bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 129: Synthetic lethality induced by loss of PKC delta and mutated Ras
Authors: Chen Changyan , Zhu Tongbo , Chen Lihua , Tsuji Takanori
Published: February 2011

Table of ContentsSynthetic Lethality Induced by Loss of PKC d and Mutated RasAbstract

Synthetic Lethality Induced by Loss of PKC δ and Mutated Ras

Tongbo Zhu,* Lihua Chen,* Wei Du, Takanori Tsuji, and Changyan Chen

Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston

Abstract

Synthetic lethal interaction between oncogenic Ha-ras and loss of PKC has been demonstrated. Recently, we reported that the concurrent knockdown of PKC α and β, via upregulating PKC δ, sensitizes cells with aberrant Ras signaling to apoptosis. As the continuation of the study, using the shRNA, we demonstrate that loss of PKC δ causes a lethal reaction in NIH3T3/Hras or prostate cancer DU145 cells that overexpress JNK. In this apoptotic process, PKC α and β are upregulated, and then associated with RACK1 (an adaptor for activated PKC) and JNK. Immunoblotting analysis shows that JNK is phosphorylated, accompanied with caspase 8 cleavage. The inhibition of JNK abrogates this apoptotic process triggered by PKC δ knockdown. Interestingly, without blocking PKC δ, the concurrent overexpression of wt- or CAT-PKCα and β is insufficient to induce apoptosis in the cells. Together with our previous findings, the data suggest that PKC α/β and δ function oppositely to maintain a balance that supports cells expressing v-ras to survive and prevents them from being eliminated through oncogenic stress-induced apoptosis.

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

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 129.
Abstract 1290: O6-methylguanine-methyltransferase modulates glioblastoma angiogenesis and invasion
Authors: Chahal Manik , Sabri Siham , Xu Yaoxian , Famulski Konrad , Murray David , Abdulkarim Bassam
Published: February 2011

Background: Glioblastoma (GBM) is the most aggressive primary malignant brain tumor in adults. Approximately 50% of GBM patients are resistant to standard therapy with temozolomide (TMZ) because their tumors express the DNA repair protein O6-methylguanine methyltransferase (MGMT), which repairs tumoral DNA damage induced by TMZ. GBM, a highly vascularized tumor, consistently displays a locally invasive phenotype, which contributes to biological aggressiveness and clinical recurrence. Therefore, use of the anti-angiogenic and anti-invasive agent sunitinib malate could be a promising strategy for GBM treatment. We showed that sunitinib-based treatment significantly improved the response of MGMT-positive, but not MGMT-negative, cells and this differential effect was reversed upon MGMT depletion with O6-benzylguanine. In the present study, we investigated the relationship between the expression of MGMT and the angiogenic and invasive profile of GBM cell lines.

Experimental Design: We performed microarray gene expression analysis and quantitative real-time RT-PCR and analyzed the differential expression of transcripts in GBM cell lines with differential expression levels of MGMT: U87 empty vector (MGMT-negative), its counterpart U87/MGMT (MGMT-positive) (stable transfection), and T98G (constitutively expressing MGMT). We also investigated the effect of MGMT expression on GBM angiogenesis by assessing vascular endothelial growth factor (VEGF) secretion and the ability of growth factors secreted in the GBM conditioned media to induce endothelial tube formation in vitro, and the effect of MGMT expression on GBM invasiveness by examining F-actin organization, and migration and invasion in vitro.

Results: The gene chip microarray showed differential mRNA expression of transcripts involved in angiogenesis, invasion, and response to sunitinib in U87/MGMT cells compared to U87/EV cells. These results were confirmed for several transcripts by quantitative real-time RT-PCR or western blotting. Additionally, we also showed that MGMT-positive cells have reduced angiogenic and invasive potential compared to MGMT-negative cells based on decreased VEGF bioavailability, decreased endothelial tube formation, altered morphology, and decreased invasion and migration in vitro.

Conclusion: Our study is the first to show a direct link between MGMT expression and decreased angiogenicity and invasiveness of GBM cells. This altered phenotype may account for the preferential in vitro efficacy of sunitinib malate in MGMT-positive GBM cell lines. Our data provide the rationale to further investigate the role of MGMT in the regulation of the angiogenic and invasive profile of GBM.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1290.
Abstract 1291: The neurotransmitter dopamine normalizes tumor neo-vessels by modulating G protein coupled receptor signaling in the cells of tumor microenvironment
Authors: Chakroborty Debanjan , Sarkar Chandrani , Yu Hongmei , Bakshi Shubham , Dasgupta Partha Sarathi , Basu Sujit
Published: February 2011

Tumor neo-vessels, unlike the normal are disorganized, tortuous and leaky. Such dysfunctional blood vessels within the tumors pose significant obstacle for the proper delivery of anticancer drugs into the tumor tissues and response to radiation. Thus normalization of these tumor neo-vessels by different approaches has shown to improve the therapeutic efficacies of conventional anti-neoplastic drugs and radiation therapy. We here show for the first time that the neurotransmitter dopamine normalizes tumor blood vessels in mice bearing orthotopic human prostate cancer by manipulating the cellular components of the tumor microenvironment through G protein coupled receptor signaling. This result is significant because dopamine or dopamine like drugs can be utilized synergistically with either anti-cancer drugs or radiation for the treatment of cancer patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1291.
Abstract 1294: Inhibition of angiogenesis in lymph node metastasis
Authors: Aki Ryoichi , Amoh Yasuyuki , Kishimoto Hiroyuki , Kimura Hiroaki , Suetsugu Atsushi , Momiyama Masashi , Katsuoka Kensei , Hoffman Robert M.
Published: February 2011

Cancer invasion of the lymphatic system and spread to draining lymph nodes is a common occurrence and is often a first step of the metastatic pathway. Suppression of tumor angiogenesis in the lymph node should inhibit cancer growth and further spread from the lymph node. In the present study, we evaluated angiogenesis inhibitors in lymph node metastasis using GFP imaging. Transgenic nude mice expressing GFP under the control of the nestin promoter (ND-GFP mice) were used as hosts. Nascent blood vessels express GFP in these mice (Cancer Res. 65, 5352-5357, 2005). B16F10-RFP mouse melanoma cells were injected into the efferent lymph vessel of the inguinal lymph node of the transgenic ND-GFP nude mice to establish experimental metastasis in the axillary lymph node. The mice were given daily i.p. injections of doxorubicin or vehicle controls at days 0, 1, and 2 after implantation of tumor cells. The axillary lymph nodes of the mice were observed at day 8 after injection of tumor cells. Angiogenesis was quantified in the tumor-involved lymph node by measuring the total length of ND-GFP nascent blood vessels. The number of ND-GFP-expressing blood vessels in the tumor-involved lymph nodes was significantly less in the doxorubicin-treated animals than in vehicle control mice. Treatment with doxorubicin significantly decreased the volume of the metastatic axillary-node tumor as well as nascent blood vessel formation. These results show the utility of the dual-color ND-GFP-mouse and RFP-cancer-cell model to visualize and quantitate angiogenesis in tumor-involved lymph nodes and to screen for angiogenesis inhibitors at this site.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1294.
Abstract 1297: IL-1a promotes neovascularization in prostate cancer via activated macrophages by BMP-6
Authors: Lee Geun Taek , Kwon Seok Joo , Lee Jae-ho , Jung Yeon Suk , Kim Isaac Yi
Published: February 2011

Prostate cancer is the most commonly diagnosed non-cutaneous cancer in men in the US. Active surveillance, radiation, and surgery are the recommended treatments for localized prostate cancer. However, approximately 35% of patients eventually develop biochemical recurrence within 5 years after the initial treatment. In the present study, we have investigated the significance of prostate cancer derived BMP-6 in the context of macrophages. BMP-6 activated macrophages and dramatically increased the production of interleukin-1α (IL-1α) in vitro. Mechanistically, optimal ligand/receptor/Smad combination for IL-1α induction was BMPRII (BMP type II receptor), ALK3 (BMP type I receptor), and Smad 1(R-Smad)/4(Co-Smad). We used a serial promoter deletion assay and found that −1,856 −1,655 bp region of IL-1α promoter contained the BMP-6 responsive element. Surprisingly, this region did not have the consensus Smad binding element. Additional analysis of this 200 bp region demonstrated multiple NF-κB binding sites. When NF-κB was inhibited, IL-1α induction was completely blocked. Moreover, BMP-6 increased NF-κB reporter vector activity and phosphorylated p50 and p65, suggesting NF-κB's role in induction of IL-1α [[Unsupported Character - ]]by BMP-6. Immunoprecipitation (IP) and chromatin immunoprecipitation (ChIP) showed that Smad1 binds to the 200 bp region and interacts with NF-κB directly. Finally, VEGF expression increased in TRAMP-C2 prostate cancer cells co-cultured with macrophages. Based on these results, we propose that tumor-derived BMP-6 induces IL-1α expression in macrophages and that this induction requires Smad and NF-κB pathways; IL-1α, in turn, induces angiogenesis in prostate cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1297.
Abstract 13: Regulation of breast cancer stem cells by miR-93
Authors: Liu Suling , Ginestier Christophe , Ibarra Ingrid , Patel Shivani H. , Heath Amber , Ou Sing J. , Jagirdar Raj , Clouthier Shawn G. , Korkaya Hasan , Charafe-Jauffret Emmanuelle , Hannon Gregory J. , Birnbaum Daniel , Wicha Max S.
Published: February 2011

There is increasing evidence that the growth and metastasis of many tumors, including breast cancer, are driven by a cellular population displaying stem cell properties. Like their normal counterparts, these breast cancer stem cells may be regulated by MicroRNAs (miRNAs). We have previously demonstrated that breast cancer cell lines contain subpopulations with stem cell properties that can be isolated by virtue of their expression of Aldehyde dehydrogenase (ALDH) as assessed by the Aldefluor assay. We compared miRNA expression in Aldefluor-positive and Aldefluor-negative populations in a series of five breast cancer cell lines. We identified specific miRNA expression profiles for each population. Among the differentially expressed miRNAs was miR-93 whose expression was significantly increased in Aldefluor-negative compared to Aldefluor-positive populations. To confirm the regulation of miR-93 during cell differentiation we constructed a miR-93 sensor tagged with GFP and demonstrated that sensor-positive (miR-93-negative) cells had significantly increased tumor initiating capacity in NOD/SCID mouse xenografts compared to sensor-negative (miR-93-positive cells). Furthermore, miR-93-negative cells gave rise to tumors containing both miR-93-negative and miR-93-positive cell populations. Utilizing a tetracycline inducible lentivirus driving miR-93 expression, we found that induction of miR-93 expression decreased the ALDH-positive population in vitro as well as in mouse xenografts where this reduction was associated with decreased tumor growth. Furthermore, induction of miR-93 expression immediately upon orthotopic implantation or intracardiac injection completely blocked subsequent tumor growth and metastasis formation. These studies demonstrate that miR-93 plays a functional role in the self-renewal and differentiation of breast cancer stem cells. Furthermore, the TET-inducible miR-93 system allows for the controlled regulation of cancer stem cell function providing a valuable model to simulate the effects of CSC-directed therapies on breast cancer growth and metastasis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 13.
Abstract 1300: Vascular endothelial growth inhibitor (VEGI; TNFSF15) inhibits bone marrow-derived endothelial progenitor cell incorporation into Lewis Lung cancer (LLC) tumors
Authors: Liang Paulina H. , Tian Fang , Lu Yi , Stolz Donna B. , Li Lu-Yuan
Published: February 2011

Bone marrow-derived endothelial progenitor cells (EPC) have been shown to be a critical component of supporting tumor neovascularization. We have shown in a variety of cancer and angiogenesis models that recombinant vascular endothelial growth inhibitor (VEGI; TNFSF15) inhibits angiogenesis and tumor growth via specific elimination of mature endothelial cells (EC) in established tumor vasculature. We report here that VEGI treatment leads to a significantly decreased population of EPC in tumors. We transplanted whole bone marrow from green fluorescent protein (GFP) transgenic mice into C57BL/6 recipient mice. The mice were then inoculated with Lewis Lung cancer (LLC) cells and treated with VEGI or vehicle. In the tumors of the VEGI-treated group, we saw both a significant inhibition of tumor growth and decreased density of CD31+ vasculature compared to vehicle treated mice. Flow cytometric analysis of the peripheral blood of tumor bearing mice revealed an initial increase of EPCs derived from the bone marrow (GFP+Flk1+) prior to the timeframe when EPCs are incorporated into the tumor. After this timeframe, the EPC in peripheral blood decreased markedly. Monitoring for EPC by GFP+CD31+ immunostaining, we found that the number of EPC in VEGI treated tumors notably decreased compared to that of the untreated group. EPC incorporation into the tumor vasculature of VEGI-treated mice was also less than that in the untreated group. TUNEL staining of the tumor sections showed a large extent of apoptosis overall in VEGI-treated tumors. These findings indicate that VEGI plays an important role in modulating bone marrow-derived EPC incorporation into LLC carcinomas, resulting in the inhibition of EPC supported tumor vasculogenesis and tumor growth.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1300.
Abstract 1301: Involvement of VEGFR-1 signal in solid tumor growth and tumor angiogenesis via recruitment of macrophage-lineage cells
Authors: Muramatsu Masashi , Yamamoto Seiji , Osawa Tsuyoshi , Shibuya Masabumi
Published: February 2011

VEGF/VEGFR signaling is essential for pathological angiogenesis. It has been reported that Flt-1 (VEGFR-1) is expressed not only in vascular endothelial cells but also in monocyte/macrophages. Previously, we and others reported that bone marrow derived cells which expressed Flt-1 were involved in the formation of pre-metastatic niche in mouse tumor model. However, the role of Flt-1 signal in tumorigenesis is not fully understood.

To clarify the involvement of Flt-1 in solid tumor growth, we used wild type (Wt) mice and Flt-1 tyrosine kinase-deficient (Flt-1 TK-/-) mice which developed basically normal blood vessels and survived. In allograft cancer model, tumor growth was significantly slower in Flt-1 TK-/- mice compared with that in Wt mice. Survival rate of Flt-1 TK-/- mice were also higher than Wt mice under tumor-bearing condition. The degree of tumor angiogenesis and infiltration of macrophage-lineage cells in tumor tissues were remarkably suppressed in Flt-1 TK-/- mice compared with those in Wt mice. Surprisingly, endothelial cells and macrophage-lineage cells at the surrounding tissue of tumors were similar in both mice. The number of pericyte marker-positive cells covered with vascular endothelial cells was reduced in tumor tissue in Flt-1 TK-/- mice compared with that in Wt mice.

To distinguish which Flt-1 signal on the vascular endothelial cell or on the macrophage is important, we performed bone marrow transplantation. Tumor volume in Wt mice implanted with bone marrow derived from Flt-1 TK-/- mice was significantly smaller than that in Wt mice implanted with bone marrow derived from Wt mice. In tumor tissue inoculated in Wt mice implanted with Flt-1 TK-/- bone marrow, angiogenesis and infiltration of macrophage-lineage cells were significantly decreased compared with Wt mice implanted with Wt bone marrow. Based on these results, we suggest that, in some tumors, Flt-1 signaling is important for bone marrow-derived macrophage-migration into tumors which promotes tumor angiogenesis and tumor growth.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1301.
Abstract 1303: Inhibition of human breast cancer cell (MDA-MB231) migration, neoangiogensis and tumor growth by selective disruption of annexin II function: in vitro and in vivo studies
Authors: Sharma Mahesh C. , Ownbey Robert T. , Blackman Marc R. , Sharma Meena
Published: February 2011

Annexin II, an abundant phospholipid binding cell surface protein, binds tissue plasminogen activator (tPA) and functions as a regulator of fibrinolysis. Annexin II also mediates angiogenesis and enhances tumor growth and metastasis. However, the mechanism supporting this role is not known. Using a human breast cancer model we found that invasive human breast cancer cells (MDA-MB231) synthesize annexin II and tPA. Annexin II interacted with tPA in vitro leading to conversion of zymogen plasminogen to the reactive enzyme plasmin. Cell surface generated plasmin inhibited the migration of MDA-MB231 cells. Silencing of the annexin II gene in mDA-MB231 cells abolished tPA binding thereby inhibiting tPA dependent plasmin generation. Moreover these annexin II suppressed MDA-MB231 cells exhibited reduced motility. Immunohistochemical analysis of prediagnosed clinical specimens showed abundant secretion of tPA and expression of annexin II on the surface of invasive cancer cells which correlates with neovascularization of the tumor. Intravenous administration of the anti-annexin II antibody to mice bearing MDA-MB231 tumors significantly inhibited tumor growth possibly due to blocked neoangiogenic activity. Taken together, these data suggest that annexin II dependent plasmin generation may be an early event switching breast cancer from the prevascular phase to the vascular phase with the possibility of metastasis. Annexin II may be an attractive target for therapeutic strategies aimed to inhibit neoangiogenesis and breast cancer metastasis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1303.
Abstract 1304: Induction of pericyte migration by autotaxin in angiogenesis
Authors: Lin Chi-yen , Leinwand Joshua , Maier Cheryl , Pober Jordan , Chang William , Braddock Demetrios
Published: February 2011

Angiogenesis is essential for tumor cell growth and progression. Recent studies have shown that autotaxin (ATX) is a critical angiogenic factor since ATX knock-out mice exhibit severe vascular defects. ATX also has the ability to stimulate tumor cell motility by enzymatically generating lysophosphatidic acid (LPA). To form a new and stable blood vessel, interactions between pericytes and endothelial cells play important roles. A previous study has shown that endothelial cells can be induced to generate ATX; however little is known about how ATX and LPA are involved in these interactions. The focus of our study is to define the role of ATX in angiogenesis by examining pericyte and endothelial cell response to ATX.

To test the effects on cell migration, we treated pericytes and endothelial cells with ATX and other angiogenic factors (including Vascular Endothelial Growth Factor (VEGF) and Platelet Derived Growth Factor (PDGF)). Pericytes responded strongly to ATX and LPA stimulation, but human umbilical vein endothelial cells (HUVEC) and skin microvescular cells (HDMEC) showed little or no effect. PDGF elicits a qualitatively similar response. In contrast, endothelial cells migrated when treated with VEGF, but pericytes showed no such stimulation. Pericytes treated with ATX and PDGF together show an additive response. Moreover, in a checkerboard assay the migratory response to ATX or LPA is chemokinetic, whereas PDGF is chemotactic.

We then utilized RT-PCR to examine the mRNA levels of four major LPA receptors in pericytes when treated with angiogenic factors. Treatment with PDGF increases LPAR1 mRNA levels and decreases LPAR4 levels. Treatment with ATX also increases LPAR1, but shows no change in LPAR4 levels. By contrast, VEGF treatment has no effect on the LPAR1 mRNA level, but increases the LPAR4 mRNA level. The amount of migration appears to correlate with an increase of LPAR1 and perhaps a decrease of LPAR4. As such, certain angiogenic factors might affect cell motility, in part, through the differential expression of various LPA receptors.

These studies suggest that the ATX/LPA signaling pathway effects the formation of mature blood vessels by promoting the pericyte coverage of immature blood vessels. The role of the ATX/LPA pathway in the formation of mature blood vessels provides the scientific rationale for targeting the pathway in the development of novel anti-angiogenic compounds.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1304.
Abstract 1305: The proangiogenic role of brain-derived neurotrophic factor in tumor development
Authors: Lam CT , Yang ZF , Fan ST , Poon RTP
Published: February 2011

Background and Aim: Hepatocellular carcinoma is a hypervascularized solid tumor which requires angiogenesis for its growth. Previous studies suggested that neurotrophins, such as brain-derived neurotrophic factor (BDNF), may serve as angiogenic factors. The involvement of BDNF in tumor angiogenesis, however, remains unclear. The present study aimed at elucidating the role of BDNF in regulating angiogenesis and tumor development. Materials and Methods: BDNF was overexpressed in a normal mouse endothelial cell line by transfection. The angiogenic properties, including proliferation, cell motility, invasiveness and cell survival, of the transfectants were subsequently assessed by MTT, cell migration, invasion and Annexin V labeling assays, respectively. Microarray analysis was performed to explore the BDNF-mediated angiogenic pathway. An in vivo cell co-injection model, using a mouse transformed hepatocyte cell line and BDNF transfectants, was used to study the role of BDNF in tumor development. Results: Firstly, overexpression of BDNF could promote endothelial cell proliferation, migration, invasion and survival. Secondly, the gene expression profiling data suggested that the BDNF-induced angiogenic effects could be attributed to a dozen of genes including transcription factors, cell adhesion molecules, chemokines and growth factors. Thirdly, the in vivo cell co-injection experiment showed that high BDNF-expressing endothelial cells are able to promote growth of tumors with significantly higher number of microvessels. On the other hand, knock-down of BDNF in a tumor-derived endothelial cell line by shRNAs impairs such tumor promoting effect. Conclusion: The present study showed that BDNF is crucial for tumor angiogenesis and may serve as a potential target for anti-angiogenic treatment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1305.
Abstract 1308: Cytogenetic abnormalities of tumor endothelial cells in human malignant tumors
Authors: Hida Kyoko , Akino Tomoshige , Hida Yasuhiro , Tsuchiya Kunihiko , Freedman Deborah , Muraki Chikara , Ohga Noritaka , Matsuda Kouhei , Akiyama Kosuke , Kurosu Takuro , Kondo Miyako , Maishi Nako , Harabayashi Toru , Shinohara Nobuo , Nonomura Katsuya , Klagsbrun Michael , Shindoh Masanobu
Published: February 2011

An important concept in tumor angiogenesis has been that tumor blood vessels contain genetically normal and stable endothelial cells (ECs), unlike tumor cells, which typically display genetic instability. Chromosomal aberration in human tumor endothelial cells (hTECs) in carcinoma has not yet been investigated. Here we isolated TECs from 20 human renal cell carcinomas (RCCs) and analyzed their cytogenetic abnormalities. The degree of aneuploidy was analyzed by fluorescence in situ hybridization using chromosome 7 and chromosome 8 DNA probes in isolated hTECs. In human RCCs, 22-58% (median 33%) of uncultured hTECs were aneuploid, whereas normal ECs were diploid. The mechanisms of TEC aneuploidy were studied using mouse TECs (mTECs) isolated from xenografts of human epithelial tumors. To investigate the contribution of progenitor cells to aneuploidy in mTECs, CD133 positive and CD133 negative mTECs were compared for aneuploidy. CD133 positive mTECs showed aneuploidy more frequently than CD133 negative mTECs. This is the first report showing cytogenetic abnormality of hTECs in carcinoma, contrary to traditional belief. It is suggested that cytogenetic alterations in tumor vessels of carcinoma can occur and may play a signicant role in modifying tumor-stromal interactions.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1308.
Abstract 1319: Proton irradiation exerts antiangiogenic and other tumor progression-limiting effects
Authors: Girdhani Swati , Lamont Clare , Hahnfeldt Philip , Weremowicz Janusz , Anaya Zachary , Beheshti Afshin , Abdollahi Amir , Hlatky Lynn
Published: February 2011

The precise targeting and energy deposition characteristics of proton theapy have attracted considerable attention in the clinical oncology community. An increasing number of proton facilities are being established to exploit the physical advantages of this radiation for cancer treatment. However, the fact that there may also be biologically-based advantages for the use of protons has essentially been overlooked. We here demonstrate that proton irradiation inhibits expression of major pro-angiogenic factors and multiple angiogenesis-associated processes, including invasion and endothelial cell proliferation, which are critical to cancer progression. Dose-dependent suppression of angiogenic signaling was demonstrated for human tumor, fibroblasts and microvascular endothelial cells. Pan-genomic microarray analysis and RT-PCR revealed that post-irradiation (0.5, 1.0 2.0 Gy) critical pro-angiogenic signaling factors, including vascular endothelial growth factor (VEGF), interleukin 6 and 8 (IL-6, IL-8) and the hypoxia-inducible factor-1 alpha (HIF-1α), were significantly downregulated. Co-culture studies demonstrated proliferation and invasion of endothelial cells was inhibited by irradiation of both tumor and fibroblast cells, suggesting that proton irradiation may contribute to angiogenesis suppression through paracrine signalings from targeted cells as well as by direct action. Addition of recombinant IL-8 or VEGF partially restored these functions, once again indicating that suppression of these factors plays a functional role in inhibition of tumor progression. Finally an attenuated growth rate and a reduced invasive capacity was demonstrated in vivo for proton-irradiated human lung cancer, A549. Taken together, these findings provide novel preclinical evidence that proton irradiation may, in addition to its physical advantages, have important biological ramifications that should be a consideration in the optimization of proton therapy.

This work was supported by NASA NSCOR grant #NNJ06HA28G

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1319.
Abstract 1327: Study the effect of IGFBP5 in human fibroblast cells
Authors: Lin Hsin-Ying , Shen Cheng-Ying , Lai Liang-Chuan , Chuang Eric Y. , Tsai Mong-Hsun
Published: February 2011

Retinoblastoma (Rb) is a rapidly developing cancer which develops in the cells of the retina, the light detecting tissue of the eye. It is the most common primary ocular malignancy (eye cancer) of childhood. There are both hereditary and sporadic forms of retinoblastoma. The hereditary form of retinoblastoma is associated with a germ line mutation in one of the RB genes and is characterized by the occurrence of multiple, bilateral Rb tumors and a predisposition to the development of second cancers. These second tumors often occur in patients treated by radiation therapy. In the previous study, an unexpected hypersensitivity to ionizing radiation in skin fibroblasts derived from unaffected parents of children with hereditary Rb was observed. By use of the prediction analysis for microarrays (PAM) and principal component analysis (PCA), 42 significantly differential expressed genes were selected and identified as radiosensitive genes. Therefore, one of these 42 genes, IGFBP5 (insulin-like growth factor binding protein 5) was chosen to further investigate the role in radiosensitivity in fibroblast cells. IGFBP5 was overexpressed or knocked down in HS68 (the human foreskin fibroblast) cells. The cells were irradiated with γ -ray, and then counted and seeded into the plates for colony formation assays. According to the results, IGFBP5-ovexpressed cells seemed to be more resistant to radiation. Also, IGFBP5 knocked down cells might be more sensitive to radiation under low dose of radiation. In summary, these results suggested that IGFBP5 may play a role in radiosensitivity in human fibroblast cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1327.
Abstract 1328: p53 represses microRNAs let-7a and let-7b in response to ionizing radiation
Authors: Saleh Anthony D. , Savage Jason E. , Ly David , Nguyen Phuongmai , Cao Lui , Soule Benjamin P. , Dan Tu D. , DeGraff William , Finkel Toren , Harris Curtis C. , Mitchel James B. , Gius David , Simone Nicole L.
Published: February 2011

Radiation-induced free radicals damage cells resulting in a stress response that involves large scale changes in gene expression including microRNAs (miRNA). Specifically, decreased expression of the let-7 family of miRNA has been reported. let-7 regulates cell cycle and DNA repair proteins such as Ras, cyclin D1, Rad 18 and Rad 51, which can all effect the radiation response. However, the precise role of the let-7 family and the mechanisms regulating this repression has not been elucidated. In this study, we demonstrate a mechanism underlying the let-7a3 and let-7b response to radiation exposure. Using real time RT-PCR we demonstrate that let-7a and b expression decreases by 80-90% in response to 0.5-10 Gy doses of IR in cultured HCT116 colon cancer cells. This effect is also observed by RT-PCR in response to other sources of geneotoxic stress such as etoposide or UV irradiation suggesting that a reduction in let-7 is common among DNA damaging events. We also show that this decrease in expression of let-7a and b is dependent on p53 as the decrease is absent in HCT116 p53 knockout cells. Further analysis of the effect of p53 on let-7a3 and b expression by CHIP showed an association of p53 with a predicted binding site in the host gene promoter. After treatment of mice with radiation a decrease in let-7a and b expression is observed in radio sensitive tissues. However no decrease was observed in tissues collected from p53 KO mice. Transfection of additional let-7a or let-7b into HCT116 cells decreased survival after radiation treatment, but survival is unchanged by increased let-7 in HCT116 p53 knockout cells. These results suggest that p53 can directly repress let-7 expression. Also, let-7 may play an important role in the regulation and fine-tuning of the p53 cell stress response to radiation. Furthermore, therapeutics aimed at inducing over expression of let-7 may be useful as radiation sensitizers.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1328.
Abstract 1329: Study the novel function of Semaphorin 6A in lung adenocarcinoma
Authors: Shen Cheng-Ying , Lu Tzu-Pin , Li Yung-Hua , Feng-Chang Wu , Lai Liang-Chuan , Chuang Eric Y. , Tsai Mong-Hsun
Published: February 2011

In Taiwan, most of female lung cancer patients are non-smoker (>90%) and most of the tumor types are adenocarcinoma. The previous studies indicated that cooking oil fumes, late menopausal ages and HPV infections have been associated with the risk of lung adenocarcinoma among non-smoking female. In order to discover novel molecular targets of non-smoking female lung cancer patients in Taiwan, the paired normal and tumor tissues were collected from 60 female non-smoking lung cancer patients for microarray analysis. From the microarray results, we found that several genes in the semaphorin family, such as SEMA3B, SEMA3G, SEMA5A, SEMA6A, and SEMA6D, were significantly down-regulated in the tumor tissues. However, the specific mechanisms of the genes in lung cancer are still unclear. Among the semaphorin genes, SEMA6A was chosen for further study. The results of quantitative real-time PCR and immunohistochemistry showed significant down-regulation of SEMA6A in lung cancer tissues. Moreover, the A549 cell line was used to further investigate the function of SEMA6A. A549 cells with overexpressed SEMA6A were identified and purified the cell membrane fraction for western blotting. A wound healing assay was also performed to address the migration ability. The result was shown that the A549 cells with over expressed SEMA6A exhibited a lower capacity to fill the gap during 24 hours incubation compared with A549 cells. In summary, our data indicated that normal lung tissues express more SEMA6A protein which might inhibit the cell migration than the cancer lung tissues.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1329.
Abstract 1330: Low dose irradiation reverts chemoresistance towards fluorouracil in human pancreatic adenocarcinoma cells by upregulation of thymidine phosphorylase
Authors: Seeliger Hendrik , Xin Ying , Camaj Peter , Ischenko Ivan , Schaffer Moshe , Jauch Karl-Walter , Bruns Christiane J.
Published: February 2011

Background: Resistance towards cytotoxic drugs and ionizing radiation (IR) accounts for poor therapy response in pancreatic cancer. The radiosensitizing effects of fluorouracil (FU) are well characterized, however, it is not known whether IR itself exerts sensitizing effects towards cytotoxic chemotherapy. Here, we hypothesize that IR induced upregulation of thymidine phosphorylase (TP) results in a reversal of resistance towards FU.

Methods: An FU resistant variant of the human pancreatic adenocarcinoma cell line AsPC-1 was developed by long term in vitro FU exposure. Proliferation of AsPC-1 and AsPC-1FUres cells was determined by an MTT proliferation assay after previous low dose (0.05 Gy) exposure of IR. To antagonize a possible effect mediated by TP upregulation, anti-TP siRNA was added in the same setting. TP expression in AsPC-1 cells before and after IR was determined by Western blotting.

Results: FU resistant AsPC-1 cells showed a 7.5-fold increase of the IC50 after FU exposure, compared to non-resistant cells. When challenging both cell lines with FU after low dose IR, no significant difference of proliferation was observed in AsPC-1 cells and their FU resistant variant, suggesting a reversal of chemoresistance by IR. This chemosensitizing effect was abrogated when blocking TP by anti-TP siRNA before IR. In cell lysates harvested from AsPC-1 cells, an increase of TP expression was seen after exposure of low dose IR (0.05 Gy) as well as high dose IR (2.0 Gy).

Conclusion: Our results suggest a TP mediated reversal of chemoresistance towards FU that is triggered by low dose IR in vitro. These results have to be confirmed in an in vivo model. In conclusion, induction of TP expression by low dose irradiation may be an interesting concept to overcome chemotherapy resistance in pancreatic cancer.

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

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1330.
Abstract 1333: Upregulation of chemokines including RANTES/CCL5 upon Toll-like receptor 3 activation in breast cancer: Association with leukocyte infiltration
Authors: Venkatesh Amritha Kalyani , Pate Michelle , Benencia Fabian
Published: February 2011

The association of leukocytes with the microenvironment of different tumors and their role in disease progression is well-documented. However, the mechanism of recruitment of leukocytes to the tumor microenvironment remains unclear. In particular, we have previously demonstrated the capability of tumor-associated dendritic cells to promote neovascularization. In this light, toll-like receptors (TLR) have recently been identified as potential players in different cancers such as malignant melanomas, thyroid cancer and pancreatic cancer. These are a family of pattern-recognition receptors that, when activated by a ligand, trigger signaling pathways involved chiefly in the inflammation process of the innate immune system. Here, we investigated the role of TLR-3 in the capability of the tumor cells to generate chemokines capable of attracting leukocytes to the tumor microenvironment. For this purpose we used the 4T1 model of breast cancer in BALB/c mouse. First, we were able to determine the expression of TLR-3 in these cells by PCR analysis. Then, 4T1 mouse breast cancer cells were treated with poly(I:C), a well-known ligand for TLR-3, and the supernatants were checked for the presence of different chemokines (RANTES, SDF-1α, MIP-3α, MIP-3β) by sandwich ELISA. These molecules were selected taking into account that their receptors are present in dendritic cells. Further, a reverse transcription quantitative PCR was performed to detect the RNA expression levels of these chemokines. In both cases, a significant increase in RANTES levels upon TLR-3 activation was observed. Further, a chemokine PCR array showed upregulation of 10 chemokines, RANTES among them, on 4T1 cells stimulated with poly(I:C). A subsequent protein array analysis of different mouse cytokines in poly(I:C)-treated 4T1 cells revealed also an increase in RANTES, IL-6 and MIP-2 and a decrease in the matrix metalloprotease inhibitor TIMP-1. Interestingly, IL-6 upregulation was further confirmed by ELISA. These results are supportive of our hypothesis that TLR-3 activation could lead to the release of chemokines that assist in leukocyte recruitment. Further in vivo studies are required to observe the effect of knocking down TLR-3 in cancer cells on the capability of the tumor to promote leukocyte infiltration.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1333.
Abstract 1338: Chemokine (CXC motif) Ligand 5 protein expression in pancreatic tumors: Exploring chemostatic gradients in the tumor microenvironment
Authors: Ustach Carolyn V. , Whatcott Clifford J. , Kingsley Christopher , Hostetter Galen , Barret Michael , Hidalgo Manuel , Diep Caroline H. , Watanabe Aprill , Demeure Michael , Han Haiyong , Von Hoff Daniel D.
Published: February 2011

Due to lack of early presenting symptoms, pancreatic cancer is often diagnosed late when the tumor is highly invasive with aggressive clinical course and conventional therapies are limited in their effectiveness. Identifying molecular targets which contribute to tumor aggressiveness and metastasis could lead to development of better therapies to treat pancreas cancer patients. To identify potential molecular targets, we applied a specialized bioinformatics Breakpoint/Gene Aberration algorithm to analyze aCGH data and microarray expression data from 16 low-passage pancreas xenograft samples. Genes which were located near a putative breakpoint and whose expression was altered in a majority of the xenografts samples were considered putative targets. We report here preliminary data concerning one target, Chemokine (CXC motif) Ligand 5 (CXCL5) and its receptor CXCR2/IL8Rb. CXCL5 is a member of the ELR+ Chemokine family, a family of small, secreted ligands which are generally involved in cellular responses to injury and inflammation, but are also implicated in cancer progression and angiogenesis. CXCL5 is up regulated in 12 out of 16 xenograft samples analyzed (75%), with an average fold increase in expression of 41 (range 18 to 59). CXCL5 over-expression was further confirmed by IHC analysis of a custom made primary pancreas tumor microarray. Median IHC score (range 0-3) for CXCL5 in adenocarcinoma epithelial cells is 1.6, versus a median of 0.8 in the matched adjacent normal samples (N=34; p<0.0007 using Mann-Whitney test). Interestingly, the receptor CXCR2 levels are increased in the stroma of tumor samples, with a median IHC score of 1.6 in the stroma surrounding ductal adenocarcinoma versus an IHC score of 0.6 in the stroma of adjacent normal ducts (N=31; p<0.0001 using Mann-Whitney test). These results suggest the stroma surrounding the adenocarcinoma is responding to the over-expression of tumor-derived CXCL5. How CXCL5-induced CXCR2 signaling in the stroma impacts tumor growth and progression is currently being studied using an in-vitro co-culture system with established pancreas epithelial carcinoma cell lines and primary pancreas cancer fibroblasts. Results from these studies will be presented.

This work was supported by NIH P01 Grant CA109552.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1338.
Abstract 1339: Inhibition of chitinase-3-like-1 protein to suppress angiogenesis and enhance immune response in a mammary tumor model
Authors: Libreros Stephania , Garcia-Areas Ramon , Iragavarapu-Charyulu Vijaya L.
Published: February 2011

The purpose of this study is to determine whether inhibition of Chitinase-3-like 1 protein reduces the expression of angiogenic chemokines in macrophages while enhancing IFN-γ production in T cells. It is known that tumors can induce the upregulation of angiogenic and extracellular matrix degrading molecules. Tumor growth is often accompanied by immunosuppression. Our previous studies using the DA-3 mammary adenocarcinoma mouse model has shown that with tumor progression there is a profound down-regulation of interferon-gamma (IFN-γ), a crucial molecule involved in anti-tumor response. Additionally, angiogenic chemokines such as Monocyte Chemoattractant Protein-1 (CCL2/MCP-1) and Matrix Metalloproteinase-9 (MMP-9) are upregulated with mammary tumor growth. CCL2/MCP-1 is a known inflammatory chemoattractant for T cells and macrophages, and we have evidence that this molecule downregulates IFN-γ by the T lymphocytes. Recent studies have linked tumor progression with upregulation of Chitinase-3-like-1 protein (CHI3L1) in human breast cancer patients and that macrophages express this molecule during inflammatory processes. Our studies using DA-3 mammary breast cancer model indicate that CHI3L1 is expressed by DA-3 tumor cells. Expression of CHI3L1 in macrophages has been linked with upregulation of angiogenic chemokine, CCL2/MCP-1. Our studies indicate that macrophages from mammary tumor-bearing mice express higher levels of CHI3L1 compared to normal control mice and also secrete CCL2/MCP-1. We hypothesize that inhibition of CHI3L1 will suppress the production of CCL2/MCP-1 and enhance IFN-γ production in mammary tumor-bearing mice. One of the known ligands for CHI3L1 is chitin microparticle. Our in vitro studies of cultured splenocytes from normal and 5-week mammary tumor-bearing mice with chitin micro-particles revealed down-regulation of CHI3L1 and upregulation of IFN-γ. Therefore, it is expected that in vivo blocking of CHI3L1 with chitin microparticles will have a favorable anti-tumor response by inhibiting the production of angiogenic chemokines, metalloproteinase, MMP-9 and enhancing the production of IFN-γ. The results from these studies may provide new avenues for cancer treatment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1339.