tumors become hypoxic as they get larger, with the interior of the tumor becoming extremely hypoxic. This makes therapeutic immune responses difficult, as lymphocytes do not typically infiltrate the interior regions of large tumors due to the hypoxic nature of these tumors. Initial observations in preclinical studies after Listeria monocytogenes (Lm)-LLO based immunotherapy specifically targeted to the hypoxia marker CA9 show the hypoxic nature of the interior of the tumor microenvironment is altered. Initially the tumors are extremely hypoxic (as evidenced by HIF-1α and HIF-2α staining) however, after Lm-LLO-CA9 vaccination they show reduced expression of these hypoxic markers. This change in hypoxia is seen with irrelevant Lm vaccines that overexpress listeriolysin O (LLO) and also with Lm vaccines targeting CA9 fused to LLO: however the change is much more dramatic and complete in the case of the Lm-LLO-CA9 immunotherapy. The irrelevant Lm-LLO effect may be seen due to the fact that the irrelevant vaccine is actually specific for a tumor associated antigen being expressed by the tumor. This does limit the growth of the tumor and may not allow it to reach the same level of hypoxia as an untreated tumor. In current studies, an additional truly irrelevant Lm-LLO vaccine is being added to see if there is a truly Listeria-driven component to the hypoxic changes being observed. As compared to the naïve untreated tumors or an irrelevant Lm-LLO control, tumors treated with the CA9 Lm-LLO immunotherapy show an increase in lymphocyte infiltration into the interior areas of the tumor. Studies are underway to determine if this increase in infiltration has an impact on the regression of these tumors by combining the Lm-LLO-CA9 vaccine with a vaccine that is specific for a tumor associated antigen. In addition, the function of these infiltrating lymphocytes are being studied under normoxic and hypoxic conditions, to determine if the infiltrating cells are functional CTLs after the tumor's hypoxic levels change. Finally, studies are being conducted to determine the exact signaling mechanism involved in the hypoxic changes of the tumor microenvironment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5243. doi:1538-7445.AM2012-5243
Abstract 5244: The Heme binding protein HRG-1 controls pH and enhances the invasive potential of cancer cells
Authors: Fogarty Fionola , Keeffe Jean O’ , Connor Rosemary O’
Published: August 2012

The Heme regulated gene (HRG)-1 encodes a 16kD transmembrane protein that is induced by IGF-1 in transformed cells and is associated with the Vacuolar - (H+) ATPase (V-ATPase) protein complex. V-ATPases pump protons across the membrane of endosomal compartments or into the extracellular environment to regulate acidification. V-ATPase expression and activity at the plasma membrane of cancer cells has been associated with an invasive phenotype. We previously showed that HRG-1 is essential for V-ATPase activity in endosomal acidification, so therefore we hypothesised that HRG-1 may also regulate extracellular pH to enhance cancer cell invasion. To test this we investigated HRG-1 expression in phenotypically distinct cancer cell lines and investigated whether it is required for V-ATPase- mediated extracellular acidification. We found that in highly invasive and migratory cell lines HRG-1 and the a1 subunit of the V-ATPase are both expressed at the plasma membrane. In less-aggressive cancer cells and in non transformed cells HRG-1 is not expressed at the cell membrane, and is restricted to the endocytic pathway. To investigate whether HRG-1 is required for V-ATPase function at the plasma membrane we stably over-expressed HRG-1 (HA-HRG-1) and empty vector (HA-EV) in MCF-7 cells and measured proton extrusion. MCF-7/HA-HRG-1 cells exhibited a greater effect on lowering the pH of the extracellular milieu compared to empty vector control cells and also exhibited a concomitant increase in cytosolic pH. The observed extracellular pH change in MCF-7/HRG-1 cells could be suppressed by the V-ATPase inhibitor Bafilomyocin A, demonstrating that this effect is V-ATPase dependent. We also observed that the expression level and activity of matrix metalloproteinase-2, a pH dependent degrading protease whose expression can be correlated with metastatic potential, was increased in MCF-7/HA-HRG-1 cells compared with control cells. Taken together, our findings suggest that HRG-1 expression at the plasma membrane of cancer cells enhances V-ATPase activity and this facilitates cell migration and invasion. Thus HRG-1 may be a suitable cell surface target for selectively disrupting V-ATPase activity and the invasive potential of cancer cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5244. doi:1538-7445.AM2012-5244
Abstract 5248: A novel ovarian cancer imaging agent based on integrin ligation
Authors: Swenson Stephen D. , Li Zibo , Minea Radu , Markland Francis S.
Published: August 2012

Ovarian cancer diagnosis and subsequent evaluation of disease progression is difficult due to the location of tumor mass and access to the organs affected. Ultrasound and surgical exposure are required to confirm disease and response to treatment. In an effort to develop a non-invasive radiologic method for both diagnostic assessment and to follow disease progression we have produced a integrin ligand, vicrostatin (VCN) that can be radiolabeled and imaged upon binding to active integrins over-expressed on the tumor. VCN displays a high affinity for integrins which are active and pivotal in tumor growth and progression. In the present studies we utilized an animal model employing the luciferase expressing cell line OVCAR-3luc. This procedure allows us to observe both the tumor itself in a living animal, and also co-register the PET image with the optical image enabling us to detect tumors in the intraperitoneal (IP) space. For these experiments we injected OVCAR-3luc cells IP and allowed the tumors to age for 28 days. At this point, with no obvious sign of disease, we imaged the tumors using the optical imaging system to detect the presence of luciferase. Since luciferase is only found in the tumor, this enables us to specifically image the tumor. Following these images 64Cu-DOTA-VCN is injected into the tail vein of the study animals and allowed to circulate for 90 minutes. At this time the animal is placed onto the mounting tray of the PET imaging instrument. There is a specific accumulation of 64Cu-DOTA-VCN in an area in the lower abdomen that correlates with a location of luciferase activity seen in the optical image. Both of these positions were confirmed by dissection and exposure of the tumor in the sacrificed animal. Experiments in which a larger number of cells are injected IP produced unusable optical images, as the tumors grew very large and coated much of the surface in the IP space yielding bright and indistinguishable images in the mouse abdomen. One problem of note is the high level of uptake by the kidneys at the 90 minute time point. The kidneys shield the ability to view an ovarian tumor via a dorsal or ventral total slice view. When viewed transversely, there is clear separation between the kidneys and the tumor mass which binds the 64Cu-DOTA-VCN. A three dimensional reconstruction yields a three dimensional model that can be rotated and rendered to observe the position of the tumor in relationship to the kidneys in space. These studies show that VCN can be utilized to image early stage OC and could possibly be used to monitor residual disease after surgical or therapeutic intervention.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5248. doi:1538-7445.AM2012-5248
Abstract 5250: Optimization of an orthotopic mouse model for in vivo fluorescent uPA imaging in breast cancer
Authors: Ides Johan , Wouters An , Messagie Jonas , Vangestel Christel , Pauwels Bea , Joossens Jurgen , Veken Pieter Van der , Augustyns Koen , Peeters Marc , Lardon Filip
Published: August 2012

The urokinase plasminogen activator (uPA) system is one of the serine protease systems involved in extracellular matrix degradation, which will facilitate tumor cell invasion and intravasation. Recently, the Medicinal Chemistry group of the University of Antwerp (UAMC) developed some very selective covalently binding uPA inhibitors. Besides its function as possible therapeutic target, uPA and its endogenous inhibitor PAI-1 are described as prognostic biomarkers for breast cancer with the highest level of evidence. Commercial ELISA and PCR kits are available for the screening of uPA in breast tumors. These ex vivo assays measure or predict the total uPA content (active and inactive). We present here a more advanced technique, which will be able to monitor the actual enzymatic uPA activity in a non-invasive manner. The technology is based on the modification of the UAMC inhibitors towards activity based probes containing a fluorescent label. Currently, we are evaluating these probes as imaging tools for uPA monitoring. The developed imaging probes will be profoundly tested in an orthotopic model of human MDA-MB-231 and MCF7 breast cancer cells since these cell lines show a high and low uPA expression respectively. We will use these models to study the potential beneficial characteristics of activity based probes in the field of bio-imaging applications targeted to uPA as a validated biomarker. The current protocol, which is still under evaluation, can be described as followed: Cell suspensions (2.106 cells/100μl) of each cell line were implanted in the mammary fat pads at both flanks of 7 mice, alternatively with and without Matrigel. Tumor size was monitored using bioluminescent imaging (BLI) and caliper measurements. When the tumor volume reached 200 - 400mm3, the rhodamine labeled uPA-inhibitor (uPA-probe) was administered (0.3 mg / kg i.v.). Fluorescent images were taken at 1, 2, 4, 6, 8, 12, 24 and 48 hours after injection. At 48 hours, the mice were sacrificed and the tumor, liver and lung were excised for ex vivo bioluminescent or fluorescent imaging and stored for histological examination. In this presentation, we describe the evaluation of the model and the first promising results. Considering the role of uPA in migration and invasion, the uPA-probe will also be tested in the future for the detection of micrometastases.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5250. doi:1538-7445.AM2012-5250
Abstract 5252: Utilization of TumorGraft technology for personalized breast cancer treatment
Authors: James Meredith , Morris Ronnie , Bruckheimer Elizabeth , Rowinsky Eric , Bacus Sarah , Sidransky David , Paz Keren
Published: August 2012

Breast cancer is the most common cancer among women with over 280,000 new annual cases and 40,000 deaths in the US alone. While hormonal-dependent breast cancer is correlated with good prognosis, an adequate solution for hormonal-independent tumors remains an unmet medical need, largely due to a significant heterogeneity within tumors and their makeover. Various personalized oncology approaches aim at tailoring the optimal regimen for these patients by better understanding their tumor distinctiveness. Champions Oncology focuses on personalizing cancer treatment through the generation of TumorGraft™ models form the patient's own tumor. The current study is focused on the attempt to identify a personalized solution for a patient with HER2-amplified metastatic breast cancer whose disease progressed on Trastuzimab (Herceptin). Using Champions novel and cutting-edge TumorGraft™ technology, the challenge was confronted empirically by preforming a personalized clinical trial in vivo. Tumor fragments, obtained from both the primary breast tumor and lung metastasis, were implanted in immune-deficient mice in a manner that preserves the biological properties of the original tumor and its supporting stroma. Once the TumorGraft™ models were established, panel of drugs and drug combinations were evaluated to help identify treatment regimens that are likely to be most effective for the patient clinically. The drug study encompassed 7 different treatment groups including FDA-approved, as well as investigational agents. Of the 7 treatment arms, 3 demonstrated a significant anti-tumor response with tumor growth inhibition values of 80, 93 and 97% for Lapatinib+Capecitabine, Gemcitabine+Carboplatin and Ixabepilone+Capecitabine, respectively. Molecular analysis of the tumor revealed a truncated HER2 (p95) and a robust HER2 translocation to the nucleus. Additional molecular characterization is ongoing to determine other potential signatures of response. Results from the TumorGraft™ study were recommended to the patient and her medical oncologist and new lines of treatment were commenced subsequently. Herein, we describe the data from the TumorGraft™ drug study, the molecular exploration and the pathway analysis of the primary and the metastatic tumor and the consequences correlation to the clinical outcome.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5252. doi:1538-7445.AM2012-5252
Abstract 5257: Manipulating the Noxa/Bim axis undermines the multicellular resistance of 3D tumor spheroids
Authors: Barbone Dario , Cheung Priscilla , Yang Tsung-Ming , Jablons David M. , Sugarbaker David J. , Bueno Raphael , Fennell Dean A. , Letai Anthony G. , Broaddus Courtney V.
Published: August 2012

When grown in 3D cultures as spheroids, cancer cells acquire a multicellular resistance to apoptosis that resembles that of solid tumors. We have previously found in lung cancer and mesothelioma that multicellular resistance lies at the level of mitochondria and can be reduced by manipulating the Bcl-2 repertoire with BH3-mimetics. One mechanism for the multicellular resistance to apoptosis is the lack of up-regulation of the pro-apoptotic sensitizer Noxa upon treatment; Noxa displaces pro-apoptotic Bim from its anti-apoptotic buffering proteins allowing the Bim to induce apoptosis. Interestingly, we have found that, at the same time that cells acquire apoptotic resistance in 3D, they also acquire a pro-apoptotic potential that has been termed “apoptotic priming,” due to an elevated Bim that is sequestered by the anti-apoptotic Bcl-2 proteins. We found that multicellular resistance could be abolished either by restoring Noxa levels using a permeable Noxa peptide or by releasing Bim via inhibition of the anti-apoptotic Bcl-2 proteins using the small molecule inhibitor, ABT-737. Moreover, multicellular resistance was also abolished by the histone deacetylase inhibitor SAHA which upregulated both Noxa and Bim expression. Knockdown of Bim blocked the effect of the Noxa peptide, ABT-737 or SAHA showing the importance of Bim in the chemotherapeutic response. Our results using a clinically-relevant 3D model show that manipulation of the core apoptotic repertoire in order to release Bim may improve chemosensitivity of lung cancer and mesothelioma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5257. doi:1538-7445.AM2012-5257
Abstract 5322: SOD2 enhances cell migration and invasion of tongue squamous cell carcinoma via H2O2-dependent Snail signaling
Authors: Wang Anxun , Liu Zhonghua , Li Su , Cai Yuchen , Zheng Chaoxu , He Qianting , Zhou Xiaofeng
Published: August 2012

In previous studies, we demonstrated that dysregulation of manganese superoxide dismutase (SOD2) is a frequent event in tongue squamous cell carcinoma (TSCC) and is associated with lymph node metastasis. Here, we further investigated the mechanism of SOD2-mediated cell migration and invasion in paired TSCC cell lines with different metastatic potential. The UM1 and UM2 are paired cell lines from a single TSCC patient, where UM1 is more aggressive than UM2 in term of cell migration and invasion. Compared to UM2 cells, UM1 cells exhibited significantly higher SOD2 activity, increased intracellular H2O2 and higher levels of Snail, MMP-1 and pERK1/2 protein, and lower level of E-cadtherin protein. Upon knockdown of SOD2 by RNA interference, UM1 cells displayed significantly reduced cell migration and invasion abilities. This is accompanied by reductions in SOD2 activities, intracellular H2O2, levels of Snail, MMP-1 and pERK1/2 proteins, as well as increase in the level of E-cadtherin protein. Cell migration and invasion of the UM2 and the SOD2 shRNA-treated UM1 cells were enhanced by H2O2 treatment. This H2O2-mediated cell migration and invasion is accompanied by increased levels of Snail, MMP-1 and pERK1/2 protein, and decreased level of E-cadtherin protein. Thus, we conclude that the SOD2-dependent production of H2O2 contributes to both cell migration and invasion of TSCC via the Snail signaling pathway through increased Snail, MMP-1 and pERK1/2 expression, and the repression of the E-cadtherin.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5322. doi:1538-7445.AM2012-5322
Abstract 5324: H3K9 histone methyltransferase G9a promotes lung cancer invasion and metastasis by silencing the cell adhesion molecule Ep-CAM
Authors: Chen Min Wei , Hua Kuo-Tai , Kao Hsin-Jung , Chi Chia-Chun , Wei Lin-Hung , Hsiao Michael , Kuo Min Liang
Published: August 2012

G9a is a mammalian histone methyltransferase that contributes to the epigenetic silencing of tumor suppressor genes. Emerging evidence suggests that G9a is required to maintain the malignant phenotype, but the role of G9a function in mediating tumor metastasis has not been explored. Here, we show that G9a is expressed in aggressive lung cancer cells, and its elevated expression correlates with poor prognosis. RNAi-mediated knockdown of G9a in highly invasive lung cancer cells inhibited cell migration and invasion in vitro and metastasis in vivo. Conversely, ectopic G9a expression in weakly invasive lung cancer cells increased motility and metastasis. Mechanistic investigations suggested that repression of the cell adhesion molecule Ep-CAM mediated the effects of G9a. First, RNAi-mediated knockdown of Ep-CAM partially relieved metastasis suppression imposed by G9a suppression. Second, an inverse correlation between G9a and Ep-CAM expression existed in primary lung cancer. Third, Ep-CAM repression was associated with promoter methylation and an enrichment for dimethylated histone H3K9. G9a knockdown reduced the levels of H3K9 dimethylation and decreased the recruitment of the transcriptional cofactors HP1, DNMT1, and HDAC1 to the Ep-CAM promoter. Our findings establish a functional contribution of G9a overexpression with concomitant dysregulation of epigenetic pathways in lung cancer progression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5324. doi:1538-7445.AM2012-5324
Abstract 5326: ID3 contributes to cerebrospinal fluid seeding and poor prognosis of medulloblastoma
Authors: Phi Ji Hoon , Choi Seung Ah , Lim Sang-Hee , Wang Kyu-Chang , Cho Byung-Kyu , Park Sung-Hye , Park Chul-Kee , Lee Ji Yeoun , Kim Seung-Ki
Published: August 2012

Seeding of medulloblastoma is the most influential prognostic factor for survival, but little has been known about the molecular mechanism of seeding in medulloblastoma. Inhibitor of differentiation (ID) genes are implicated as promoter of tumor progression and metastasis in many human cancers. We investigated the expression and functional roles of ID genes in terms of seeding and prognosis of medulloblastoma. Significantly higher ID3 expression was observed in medulloblastoma with seeding than in tumors without seeding. In vitro studies using D283 medulloblastoma cells and ID3-siRNA showed that ID3 knockdown resulted in decreased proliferation, enhanced apoptosis, and suppressed migration. We made a seeding model of medulloblastoma in mice and injected a stable cell line with ID3 knockdown with shRNA. ID3 knockdown in vivo inhibited growth of primary tumor and development of leptomeningeal seeding and prolonged survival of animals compared with controls. A mRNA mini-array revealed that ID3 knockdown leads to up-regulation (TIMP3, ITGB4, COL12A1, ADAMTS8) and down-regulation (TNC, CTGF, ICAM1) of several genes related to cellular invasion and migration. High ID3 expression was associated with poor survival of patients with medulloblastoma as with seeding at presentation. These findings indicated that ID3 plays an important role in seeding of medulloblastoma and may be a target of therapeutic intervention for enhancing survival of the patients in the poor-prognostic group.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5326. doi:1538-7445.AM2012-5326
Abstract 5331: The effect of heat shock protein 27 (HSP27) on prostate cancer cell adhesion, invasion and metastasis
Authors: Voll Eric , Ogden Irene , Huang XiaoKe , Bergan Raymond
Published: August 2012

Prostate cancer (PCa) is the most commonly diagnosed form of cancer among American men, and the second leading cause of all cancer-related deaths. PCa death is caused by the process of metastasis. In order to metastasize, tumor cells must acquire a series of phenotypic alterations, collectively known as the metastatic cascade. We have demonstrated that the small heat-shock protein, HSP27, affects key early steps of the metastatic cascade, and therefore may be a regulator of human PCa metastasis. HSP27 protein expression increases during PCa progression. It is phosphorylated in response to signaling from transforming growth factor-β (TGF-β), which in turn increases matrix metalloproteinase 2 (MMP-2) and cell invasion in vitro. We now demonstrate that HSP27 does not affect human PCa cell adhesion, cell detachment or cell migration, all of which contribute to the composite function of cell invasion. We went on to show that proteolysis by MMPs is necessary for HSP27-driven cell invasion by demonstrating that it was abrogated Marimastat, a broad-spectrum MMP inhibitor. By orthotopically implanting HSP27 variant human PCa cell lines, we demonstrated that HSP27 increases metastasis in a murine model. We also found that HSP27 increased tumor size. HSP27 increases human PCa metastasis and tumor growth in vivo. These findings provide a mechanistic explanation for the poor prognosis associated with increased HSP27 expression in human prostate tissue.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5331. doi:1538-7445.AM2012-5331