bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3756: MiR-203 inhibits the proliferation and self-renewals of esophageal cancer stem-like cells by suppression stem renewal factor Bmi-1.
Authors: Yu Xiying , Jiang Xingran , Li Hongxia , Guo Liping , Jiang Wei , Lu Shih-Hsin
Published: August 2014

Recent reports have shown that cancer stem-like cells exist in many malignancies and several stem cell-related gene products and miRNAs, such as Bmi-1 and miR-203, were identified as cancer stem-like cell regulators using gene microarray or sequencing analysis. Bmi-1 is essential for the self-renewal capacity of several types of normal stem cells and cancer stem-like cells while miR-203 is involved in stem cell self-renewal regulation. Previously, we used side population (SP) sorting to enrich cancer stem-like cells from esophageal squamous cell carcinoma (ESCC) cell line EC9706. Our results demonstrated that EC9706 SP cells have the same common features of cancer stem-like cells, such as the ability of self renewal, highly proliferation and tumorigenesis. In this study, we examined expression regulation of Bmi-1 and miR-203 in ESCC SP and none-SP (NSP) cells. Our results showed that, when compared with NSP cells, Bmi-1 was up-regulated and miR-203 was down-regulated in SP cells. During the differentiation from SP to NSP cells, the expression levels of Bmi-1 were gradually decreased, suggesting that Bmi-1 might be required for maintaining the ESCC SP cells. As sequence analysis revealed that the mRNA 3’UTR of Bmi-1 has a highly conserved binding site of miR-203, we overexpressed miR-203 in EC9706 cells using lentivirus mediated gene transduction. Overexpression of miR-203 (lenti-miR-203) resulted in a significant reduction of endogenous Bmi-1 protein levels in EC9706 cells. SP and NSP analyses revealed that the SP cell fraction markedly decreased in lenti-miR-203 cells. Lenti-miR-203 cells also showed significantly reductions of the colony formation, resistant to chemotherapeutic drug treatment and tumorigenicity in nude mice. Rescue experiments demonstrated that ectopic expression of Bmi-1 in lenti-miR-203 cells increased the SP fraction and restored cell proliferation. Taken together, these results indicated that stem renewal factor Bmi-1 is a direct target of miR-203. Regulation of Bmi-1 by miR-203 may play an important role in controlling cell proliferation and self-renewals of esophageal cancer stem-like cells.

Citation Format: Xiying Yu, Xingran Jiang, Hongxia Li, Liping Guo, Wei Jiang, Shih-Hsin Lu. MiR-203 inhibits the proliferation and self-renewals of esophageal cancer stem-like cells by suppression stem renewal factor Bmi-1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3756. doi:10.1158/1538-7445.AM2013-3756
Abstract 3767: Nicotine promotes acquisition of stem cell and epithelial-to-mesenchymal properties in head and neck squamous cell carcinoma.
Authors: Yu Michael A. , Kiang Alan , Wang-Rodriguez Jessica , Rahimy Elham , Haas Martin , Yu Vicky , Ellies Lesley G. , Fan Jian-Bing , Brumund Kevin T. , Weisman Robert A. , Ongkeko Weg M.
Published: August 2014

The ability of nicotine to enhance the malignancy of cancer cells is known; however, the possibility that nicotine could regulate a cancer stem cell phenotype remains to be well-established. In this study we sought to determine whether long-term exposure to nicotine could promote cancer stem cell-like properties in two head and neck squamous cell carcinoma cell lines, UMSCC-10B and HN-1. Nicotine treatment induced epithelial-to-mesenchymal transition (EMT) in both cell lines by repressing E-cadherin expression, and led to the induction of stem cell markers Oct-4, Nanog, CD44 and BMI-1, which was reversed upon ectopic re-expression of E-cadherin. Nicotine-treated cells formed spheres at a higher efficiency than non-treated cells, formed larger tumors when injected into mice, and formed tumors with 4-fold greater efficiency compared to control cells when injected at limiting doses. Consistent with previous literature, nicotine-treated cells demonstrated a greater capacity for survival and also a higher tendency to invade. Comparison of microRNA profiles between nicotine and control cells revealed the upregulation of miR-9, a repressor of E-cadherin, and the downregulation of miR-101, a repressor of EZH2. Taken together, these results suggest that nicotine may play a critical role in the development of tobacco-induced cancers by regulating cancer stem cell characteristics, and that these effects are likely mediated through EMT-promoting, microRNA-mediated pathways. Further characterization of such pathways remains a promising avenue for the understanding and treatment of tobacco-related cancers.

Citation Format: Michael A. Yu, Alan Kiang, Jessica Wang-Rodriguez, Elham Rahimy, Martin Haas, Vicky Yu, Lesley G. Ellies, Jian-Bing Fan, Kevin T. Brumund, Robert A. Weisman, Weg M. Ongkeko. Nicotine promotes acquisition of stem cell and epithelial-to-mesenchymal properties in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3767. doi:10.1158/1538-7445.AM2013-3767
Abstract 3770: ER-α36-mediated non-genomic estrogen signaling positively regulates ER-positive breast cancer stem/progenitor cells .
Authors: Deng Hao , Zhang Xintian , Wang Molin , Guo Mingxi , Liu Lijiang , Wang Zhaoyi
Published: August 2014

Accumulating experimental and clinical evidence indicated that breast cancer may arise from mammary stem/progenitor cells that posses the features like the ability of self-renewal and tumor generation from very few cells, slow cell division, differentiation into different lineages, selective resistance to radio- and chemo-therapy, constitutive activation of anti-apoptotic pathways and induction of angiogenesis, the ability to migrate and spread in metastasis. Many signaling pathways involved in regulation of normal mammary stem cells including Hedgehog, Bmi-1, Wnt, NOTCH, HER-2, p53 and PTEN/Akt/β-catenin pathways have been identified to play roles in breast cancer stem/progenitor cells. However, the involvement of estrogen signaling, a major signaling pathway in breast cancer development, in regulation of breast cancer stem/progenitor cells has not been well established, mainly because expression of estrogen receptor-α (ER-α) in breast cancer stem/progenitor cells remains controversial. Previously, we have identified and cloned a novel variant of ER-α, ER-α36, that is mainly expressed near the plasma membrane and in the cytoplasm, and mediates non-genomic estrogen signaling such as activation of the MAPK/ERK and PI3K/AKT signaling pathways. In this study, we ivestigated the function of ER-α36 in regulation of breast cancer stem/progenitor cells. We found that ER-α36 was highly expressed on the plasma membrane of the breast cancer stem/progenitor cells while the original ER-α was expressed outside of the cell nuclei. We also found that the genomic estrogen signaling was down-regulated in ER-positive breast cancer stem/progenitor cells. ER-positive breast cancer cells with forced expression of recombinant ER-α36 exhibited increased populations of stem/progenitor cells and high tumor seeding efficiency. Estrogen treatment significantly increased the population of breast cancer stem/progenitor cells while failed to do so in ER-positive breast cancer cells with knocked-down expression levels of ER-α36. We further found that estrogen positively regulates breast cancer stem/progenitor cells through ER-α36-mediated non-genomic estrogen signaling pathways such as activation of the PI3K/AKT pathway. Thus, our results demonstrated that the novel variant of ER-α, ER-α36 plays an important role in estrogen regulation of ER-positive breast cancer stem/progenitor cells.

Citation Format: Hao Deng, Xintian Zhang, Molin Wang, Mingxi Guo, Lijiang Liu, Zhaoyi Wang. ER-α36-mediated non-genomic estrogen signaling positively regulates ER-positive breast cancer stem/progenitor cells . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3770. doi:10.1158/1538-7445.AM2013-3770
Abstract 3777: Low physiological fluid shear exposure induces metastatic potential of chondrosarcoma cells via MMP-12 activation.
Authors: Chen Shih-Hsun , Wang Pu , Hung Wei-Chien , Konstantopoulos Konstantinos
Published: August 2014

Interstitial flow in and around tumor tissue affects the mechanical microenvironment to modulate tumor cell metastasis. We investigated the roles of flow-induced shear stress in modulating chondrosarcoma cell metastatic potential such as cell invasiveness and motility, and the underlying mechanisms. Using human chondrosarcoma cell lines as a model system, we demonstrated that low fluid shear (2 dyn/cm2), but not high fluid shear (20 dyn/cm2), exposure for 48 h remarkably induced MMP-12 mRNA by ∼35-fold and significantly triggered it proteolytic activity among all MMP members. The molecular basis of the shear effect was further analyzed, and our data revealed that low fluid shear stress induces the rapid synthesis of VEGF-B, VEGF-D and IGF-2, which in turn transactivates MMP-12 expression via PI3K and p38 MAPK-dependent pathways. Furthermore, highly induced MMP-12 markedly enhanced chondrosarcoma cell invasion and motility by using chemtaxis transwell chambers in conjunction with time-lapse micro-channel assays. Collectively, our data suggest that the induction of IGF-2, VEGF-B, and VEGF-D under low fluid shear, which in turn upregulates MMP-12 via PI3K and p38 MAPK signaling pathways, leading to the enhanced invasive and migratory capabilities of chondrosarcoma cells. Our findings provide insights into the mechanism by which shear stress induces chondrosarcoma metastatic potential and demonstrate the importance of mechanical environments in modulating molecular signaling, gene expression and functions in tumor cells.

Citation Format: Shih-Hsun Chen, Pu Wang, Wei-Chien Hung, Konstantinos Konstantopoulos. Low physiological fluid shear exposure induces metastatic potential of chondrosarcoma cells via MMP-12 activation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3777. doi:10.1158/1538-7445.AM2013-3777
Abstract 3782: NEDD9 promotes cell invasion through modulation of Arf6-dependent endocytic recycling.
Authors: Loskutov Yuriy , McLaughlin Sarah , Kozyulina Polina , Kozyreva Varvara , Ice Ryan , Rajulapati Anuradha , Culp Mark , Wysolmerski Robert , Ivanov Alexey , Weed Scott , Pugacheva Elena
Published: August 2014

The adaptor protein, NEDD9, is an established pro-metastatic trigger in several cancers but the molecular mechanisms behind NEDD9-driven invasion remain unclear. Here we show that expression of NEDD9 protein tightly correlates with the transition of breast cancer to invasive stages. Overexpression of NEDD9 is critical for the invasion of cancer cells but depletion of NEDD9 is sufficient to block invasion. It is also well established that invadopodia are important for cell invasion. We demonstrate that NEDD9 localizes to invadopodia and its depletion leads to a deficiency in matrix degradation. NEDD9 depletion was also accompanied by an increase in surface levels of inactive proteases (MT1-MMP) and adhesion receptors (integrins). In addition, depletion of NEDD9 induced an increase in recycling and early endosomes via activation of Arf6. NEDD9 binds to the Arf6 specific GAP, ASAP3, which explains NEDD9 modulation of Arf6 activity. Inhibition of Arf6 or re-expression of NEDD9 rescue in NEDD9-deficient cells is sufficient to restore proper recycling rates and invasive properties of breast cancer cells. These results reveal the mechanism behind NEDD9-driven migration and tumor invasion.

Citation Format: Yuriy Loskutov, Sarah McLaughlin, Polina Kozyulina, Varvara Kozyreva, Ryan Ice, Anuradha Rajulapati, Mark Culp, Robert Wysolmerski, Alexey Ivanov, Scott Weed, Elena Pugacheva. NEDD9 promotes cell invasion through modulation of Arf6-dependent endocytic recycling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3782. doi:10.1158/1538-7445.AM2013-3782
Abstract 3787: ERK and AKT signaling cooperate to translationally regulate survivin expression for promotion of cell motility and metastasis in colorectal cancer.
Authors: Ye Qing , Cai Weijia , Zheng Yongbin , Evers B. Mark , She Qing-Bai
Published: August 2014

The MEK/ERK and PI3K/AKT pathways are often concurrently activated by separate genetic alterations in colorectal cancer (CRC), which is associated with CRC progression and poor survival. However, how activating both pathways is required for CRC metastatic progression remains unclear. Our recent study showed that both ERK and AKT signaling are required to activate eIF4E-initiated cap-dependent translation via convergent regulation of the translational repressor 4E-BP1 for maintaining CRC transformation. Here, we identified that the activation of cap-dependent translation by cooperative ERK and AKT signaling is critical for promotion of CRC motility and metastasis. In CRC cells with coexistent mutational activation of ERK and AKT pathways, inhibition of either MEK or AKT alone showed limited activity in inhibiting cell migration and invasion, but combined inhibition resulted in profound effects. Genetic blockade of the translation initiation complex by eIF4E knockdown or expression of a dominant active 4E-BP1 mutant effectively inhibited migration, invasion and metastasis of CRC cells, whereas overexpression of eIF4E or knockdown of 4E-BP1 had the opposite effect and markedly reduced their dependence on ERK and AKT signaling for cell motility. Mechanistically, we found that these effects were largely dependent on the increase in mTORC1-mediated survivin translation by ERK and AKT signaling. Despite the modest effect of survivin knockdown on tumor growth, reduction of the translationally-regulated survivin profoundly inhibited motility and metastasis of CRC. These findings reveal a critical mechanism underlying the translational regulation of CRC metastatic progression, and suggest that targeting cap-dependent translation may provide a promising treatment strategy for advanced CRC.

Citation Format: Qing Ye, Weijia Cai, Yongbin Zheng, B. Mark Evers, Qing-Bai She. ERK and AKT signaling cooperate to translationally regulate survivin expression for promotion of cell motility and metastasis in colorectal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3787. doi:10.1158/1538-7445.AM2013-3787
Abstract 3788: Alpha-2 Heremans Schmid glycoprotein (AHSG) promotes migration in head and neck squamous cell carcinoma (HNSCC) cell lines.
Authors: Thompson Pamela D. , Watson Kurt , Sakwe Amos , Ochieng Josiah , Marshall Dana
Published: August 2014

Head and neck squamous cell carcinoma (HNSCC) is the eighth most common tumor in the world. Approximately 30,000 new cases of HNSCC are reported each year and 8,000 related deaths, in the United States. HNSCC of the oral cavity is highly metastatic and has a recurrence rate of 20%-50%, with a dismal 5-year survival rate of ∼50%. African-American males have a 5-year survival rate of ∼42% while the survival rate for Caucasian males is 52%. To date, there is no consistent means for early detection or a reliable biomarker for HNSCC, thus perpetuating the lack of improvement in mortality despite advances in delivery of treatment and surgical reconstruction. In a proteomic study seeking possible biomarkers for HNSCC, AHSG was detected at elevated levels in serum. Interestingly, we have found that some HNSCC cell lines synthesize and secrete AHSG. We have also shown in earlier studies, that the lack of AHSG reduces mammary tumor incidence and prolongs tumor latency in a mouse model for breast cancer. Together, this suggests that this hepatocellular glycoprotein that is found in most body fluids including serum could be a potential biomarker for HNSCC. AHSG is a negative acute phase response protein, which makes the findings of elevated levels of AHSG in the proteomic study most intriguing. In order to evaluate the role of AHSG in HNSCC progression (proliferation and metastatic properties) in vitro, we depleted AHSG in the HNSCC cell lineSQ20B. Thus far, we show that depletion of AHSG does not affect the proliferation of SQ20B cells. However, the depletion of AHSG mediates a decrease in migration and invasion of SQ20B cells. We are currently elucidating the molecular mechanisms of AHSG-mediated motility.

This work was supported by grants from the NIH-NCI-Score 1 SC1 CA134018-01 (JO); DOD W81XWH-07-1-0254 (J.O); 5 T32 HL007735-15 (SA) and U54 CA091408 (subproject, DM).

Citation Format: Pamela D. Thompson, Kurt Watson, Amos Sakwe, Josiah Ochieng, Dana Marshall. Alpha-2 Heremans Schmid glycoprotein (AHSG) promotes migration in head and neck squamous cell carcinoma (HNSCC) cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3788. doi:10.1158/1538-7445.AM2013-3788
Abstract 3807: Integrative genomic profiling of pilocytic astrocytomas reveals location specific differential methylation and expression of brain developmental genes.
Authors: Lambert Sally R. , Witt Hendrik , Hovestadt Volker , Zucknick Manuela , Kool Marcel , Pearson Danita , Korshnov Andrey , Pfister Stefan , Collins V. Peter , Jones David T. W.
Published: August 2014

Pilocytic astrocytomas (PA) are the most common brain tumor in pediatric patients and cause significant morbidity, particularly related to chronic neurological deficiencies. They are characterized by activating alterations in the mitogen activated protein kinase (MAPK) pathway, but little else is known about their development. To map the methylation profile of these tumors, we have performed a global DNA methylation analysis on 62 PAs and 7 normal cerebellum samples using Illumina 450K microarrays. Based on these data we have found two subgroups of PA that separate according to tumor location (infratentorial versus supratentorial), and have identified key neural developmental genes that are differentially methylated between the two groups, including NR2E1 and EN2. Integration with gene expression microarray data revealed significant expression differences that were typically associated with a strong positive correlation between methylation and expression. Differential methylation was most commonly identified within the gene body and/or 10 kb upstream/downstream of the gene body, and was, in part, accounted for by differences in the level of 5-hydroxymethylcytosine. We also identified a large number of differentially methylated genes between cerebellar PAs and normal cerebellum, which were again enriched for developmental genes. In addition, we found a significant association between differential methylation and SUZ12 binding sites, suggesting potential disruption of the polycomb repressor complex 2 (PRC2) in PA development. Taken together these data suggest the methylation profile of PA may reflect the cell of origin from which the tumors are derived, and highlights the potential disruption of key developmental regulators during tumorigenesis. These findings have implications for future clinical trials, as they suggest drug sensitivity and response may differ according to tumor location.

Citation Format: Sally R. Lambert, Hendrik Witt, Volker Hovestadt, Manuela Zucknick, Marcel Kool, Danita Pearson, Andrey Korshnov, Stefan Pfister, V. Peter Collins, David T. W. Jones. Integrative genomic profiling of pilocytic astrocytomas reveals location specific differential methylation and expression of brain developmental genes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3807. doi:10.1158/1538-7445.AM2013-3807
Abstract 381: VEGF-A and VEGF-C: potential coregulators in angiogenic event in thyroid cancer.
Authors: Salajegheh Ali , Smith Robert A. , Pakneshan Sahar , Gopalan Vinod , Lam Alfred KY
Published: August 2014

Vascular endothelial growth factor (VEGF) promotes growth of blood or lymphatic vessels, termed angiogenesis and lymphangiogenesis. These share several basal mechanisms, including cross-talk between VEGF-A, C and their receptors, which may be important in the development of malignancies. Study aimed to identify relationships between VEGF-A, VEGF-C and their impact in angiogenesis and metastases in thyroid cancers.We studied VEGF-A and VEGF-C mRNA expression in 123 thyroid cancers, 40 lymph node metastases and 7 non-malignant thyroid control tissues using qPCR. Protein expressions for both VEGFs were determined by immunohistochemistry.VEGF-A and VEGF-C mRNA over-expression was noted in 51% (n=62) and 27% (n=33) of the thyroid cancers respectively. VEGF-A and VEGF-C protein over-expression was also identified in 70% (n=86) and 62% (n=76) of the thyroid cancers. ANOVA showed significant expression differences for VEGF-A and VEGF-C in subtypes of thyroid carcinoma, lymph node metastasis and normal controls (P=1x10-6 and 1x10-5 respectively). VEGF-A mRNA was significantly higher in cancers with lymph node metastases compared to non-metastatic cancers (p=0.001) however, most metastatic tumors under-expressed VEGF-C (p=0.0002). Similarly, VEGF-A protein expression was higher in metastases and VEGF-C protein was higher in non-metastatic lesions, though not significantly. Finally, we used linear regression analysis to test previous proposals about physiological interactions of VEGF-A and VEGF-C expression. Our model indicated that in metastatic cancers, 32% of the expression of VEGF-A could be predicted by a positive influence of VEGF-C production (model p=0.0002). These findings add weight to previous hypotheses concerning VEGF-A and C interaction. Early lymphangiogenic events were proposed to regulate angiogenesis, and changes to VEGF expression in this cancer population bear this out, with VEGF-C in early tumors giving way to VEGF-A in metastases. The commonality of local metastasis in thyroid cancer makes it a useful model to further examine mechanisms of angiogenesis and lymphangiogenesis in cancer metastasis. Keywords: thyroid cancer, VEGF-A, VEGF-C

Citation Format: Ali Salajegheh, Robert A. Smith, Sahar Pakneshan, Vinod Gopalan, Alfred KY Lam. VEGF-A and VEGF-C: potential coregulators in angiogenic event in thyroid cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 381. doi:10.1158/1538-7445.AM2013-381
Abstract 3812: Target identification in rhabdomyosarcomas induced in mouse skeletal muscle.
Authors: Hettmer Simone , Schinzel Anna , Hahn William C. , Wagers Amy
Published: August 2014

Soft-tissue sarcomas are a group of cancers arising in non-hematopoietic, mesodermal tissues. Rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in children and adolescents, represents a biologically heterogeneous category of mesodermal, non-hematopoietic malignancies, which exhibit evidence of skeletal muscle differentiation. This study builds on the premise that ex-vivo induction of sarcomas by cell-specific activation of sarcoma-relevant pathways can be used to model biologically distinct sarcoma categories in mice and identify novel downstream targets. Up to 35% of human RMS tumors carry Ras mutations, and we have demonstrated Ras/ Raf/ Mek/ Erk pathway activation in approximately 50% of human RMS tumors surveyed. We showed that Kras-expressing; p16p19null muscle satellite cells gave rise to mouse pleomorphic RMS, whereas Kras-expressing; p16p19null non-myogenic progenitors typically induced pleomorphic non-myogenic sarcomas (NMS). Transcriptional profiling demonstrated that myogenic and non-myogenic Kras; p16p19null sarcomas recapitulate gene expression signatures of human RMS and share a cluster of concordantly upregulated genes (many associated with Ras pathway activation) with human RMS. We used a customized RT² Profiler™ PCR Array (SA Biosciences) to confirm that 105 out of these 141 candidate genes were indeed upregulated in Kras;16p19null sarcomas compared to mouse skeletal muscle. The functional contributions of individual candidate genes to sarcoma proliferation were evaluated using a customized shRNA-based proliferation screen in cell lines established from Kras; p16p19null sarcoma explants. These comprehensive experiments identified a small group of high-priority candidates that contributed significantly to the growth of Kras; p16p19null NMS (n=11 genes) and RMS (n=10 genes) (q<0.05). The effects of shRNA mediated silencing of selected candidate genes in human sarcoma cells were verified using human sarcoma cell lines. We conclude that these genes have potential broad importance in sarcoma biology and anti-sarcoma therapy. Future studies will help to illuminate their roles in promoting RMS growth and may suggest new approaches for therapeutic intervention.

Citation Format: Simone Hettmer, Anna Schinzel, William C. Hahn, Amy Wagers. Target identification in rhabdomyosarcomas induced in mouse skeletal muscle. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3812. doi:10.1158/1538-7445.AM2013-3812
Abstract 3863: Transgenic model for early prostate metastasis to the lymph nodes.
Authors: Ko Hyun-Kyung , Akakura Shin , Peresie Jennifer , Goodrich David W. , Foster Barbara A. , Gelman Irwin H.
Published: August 2014

Background: The emergence of recurrent, metastatic prostate cancer following the failure of androgen-deprivation therapy represents the lethal phenotype of this disease. However, little is known regarding the genes and pathways that regulate this metastatic process, and moreover, it is unclear whether metastasis is an early or late event. The SSeCKS/Gravin/AKAP12 (“SSeCKS”) gene, previously shown to suppress prostate cancer metastasis upon re-expression (1,2), as well as the tumor suppressor gene, Rb, are either transcriptionally downregulated or deleted in human prostate cancers (3,4). Mouse models deficient in either SSeCKS or Rb exhibit prostatic hyperplasia (5,6). Moreover, SSeCKS-null fibroblasts as well as SSeCKS-null prostates display Rb-dependent premature senescence markers (7). Methods: In order to determine whether the combined loss of AKAP12 and Rb in the prostate synergizes to induce oncogenic progression, Akap12-/- mice were crossed with Pb4-Cre;RbloxP/loxP mice to generate Akap12-/-;RbPE-/− progeny, with the Probasin-Cre passed only through males. Results: The combined loss of SSeCKS and Rb results in prostatic intraepithelial neoplasia (PIN) starting at 6 months of age that fails to progress to malignancy (adenocarcinoma) after 18 months. The PIN lesions were marked by increased Ki-67 proliferation of cytokeratin 8 (CK8), p63-negative luminal cells as well as p63-positive basal cells. Interestingly, these lesions also had increased numbers of androgen-receptor (AR)-positive, p63-positive, CK5-negative cells. There was evidence of reactive stroma including mural hyperplasia and inflammatory cell infiltration. Strikingly, 83% of mice with PIN lesions exhibited metastases to draining lymph nodes (LN), marked by well-differentiated tumors cells expressing markers of basal (p63, CK14) and luminal (CK8 and AR) epithelial cells, although none expressed the basal marker, CK5. PCR-based tests for the deleted floxed Rb allele from laser capture microdissected LN lesions confirmed the prostatic origin of these metastatic cells. The LN lesions showed very limited inflammation, based on very few cells staining with the histiocyte marker, CD68. Conclusions: Taken together, these data suggest that in the context of Rb loss, SSeCKS suppresses the oncogenic proliferation and early metastatic spread of transitional, basal-luminal prostate tumor cells.

Citation Format: Hyun-Kyung Ko, Shin Akakura, Jennifer Peresie, David W. Goodrich, Barbara A. Foster, Irwin H. Gelman. Transgenic model for early prostate metastasis to the lymph nodes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3863. doi:10.1158/1538-7445.AM2013-3863
Abstract 387: Intermittent hypoxia stimulates capillary growth and branching in an in vitro model of tumor angiogenesis.
Authors: Ehsan Seema M. , Mendoza Nicole L. , George Steven C.
Published: August 2014

Introduction: The complex heterogeneity of the tumor microenvironment, including a structurally and functionally abnormal vasculature, impacts oxygen tension leading to intermittent hypoxia (IH). While it is known that chronic hypoxia modulates tumor development in vivo, including mechanisms of angiogenesis and resistance to radiotherapy, our understanding of IH remains limited. We hypothesized that IH would stimulate vasculogenesis and lead to a larger and more branched network of vessels consistent with the vessel network in tumors. Using a fabricated oxygen control system, we explored the effects of chronic and intermittent hypoxia on in vitro vascularized tissues.

Materials and Methods: A co-culture of endothelial colony forming cell-derived endothelial cells (ECFC-ECs) from cord blood and normal human lung fibroblasts was distributed in a fibrin gel, and incubated for 7 days at constant 20% O2 (ambient condition), constant 5% O2 (physiological condition), constant 1% O2 (hypoxia conditions), and multiple IH conditions all of which had a mean value of 5% O2. The resulting vessel networks were quantified for total vessel network length and number of junctions. Vascular endothelial growth factor (VEGF) production was measured over the course of the 7 days.

Results and Discussion: Decreasing oxygen tension from 20% to 5% O2 limits vessel formation in vitro, and 1% O2 stopped vessel formation completely. Tissues exposed to IH, however, exhibited significantly more developed and branched vascular networks compared to the constant 5% O2 counterparts (total vessel network length: 5%: 5.4 ± 0.8 mm, IH: 10.7 ± 1.6 mm; (number of junctions: 5%: 32.7 ± 10.6 mm, IH: 99.2 ± 23.1), suggesting a role in tumor angiogenesis. Indeed, VEGF production was upregulated as well in the IH conditions, which may contribute to the enhanced blood vessel formation. We conclude that IH conditioning generates vascular networks in vitro that more structurally and functionally resemble tumor vascular networks in vivo.

Citation Format: Seema M. Ehsan, Nicole L. Mendoza, Steven C. George. Intermittent hypoxia stimulates capillary growth and branching in an in vitro model of tumor angiogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 387. doi:10.1158/1538-7445.AM2013-387
Abstract 3872: A lineage-selective transcriptional pathway linking lung differentiation and cancer metastasis.
Authors: Cheung William K.C. , Zhao Minghui , Liu Zongzhi , Cao Paul D. , Stevens Laura E. , Westbrook Thomas F. , Nguyen Don X.
Published: August 2014

Lung cancer is the leading cause of cancer-related deaths worldwide. This poor prognosis is due to the rapid metastatic progression of this disease, yet the mechanisms that govern lung cancer dissemination and colonization are unclear. Through an integrated approach, we identified an alveolar cell-selective gene signature that stratifies lung adenocarcinoma (ADC) into molecular subtypes of distinct prognosis and differentiation states. This transcriptional program is regulated in part by the cell fate transcription factors, GATA6 and HOPX, which are normally required for proper alveolar differentiation. In an experimental model, suppression of GATA6 and HOPX cooperatively enhances metastatic competence of lung ADC cells. Whole genome RNA sequencing reveals that this transcriptional network can modulate specific target genes and pathways involved in airway epithelial differentiation and invasion. Our findings identify a novel cell lineage molecular program whose perturbation enhances lung cancer metastasis.

Citation Format: William K.C. Cheung, Minghui Zhao, Zongzhi Liu, Paul D. Cao, Laura E. Stevens, Thomas F. Westbrook, Don X. Nguyen. A lineage-selective transcriptional pathway linking lung differentiation and cancer metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3872. doi:10.1158/1538-7445.AM2013-3872
Abstract 3883: Robo1, a new target, by Wnt and androgen receptor signaling in castration resistant prostate cancer.
Authors: Yokoyama Noriko N. , Sun Zheng , Sakai Toshinori , Hoang Bang H. , Zi Xiaolin
Published: August 2014

Androgen receptor (AR) and β-catenin/TCF-4 signaling both play a critical role during prostate cancer progression to castration-resistance and cross-talk with each other. Therefore, identifying novel down-stream targets that are co-regulated by AR and β-catenin/TCF-4 signaling is important for understanding the development of castration-resistant prostate cancer (CRPC). Robo1, a transmembrane receptor of Slit, is known for its roles in neurogenesis and angiogenesis. Previous studies have shown that Robo/Slits are aberrantly expressing in different cancers. We have shown that Robo1 is over-expressed in androgen-insensitive prostate cancer cell lines (C4-2B, PC3, 22RV1) compared to androgen-sensitive lines (LNCaP and LAPC-4), and absent in normal prostate epithelial cells. Androgen deprivation resulted in enhanced expression of Robo1 and addition of synthetic androgen R1881decreased Robo1 expression in LNCaP cells. In addition, Wnt 3A and 5A conditioned medium stimulation increased Robo1 mRNA expression in LNCaP cells. Inhibition of Wnt signaling by overexpression of secreted Wnt antagonists Wnt-inhibitory factor-1 (WIF-1) and Frzb/sFRP3 in two CRPC cell lines, decreased the expression of Robo1. Using GenBank and MatInspector software, we have identified potential binding sites for TCF-4 in the 5’ flanking promoter region of the ROBO1 gene. Chromatin immunoprecipitation followed by real-time PCR analysis revealed that TCF-4 binds to the ROBO1 promoter at two predicted sites, and that WIF-1 and Frzb/sFRP3 expression inhibited its promoter binding. Overexpression of WIF-1 and Frzb/sFRP3 also inhibited the promoter activity of Robo1. Knock-down of Robo1 by short-hairpin RNA in PC3 cells resulted in dramatic morphological changes with increased cell aggregation and adhesion. Taken together, our results indicate that Robo1 is a co-target of Wnt and AR signaling. Further studies are in progress to define the functional role of Robo1 in CRPC.

Citation Format: Noriko N. Yokoyama, Zheng Sun, Toshinori Sakai, Bang H. Hoang, Xiaolin Zi. Robo1, a new target, by Wnt and androgen receptor signaling in castration resistant prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3883. doi:10.1158/1538-7445.AM2013-3883
Abstract 3886: Activated insulin-like growth factor (IGF) signaling pathway is a potential therapeutic target for brain metastasis from lung cancer.
Authors: Wu Pei-Fang , Lin Ching-Hung , Huang Wen-Chang , Feng Wen-Chi , Lu Yen-Shen , Yang Chih-Hsin , Cheng Ann-Lii
Published: August 2014

Background: Brain metastasis is a formidable challenge in lung cancer treatment. The incidence has been increasing recently because of the advances of treatment which leads to prolonged survival of many patients.

Methods: We established xenograft model of brain metastasis. Repeated rounds of in vivo selection and ex vivo primary cultures of the brain metastases resulted in a cell line with a high propensity to metastasize to the brain. Brain-tropism was confirmed by bioluminescence study. Gene expression profiles between parental lung cancer cells (PC9) and brain-tropic cells (PC9-Br) was compared by Agilent Gene Expression Microarray followed by Ingenuity Pathway Analysis. The biologic effects and clinical implications of the candidate molecules were further investigated.

Results: Significant activation of IGF and WNT signaling pathways was detected in PC9-Br, as compared with the parental cells. We selected IGF pathway as the first step to explore the potential therapeutic implication. Western blot and RT-PCR validated the results of IGF2 overexpression and activated IGF1R (pIGF1R) in PC9-Br cells. In vitro drug sensitivity tests showed that PC9-Br, in comparison to parental cells, was more resistant to erlotinib, an EGFR TKI (IC50: 30nM for PC9 and 189nM for PC9-Br). Knocking down IGF2 expression in PC9-Br reversed the drug resistance, while adding IGF2 to PC9 enhanced the resistance. In clinical samples, there was a significantly higher expression of pIGF1R in 26 brain metastases as compared to their paired primary lung tumors (mean of H score differences=40.96, 95% C.I.=10.96∼70.96, P=0.0094).

Conclusions: IGF signaling pathway is activated in brain-tropic lung cancer cells and could potentially be a therapeutic target for brain metastasis.

Acknowledgement: This study was supported by Research Grant 101-M2002 from the National Taiwan University Hospital.

Citation Format: Pei-Fang Wu, Ching-Hung Lin, Wen-Chang Huang, Wen-Chi Feng, Yen-Shen Lu, Chih-Hsin Yang, Ann-Lii Cheng. Activated insulin-like growth factor (IGF) signaling pathway is a potential therapeutic target for brain metastasis from lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3886. doi:10.1158/1538-7445.AM2013-3886
Abstract 3889: GRM1 over-expression in melanoma cells promotes microvesicle formation which functions in stimulating angiogenesis.
Authors: Koo Jasmine J. , Wen Yu , Li Jiadong , Chen Suzie , Goydos James
Published: August 2014

Our group has reported that metabotropic glutamate receptor 1 (GRM1) as an oncogene and a potential therapeutic target for melanoma. More than 60% of human melanoma expresses GRM1, while normal melanocytes do not. In a Phase-0 trial of riluzole (an oral inhibitor of GRM1) in patients with advanced melanoma we showed suppression of the PI3K/AKT and MAPK pathways in melanoma specimens in 34% of the patients.

Our group found that enhanced expression of GRM1 in a subclone of a melanoma cell line (UACC903) that normally expresses low levels of GRM1 stimulated angiogenesis via activation of the AKT-mTOR-HIF1-IL8, VEGF signaling pathway. Increasing GRM1 expression promoted membrane blebbing (a phenomenon that appears to lead to microvesicle formation) and riluzole treatment inhibited this effect. Suppression of GRM1 expression using an inducible siRNA construct inhibits the blebbing phenomena and microvesicle formation. Therefore, we hypothesize that GRM1 expression promotes microvesicle formation that may be involved in stimulating angiogenesis and tumor progression. Through electron-microscope and flow cytometry analysis, we have confirmed that increased GRM1 expression enhanced microvesicle secretion into the medium in cell culture and that knocking down GRM1 expression significantly reduced the quantity of microvesicle secretion. Furthermore, intra-tumor injection of collected microvesicles into two different melanoma xenograft tumors (UACC903 and C8161) demonstrated that the microvesicles formed from high GRM1 expressing cells promoted tumor growth and angiogenesis. We also showed that microvesicles from high GRM1 expressing cells increased endothelial cell proliferation exhibited measured using a BrdU incorporation assay.

Our preliminary data demonstrate that GRM1 expression promotes microvesicle formation and that these microvesicles are associated with angiogenesis. Research on the molecular events associated with the phenomenon is ongoing.

Citation Format: Jasmine J. Koo, Yu Wen, Jiadong Li, Suzie Chen, James Goydos. GRM1 over-expression in melanoma cells promotes microvesicle formation which functions in stimulating angiogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3889. doi:10.1158/1538-7445.AM2013-3889
Abstract 3892: BATF3 is a novel regulator of breast cancer cell invasion.
Authors: Stein Elliot , Jia Di , Moses Marsha
Published: August 2014

The escape from tumor dormancy has been shown to be mediated by the acquisition of the angiogenic phenotype. Utilizing an in vivo model that reliably recapitulates the transition from the preangiogenic to the angiogenic phenotype, we have identified and validated the differential expression of a number of genes including a basic leucine zipper transcription factor, ATF-like-3 (BATF3). We further found that the mRNA level of BATF3 is significantly upregulated in angiogenic tumor lesions compared to preangiogenic lesions. In order to investigate the role of BATF3 in breast cancer neovascularization, we established a panel of ER+ and ER- breast cancer cell lines, MCF7 and MDA-MB-231, respectively, which stably overexpress BATF3 by lentiviral transduction. Cells overexpressing BATF3 exhibited significantly increased levels of VEGF (vascular endothelial growth factor) compared to their wild type counterparts. Overexpression of BATF3 also significantly increased cell invasion in both cell lines. Moreover, MMP-9 (Matrix Metalloproteinase-9) levels and activity were significantly increased in MDA-MB-231-BATF3 cells. This latter observation suggested that the increased invasiveness observed might be due to elevated levels of MMP-9, which has been shown to regulate tumor cell invasion by remodeling the surrounding extracellular matrix. TIMP-1, an endogenous inhibitor of MMP-9 activity, was also significantly increased in MDA-MB-231-BATF3 cells, however the ratio of MMP-9 to TIMP-1 is dramatically higher in these cells as compared with control cells. Interestingly, BATF3 also significantly suppressed cell proliferation in both the MDA-MB-231 and MCF7 cell lines. Taken together, these data support the conclusion that BATF3 may play a dual role in the regulation of tumor angiogenesis, growth and progression. (Supported by NIH P01 CA045548, The Breast Cancer Research Foundation, The Advanced Medical Research Foundation, and the Harvard College Research Program).

Citation Format: Elliot Stein, Di Jia, Marsha Moses. BATF3 is a novel regulator of breast cancer cell invasion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3892. doi:10.1158/1538-7445.AM2013-3892
Abstract 3903: AltaBluTM and Microfil 3D imaging of tumor vasculature.
Authors: Small Daniel , Weinstein David , O'Day Christine , Knitter Glenn , Ouellette Naveen , Pinkos Liane , Vasquez Sergio X. , Shah Neha
Published: August 2014

As angiogenesis inhibitors have proven to be valuable agents in oncology there is a need to measure changes in vasculature for these and other related agents. Many of the angiogenesis inhibitors, though reducing tumor volume by reducing nutrient access to the tumor, do not always show decreased density of vasculature. This paradox, however, may be attributable to the difficulties of measuring the vasculature. One reason for this problem has been the heterogeneity of vessels in the tumor. With the ability to now scan 360o by 3D microCT imaging of the tumor, all image captured vessels can be measured. Another problem is the limitation of vessel size measured by microCT scanning resolution. Vessels smaller than ∼20 microns cannot be distinguished by size at scan parameters of 10 micron voxel resolution. In addition, agents used for imaging have their own limitations. Microfil is a polymer based radiopaque imaging reagent whereas AltaBlu is a proprietary formulation of a non-soluble radiopaque colloid suspension. For reasons not entirely clear, we do not believe Microfil enters capillaries since we have not observed it in the venous side of an arterial infusion. We believe AltaBluTM, can pass into smaller vessels since we have occasionally seen small amounts on the venous side, but these smaller vessels cannot be adequately measured in diameter by microCT scanning. We hypothesized that even though we may not be able to see smaller vessels by AltaBluTM, we might be able to see more of the smaller vessels due to improved reagent penetration and would therefore be more sensitive to changes in small vessel populations. Herein, we have compared perfusion of tumors via AltaBluTM and Microfil to determine the benefits/detractions of the various agents and used a variety of measuring parameters such as vessel radius, average vessel length, vessel density, tortuosity, and vessel branching factors in AltaViewerTM software to determine the sensitivity of each agent to vascular imaging.

Citation Format: Daniel Small, David Weinstein, Christine O'Day, Glenn Knitter, Naveen Ouellette, Liane Pinkos, Sergio X. Vasquez, Neha Shah. AltaBluTM and Microfil 3D imaging of tumor vasculature. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3903. doi:10.1158/1538-7445.AM2013-3903