bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1424: Enhanced pyruvate carboxylation is crucial to non-small cell lung cancer proliferation and anabolism
Authors: Sellers Katherine E. , Fox Matthew P. , Bousamra Michael , Yan Jun , Yuneva Mariia , Higashi Richard M. , Lane Andrew N. , Fan Teresa W.
Published: May 2015

Proliferating cancer cells require an active Krebs cycle for generating anabolic precursors, in addition to energy production. Diversion of the Krebs cycle intermediates to meet anabolic demands cannot be sustained without anaplerosis. The two major anaplerotic pathways are the deamidation of glutamine to form glutamate catalyzed by glutaminase (GLS) or glutaminolysis, and the carboxylation of pyruvate to oxaloacetate by pyruvate carboxylase (PC).

We determined the expression of PC and GLS proteins in fresh tumor tissue and paired adjacent benign tissue from 86 human NSCLC patients. PC was elevated (median 8-10 fold) in 94% of the tumor tissues, whereas GLS expression did not differ significantly between tumor and paired non tumorous lung tissue. Using 13C6 glucose as tracer and stable isotope-resolved metabolomics (SIRM), we also observed elevated PC activity in vivo in human patients with early stage NSCLC. To examine the importance of PC in the growth and survival of NSCLC, 5 NSCLC cell lines were transduced with a lentiviral vector containing an shRNA against PC. PC knockdown slowed proliferation, induced multinucleation, and reduced colony formation in soft agar. To validate attenuated PC activity and to profile the metabolic effect of PC knockdown, A549 cells were grown in 13C6 glucose or 13C5 glutamine and the incorporation of 13C into various metabolic pathways was measured by NMR and MS by SIRM. We found reduced entry of both glucose and glutamine carbon into the Krebs cycle metabolites, fatty acyl chains of lipids, and nucleotides, suggesting that both energy production and anabolic pathways were hindered and blocking the PC pathway was not compensated by GLS activity. In addition, glutathione biosynthesis and homeostasis were perturbed by PC suppression, leading to compromised anti-oxidation capacity. We further found that PC knockdown in A549 cells reduced their growth rate in mouse xenografts, and recapitulate the metabolic perturbations seen both in cell culture and in human patients. Together, these results suggest that PC is indispensible for the growth and anabolism of NSCLC.

This work was funded by 5P20RR018733, 1R01CA118434-01A2, 1P01CA163223-01A1, 1R01ES022191-01, and 3R01CA118434-02S1; and the KLCRP, CTSPGP, and the KY Challenge for Excellence.

Citation Format: Katherine E. Sellers, Matthew P. Fox, Michael Bousamra, Jun Yan, Mariia Yuneva, Richard M. Higashi, Andrew N. Lane, Teresa WM Fan. Enhanced pyruvate carboxylation is crucial to non-small cell lung cancer proliferation and anabolism. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1424. doi:10.1158/1538-7445.AM2014-1424
Abstract 1431: Gene expression analysis of argininosuccinate synthetase loss and the effects of pegylated arginine deiminase in malignant pleural mesothelioma
Authors: Cutts Rosalind , Jithesh Puthen V. , Delage Barbara , Luong Phuong , Thomas Gareth , Chelala Claude , Szlosarek Peter W.
Published: May 2015

Malignant pleural mesothelioma (MPM) is a devastating asbestos-related malignancy that is increasing in many countries worldwide with few systemic treatment options beyond platinum and antifolate chemotherapy. Deficiency of the arginine biosynthetic enzyme argininosuccinate synthetase (ASS1) occurs in up to 50% of MPM cell lines and primary tumors and is being validated as a biomarker in patients treated with the arginine-depleting agent, pegylated arginine deiminase (ADI-PEG20). To understand the role of ASS1 loss and the effect of the ADI-PEG20 in MPM we used pathway analysis tools on microarray gene expression data from a representative panel of MPM cell lines. First, we identified that ASS1 loss was linked to several protumorigenic functions including increased cell invasiveness and migration, which were confirmed subsequently using invasion assays and organotypic modelling, respectively. We also detected a large number of enriched pathways connected to the immune response including communication between innate and adaptive immune cells and interferon signalling. Furthermore, ASS1 deficiency was linked to worse outcome with a median survival of 6 months in ASS1 low expressors compared to 12 months for ASS1 high expressors using a retrospective dataset (n=41; p=0.003). Second, ADI-PEG20 treatment modulated numerous pathways in ASS1-deficient cells including suppression of mTOR and folate metabolism, while promoting stress, oxidant and amino acid signalling. Third, bioinformatics analyses of drug interactions using Connectivity map revealed that arginine deprivation using ADI-PEG20 may potentiate several chemotherapeutic and targeted agents in the clinic. Taken together, our bioinformatics approach links loss of ASS1 in MPM cells to a more aggressive phenotype and identifies several potential combination strategies with ADI-PEG20 for further clinical investigation.

Citation Format: Rosalind Cutts, Puthen V. Jithesh, Barbara Delage, Phuong Luong, Gareth Thomas, Claude Chelala, Peter W. Szlosarek. Gene expression analysis of argininosuccinate synthetase loss and the effects of pegylated arginine deiminase in malignant pleural mesothelioma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1431. doi:10.1158/1538-7445.AM2014-1431
Abstract 1445: Thyroid hormone receptor represses microRNA-130b to enhance cell metastasis
Authors: Lin Yang Hsiang , Wu Meng-Han , Yeh Yung-Hsin , Chao Angel , Lin Kwang-Huei
Published: May 2015

Thyroid hormone (T3) and its receptor (TR) are involved in cell growth and cancer progression. Although deregulation of miRNA expression has been detected in many tumor types, the mechanisms underlying functional impairment and specific involvement of miRNA in tumor metastasis remain unclear. We proposed that deregulation of specific miRNAs and target gene expressions mediated by T3/TR constitute critical steps of cancer progression. In the current study, we demonstrated that T3/TR negatively regulates microRNA-130b (miR-130b) expression, both in vitro and in vivo. Overexpression of miR-130b markedly inhibited cell migration and invasion in vitro and in vivo, which was mediated via suppression of interferon regulatory factor 1 (IRF1). Cell migration ability was promoted by T3, but partially suppressed upon miR-130b overexpression. Furthermore, miR-130b suppressed the expression of epithelial-mesenchymal transition (EMT)-related genes, matrix metalloproteinase (MMP)-9, p-AKT and p-signal transducer and activator of transcription (STAT)-3. Notably, miR-130b was downregulated in hepatoma samples and inversely correlated with TR and IRF1. Our data collectively highlight a novel pathway interlinking T3/TR, miR-130b, EMT-related genes, MMP9, p-AKT and p-STAT3 cascade that regulates the motility and invasion of hepatoma cells. Thus, miR-130b activation may be a useful strategy for cancer treatment in metastatic HCC.

Citation Format: Yang Hsiang Lin, Meng-Han Wu, Yung-Hsin Yeh, Angel Chao, Kwang-Huei Lin. Thyroid hormone receptor represses microRNA-130b to enhance cell metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1445. doi:10.1158/1538-7445.AM2014-1445
Abstract 1452: MicroRNA-375 impairs head and neck squamous cell carcinoma invasion by suppressing invadopodia activity
Authors: Jimenez Lizandra , Sharma Ved P. , Lim Jihyeon , Angeletti Ruth , Condeelis John , Harris Thomas , Prystowsky Michael B. , Childs Geoffrey , Segall Jeffrey E.
Published: May 2015

Head and neck squamous cell carcinoma (HNSCC) is a highly invasive cancer, with a five-year survival rate of around 50%. Our goal is to improve patient outcome through understanding the mechanisms determining HNSCC invasiveness. Our research group previously reported that miR-375 expression levels correlate with patient survival, recurrence and distant metastasis. We have previously observed diminished HNSCC invasion in vitro correlates with increased miR-375 expression. To assess whether the miR-375 over-expressing transductant lines have reduced matrix degradation properties, a fluorescent matrix degradation assay was used. We have observed that miR-375 over-expression in HNSCC cells suppresses extracellular matrix degradation. We further determined whether miR-375 over-expressing cells show reduced invadopodium formation and maturation. We observed miR-375 expression in HNSCC cell lines reduces the number of mature invadopodia, but does not affect levels of invadopodium precursors. We examined the phosphorylation and total levels of key invadopodia components (Tks5 and cortactin) to determine whether changes in regulation or expression of these components may lead impaired matrix degradation properties. We determined that miR-375 expression in HNSCC cell lines does not reduce cellular levels of cortactin and Tks5, nor is tyrosine phosphorylation of cortactin altered. To identify candidate proteins regulated by miR-375, we are utilizing stable isotope labeling of cells in cell culture (SILAC). Candidate target proteins are being validated as targets of miR-375 and tested for involvement in the diminished invasive properties of HNSCC as a result of miR-375 expression. In conclusion, increased miR-375 expression may suppress the invasive properties of HNSCC through diminished invadopodia activity.

Citation Format: Lizandra Jimenez, Ved P. Sharma, Jihyeon Lim, Ruth Angeletti, John Condeelis, Thomas Harris, Michael B. Prystowsky, Geoffrey Childs, Jeffrey E. Segall. MicroRNA-375 impairs head and neck squamous cell carcinoma invasion by suppressing invadopodia activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1452. doi:10.1158/1538-7445.AM2014-1452
Abstract 1454: MiR-517a mediates cisplatin sensitivity in bladder cell carcinoma
Authors: Ibrahim Ahmed , Chakravarti Arnab , Lautenschlaeger Tim
Published: May 2015

Cisplatin-containing neoadjuvant chemotherapy is the standard of care treatment for patients with muscle-invasive bladder cancer. We sought out to assess if miR-517a modulates sensitivity to cisplatin in bladder cancer cell lines. We used four transitional cell carcinoma cell lines T24, J82, UM-UC3 and TCCSUP. Cells were transfected with miR-517a mimic or inhibitor using lipofectaime according to manufacturer's instruction. Mtt assay was performed to assess viability after 96 hrs and clonogenic survival assays were performed to assess colony forming ability of cell lines.

We found that miR-517a reduced proliferation and increased cisplatin sensitivity. Overexpression of miR-517a resulted in reduction of proliferation by 53%, 45%, 62% and 67% respectively (t-test p<0.05 for comparison with respective control cell lines). Transfection with miR-517a followed by cisplatin treatment resulted in a dramatic reduction in viability and colony forming ability of cells compared to cisplatin treatment alone. This reduction in viability was not observed after transfection with the miR-517a inhibitor. To our knowledge, this is the first report indicating that miR-517a functions as a sensitizer to cisplatin therapy in bladder cancer. Bioinformatics and microRNA target prediction databases identified several candidate genes.

Note: This abstract was not presented at the meeting.

Citation Format: Ahmed Ibrahim, Arnab Chakravarti, Tim Lautenschlaeger. MiR-517a mediates cisplatin sensitivity in bladder cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1454. doi:10.1158/1538-7445.AM2014-1454
Abstract 1469: Functional roles of SCAL1 expression in lung cancer and progression
Authors: Statt Sarah , Thai Philip , Chen Ching-Hsien , Cheng Ya-Wen , Belinsky Steven , Wu Reen
Published: May 2015

Our group has identified a novel long non-coding RNA (lncRNA), SCAL1 (smoke and cancer-associated lncRNA 1), whose expression is associated with smoking and related to cancer malignancy (Thai et al., 2013, AJRCMB). Currently, we have found that SCAL1 expression is necessary in lung epithelial cells during injury in order to initiate repair after exposure to smoke. In lung cancer cells, SCAL1 expression is needed for cancer cells to combat DNA double stranded breaks associated with chemotherapy treatment, especially cisplatin, doxyrubcin, and possibly other DNA damaging reagents. Most interestingly, the induction of SCAL1 expression is regulated by NRF-2. While NRF2 may protect normal cells from injury, it becomes persistently activated in some cancers and may enhance their malignant characteristics. Therefore, SCAL1 expression is an early marker for lung cellular injury and may be involved in lung cancer progression. We believe that smoke-induced SCAL1 expression leads to epithelial repair and enhances cancer cell resistance to chemotherapy. Our hypothesis is that SCAL1 expression in lung cancer is associated with increased malignant potential and is a poor prognostic marker.

Citation Format: Sarah Statt, Philip Thai, Ching-Hsien Chen, Ya-Wen Cheng, Steven Belinsky, Reen Wu. Functional roles of SCAL1 expression in lung cancer and progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1469. doi:10.1158/1538-7445.AM2014-1469
Abstract 1497: A novel long non-coding RNA associated with poor prognosis in epithelial ovarian cancer
Authors: Lawrenson Kate , Spindler Tassja , Gayther Simon A.
Published: May 2015

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy in the Western world. In the US, there are ∼22,000 new diagnoses and ∼15,500 deaths from the disease each year. Identifying novel biomarkers associated with disease outcome, which may represent novel therapeutic targets, is critical to improve survival rates and reduce mortality from the disease. Research in this area has traditionally focused on protein-coding genes, and while this has yielded many insights into tumor biology, prognoses for women diagnosed with EOC have improved very little over the last four decades. However, protein-coding genes make up only ∼2% of the human genome. Recent studies show that the 98% non-coding ‘dark matter’ of the genome contains a myriad of RNA transcripts that are never translated, including long non-coding RNAs (lncRNAs).

The existence and potential functional significance of lncRNAs is a relatively recent discovery, and the lncRNA transcriptome remains poorly characterized for most human diseases. In this study, we used RNA sequencing to profile lncRNAs in ovarian cancer and normal ovarian epithelial cells. We identified 294 significantly lncRNAs that were differentially expressed between the two cell types, the majority of which were novel antisense or intergenic RNAs. One of the most highly overexpressed lncRNAs in EOC was UCA1, which was expressed in cell lines representing the major subtypes of EOC (n=39) but not in normal ovarian or fallopian tube epithelial cells (n=10). We validated UCA1 overexpression in primary EOCs using data from 489 ovarian cancers analyzed by The Cancer Genome Atlas (TCGA) compared to expression in 8 normal fallopian tube tissue specimens (P=4.66x10-8). Overexpression or amplification of UCA1 was associated with a significantly worse prognosis, both overall survival (P=5.60x10-4) and disease-free survival (P=4.79x10-4). Using TCGA data, we also found that UCA1 amplification/overexpression occurred exclusively in patients without either germline or somatic BRCA1 mutations (P=6x10-4), suggesting functional redundancy between UCA1 and a failure in homologous recombination (HR) and defective double-stranded DNA break repair. This was supported by in silico analyses of HR and EOC associated susceptibility genes and the finding that, in contrast to UCA1, EOC patients with mutations in BRCA1 and BRCA2 have improved survival. Finally, functional knockdown of UCA1 in EOC cell lines caused a significant reduction in proliferation and migration in vitro, further suggesting that UCA1 plays and important role in EOC development.

Citation Format: Kate Lawrenson, Tassja Spindler, Simon A. Gayther. A novel long non-coding RNA associated with poor prognosis in epithelial ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1497. doi:10.1158/1538-7445.AM2014-1497
Abstract 152: Galectin-7 increases the invasive behavior of ovarian cancer cells and promotes tumor escape
Authors: Labrie Marilyne , Vladoiu Maria Claudia , Grosset Andrée-Anne , Gaboury Louis , St-Pierre Yves
Published: May 2015

Ovarian cancer is the fifth leading cause of cancer-related deaths in the Western world, the second most common gynecological cancer and the leading cause of death from gynecological malignancies. Epithelial ovarian cancer (EOC) accounts for the majority of ovarian cancers. Here, we have investigated the role of galectin-7 in EOC. Galectin-7 is an epithelial cell marker which has been shown to play an important role in the progression of many types of cancer, including breast cancer and colon cancer. Galectins play an important role in several physiological processes, including cell migration, programmed cell death and regulation of the immune response. They are also involved in a number of pathological conditions, including cancer. In fact, galectins have been shown to create an immunosuppressive tumor microenvironment by killing helper and effector T cells. They can also bind to the surface of tumor cells to form cell surface lattice that stabilizes the expression of key growth factor receptors, thereby increasing cell growth, motility, and production of matrix metalloproteinases. Using tissue microarrays, we found that gal-7 expression increases in malignant tumors compared to normal tissues and benign tumors. Detection of galectin-7 was also more frequent in tissue samples with metastasis as compared to non-metastatic primary tumor samples. Western blot analysis showed that OVCAR-3 EOC cells secrete gal-7, while no expression was detected in COV-434, A2780 and SK-OV-3 cell lines. Treatment of EOC cells with recombinant gal-7 increased their invasive behavior through matrigel and their ability to express MMP-9, an extracellular protease which confers an invasive phenotype to cancer cells. Treatment of freshly isolated human PBMCs with gal-7 also showed gal-7 increased apoptosis of monocytes, T CD4+ and T CD8+ cells. Overall, these data provide impetus for further studies to determine the potential of gal-7 as a therapeutic target for the treatment of EOC.

Citation Format: Marilyne Labrie, Maria Claudia Vladoiu, Andrée-Anne Grosset, Louis Gaboury, Yves St-Pierre. Galectin-7 increases the invasive behavior of ovarian cancer cells and promotes tumor escape. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 152. doi:10.1158/1538-7445.AM2014-152
Abstract 1571: The tumor suppressor Liver Kinase B1 inhibits triple-negative breast cancer cell metastasis via regulation of AP-1 signaling
Authors: Rhodes Lyndsay V. , Tate Chandra R. , Burks Hope E. , Hoang Van T. , Gilliam Diari , Martin Elizabeth C. , Elliott Steven , Miller David F. , Buechlein Aaron , Rusch Douglas , Tang Haixu , Nephew Kenneth P. , Burow Matthew E. , Collins-Burow Bridgette M.
Published: May 2015

The basal sub-type, which shares features with triple-negative breast cancer (TNBC), is among the most lethal breast cancer subtype, characterized by a highly aggressive and metastatic phenotype. Although pathways that may represent targets for novel therapeutic intervention for basal like breast cancer (BLBC) have begun to be elucidated, the ability to define and selectively target the invasive and metastatic phenotype of basal-type/TNBC remains a major challenge facing the breast cancer field.

Liver kinase B1 (LKB1), also known as serine/threonine kinase 11 (STK11), is a known tumor suppressor in many cancers including breast. Low LKB1expression has been observed in breast cancer patients and we report a significant association between loss of LKB1 expression and poor prognosis specifically in the basal sub-type of breast cancer. Induction of LKB1 expression in BLBC cell lines inhibited invasiveness in vitro as well as lung and brain metastatic burden in an orthotopic xenograft tumor model. Further analysis of BLBC cell lines overexpressing LKB1 by next generation sequencing (RNA-seq) revealed striking regulation of metastasis-associated pathways, including cell adhesion, extra cellular matrix remodeling, and epithelial-to-mesenchymal transition (EMT). We further demonstrated marked inhibition of matrix metalloproteinase 1 (MMP-1) expression and activity via regulation AP-1 family member cJun. Additionally, LKB1 overexpression inhibited EMT-associated genes (CDH2, Vimentin, Twist) and induced the epithelial cell marker CDH1, indicating a reversal of the EMT phenotype in a triple-negative breast cancer cell line MDA-MB-231. We have demonstrated a role for LKB1 expression in the regulation of cell invasion and metastasis in addition to tumorigenesis. Taken together these data support future development of therapeutic agents to induce the LKB1 signaling pathway in BLCB/triple-negative breast cancer.

Citation Format: Lyndsay V. Rhodes, Chandra R. Tate, Hope E. Burks, Van T. Hoang, Diari Gilliam, Elizabeth C. Martin, Steven Elliott, David FB Miller, Aaron Buechlein, Douglas Rusch, Haixu Tang, Kenneth P. Nephew, Matthew E. Burow, Bridgette M. Collins-Burow. The tumor suppressor Liver Kinase B1 inhibits triple-negative breast cancer cell metastasis via regulation of AP-1 signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1571. doi:10.1158/1538-7445.AM2014-1571
Abstract 1949: P38MAPK plays a tumor suppressive role through differentiation of ALDHhigh/CD44high tumor iniating cells in primary human lung adenocarcinoma
Authors: Liu Jing , Leung Lai-han , Xiao Zhi-jie , Tin Vicky P. , Wong Maria Pik
Published: May 2015

The p38 MAPK cascade regulates body responses to a wide range of stress stimuli in a context-dependent and tissue-specific manner. The isoform p38alpha (p38) mediates both tumor suppressive and tumor-supportive functions in different cancer models. However, its role in lung cancer is not clear. Since EGFR is frequently mutated in lung adenocarcinomas in non-smoking women and p38 is a downstream target of EGFR signaling, it is possible that p38 plays a tumor-supportive role. Using immunohistochemistry to analyze nuclear activated p38 expression in 90 lung adenocarcinomas, we observed p38 expression was significantly associated with well differentiated compared to poorly-differentiated carcinomas (p<0.001) and better patient outcome (p=0.019, disease-free survival; p=0.03, overall survival). In vitro p38 suppression in lung cancer cell lines did not lead to enhanced cell death. On the other hand, p38 suppression alone or in combination with EGFR inhibition led to increased CD44high/ALDHhigh lung tumor initiating cells (TIC); isolated lung TIC also showed higher p38 expression. Together, the data implicate p38 plays a tumor suppressive role in lung cancer by driving tumor cell differentiation. Disrupting pathways that inhibit p38 expression or enhancement of p38 expression might be useful for lung cancer treatment.

Citation Format: Jing Liu, Lai-han Leung, Zhi-jie Xiao, Vicky PC Tin, Maria Pik Wong. P38MAPK plays a tumor suppressive role through differentiation of ALDHhigh/CD44high tumor iniating cells in primary human lung adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1949. doi:10.1158/1538-7445.AM2014-1949
Abstract 1958: Interrogating hedgehog pathway and smoothened inhibition by PF-04449913 in patient-derived acute myeloid leukemia models
Authors: Jackson-Fisher Amy , Whalen Pamela , Elliott Mark , McMahon Melissa , Chen Enhong , Zheng Xianxian , Ozeck Mark , Huang Donghui , Lira Paul , Lee Joseph , Zhang Cathy , Lam Justine , Spilker Mary , Deng Shibing , Lappin Patrick , Venne Penny , Heinlein Cynthia , Schairer Annelie , McLachlan Karen , VanArsdale Todd
Published: May 2015

Aberrant activation of the Hedgehog (Hh) signaling pathway has been implicated in a variety of cancers and a small molecule inhibitor of Smoothened (SMO), Vismodegib, has been approved for the treatment of basal cell carcinoma, a disease frequently driven by Hh pathway signaling due to pathway mutations. SMO dependent Hh signaling has also been implicated in models of myeloid leukemia, primarily CML, where genetic loss of SMO or pharmacological inhibition limits disease progression in part through targeting the leukemic stem cell (LSC). Outside of BCR-ABL driven leukemia the role of Hh signaling and impact of SMO inhibition on disease progression and the LSC remains unclear. To explore the role of Hedgehog pathway signaling and interrogate responses to SMO inhibition in AML, a panel of primary AML patient-derived models was utilized to examine responses to PF-04449913, an oral small molecule SMO inhibitor currently in early phase clinical trials targeting myeloid malignancies. AML patient samples were characterized for Hh pathway expression levels and screened for responses to PF-04449913. Ex vivo treatment of AML bone marrow cells showed PF-04449913 was capable of inhibiting Hh pathway activity, reducing expression of key LSC regulators and decreasing populations of cells expressing LSC markers. Use of AML xenotransplant models to assess in vivo responses to PF-04449913 as single agent and in combination with Cytarabine have shown potential for combination efficacy of the two agents in select models suggesting patient selection strategies may be critical for SMO inhibitor-based therapies in AML.

Citation Format: Amy Jackson-Fisher, Pamela Whalen, Mark Elliott, Melissa McMahon, Enhong Chen, Xianxian Zheng, Mark Ozeck, Donghui Huang, Paul Lira, Joseph Lee, Cathy Zhang, Justine Lam, Mary Spilker, Shibing Deng, Patrick Lappin, Penny Venne, Cynthia Heinlein, Annelie Schairer, Karen McLachlan, Todd VanArsdale. Interrogating hedgehog pathway and smoothened inhibition by PF-04449913 in patient-derived acute myeloid leukemia models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1958. doi:10.1158/1538-7445.AM2014-1958
Abstract 1985: Vitronectin expression in primary lung cancers
Authors: Salazar-Pelaez Lina M. , Martinez-Forero Ivan
Published: May 2015

Rationale

Vitronectin (Vn) plays a role in extracellular matrix (ECM) remodeling during tumorigenesis. Vn is present in human bronchial submucosal glands and it is secreted by A549 lung adenocarcinoma cell line. However, whether Vn is differentially expressed in tissues of primary lung cancer has not been explored.

Methods

Lung tissue from 22 primary lung carcinoma and 36 non-lung cancer subjects were obtained from fibrobronchoscopy. Sections were analyzed by histo- immunohistochemical methods. The total area occupied by ECM, surface and gland epithelium, as well as the percentage area occupied by Vn at each of these localizations were measured. Chi-square, t-student, U-Mann Whitney and ANOVA tests were used in group comparisons. Statistical significance was tested at P<0.05.

Results

Vn expression was observed in bronchial glandular and surface epithelium, as well as in ECM . In total, the average area of ECM, surface or glandular epithelium was 0.289mm2 (± 0.032), 0.043mm2 (±0.009), 0.084mm2 (±0.031), respectively. The percentage area occupied by Vn in ECM, surface or glandular epithelium, was 4.899 (±1.186), 7.279 (±1.623), 3.285 (±1.038), respectively; with not significant statistical differences between lung cancer and non-cancer subjects or within different types of primary lung cancer (Table 1).

Conclusion

There were no statistically significant differences in Vn expression between lung cancer and non-cancer subjects, or within primary lung carcinomas.

Demographic characteristics and measurement comparisons of subjects studiedConditionNon-cancer(n=36)Cancer(n=22)PAge (Years)59.810(+/- 13.556)65.790(+/- 10.648)0.074Sex*0.752Male18 (50.0%)11 (50.0%)Female18 (50.0%)11 (50.0%)ECM area (mm2)0.311(+/- 0.204)0.424(+/- 0.246)0.059Area occupied by Vn in ECM (percentage)2.894(+/- 2.717)3.584(+/- 3.220)0.384Surface epithelium area (mm2)0.221(+/- 0.678)0.052(+/- 0.023)0.398Area occupied by Vn in surface epithelium (percentage)5.583(+/- 3.939)6.478(+/- 7.757)0.662Gland epithelium area (mm2)0.142(+/- 0.129)0.086(+/- 0.079)0.389Area occupied by Vn in glandular epithelium (percentage)4.151(+/- 2.810)1.424(+/- 0.505)0.200

Note: This abstract was not presented at the meeting.

Citation Format: Lina M. Salazar-Pelaez, Ivan Martinez-Forero. Vitronectin expression in primary lung cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1985. doi:10.1158/1538-7445.AM2014-1985
Abstract 1988: MenaINV interaction with α5β1 promotes tumor cell invasion in response to gradients of growth factors and fibronectin
Authors: Oudin Madeleine J. , Broye Liliane C. , Lussiez Alisha , Asokan Sreeja B. , Miller Miles A. , Lauffenburger Douglas A. , Bear James E. , Gertler Frank B.
Published: May 2015

To metastasize and disseminate, carcinoma cells must invade the surrounding extracellular matrix (ECM) and migrate to reach blood or lymphatic vessels. Collagen (CN) and fibronectin (FN), two components of the ECM surrounding tumors, are highly expressed in metastatic tumors and their patterns of expression can correlate with metastatic outcome in patient samples. Mena, an actin regulatory protein, is upregulated in breast cancer and is alternatively spliced to produce protein isoforms with distinct functions during tumor progression. The invasion-specific MenaINV isoform increases metastasis by potentiating tumor cell motility and invasion responses to various growth factors. All Mena isoforms bind directly to the α5 subunit of the α5β1 integrin, an FN receptor. We hypothesized that the interaction between MenaINV and α5β1 plays an important role in integrating signals from the ECM as well as growth factors to drive invasion.

MDA-MB-231 breast cancer cells expressing MenaINV showed increased adhesion to FN alone as well as mixture of FN and CN, compared to control cells. MenaINV cells also had an increased number of α5-positive adhesions as well as increased signalling at focal adhesions as measured by phospho-Paxilin, compared to control. In 3D invasion assays, addition of FN to a CN gel drove invasion of MenaINV-expressing cells even in the absence of any growth factor ligand, an effect driven by its interaction with α5 and dependent on signalling via EGFR and Met. Furthermore, addition of FN caused an even greater potentiation of EGF-induced invasion by MenaINV, an effect that was dependent upon its interaction with α5. Next, we examined how MenaINV affects tumor cell responses to a gradient of FN within a 3D collagen gel. Control cells failed to respond to an FN gradient, while cells expressing MenaINV migrated preferentially towards FN and significantly reorganized both the collagen and FN surrounding them. Deletion of the α5 binding site or treatment with an α5 function blocking antibody ablated the effects MenaINV on movement towards a FN gradient as well ECM reorganization.

Overall, these results suggest that some aspects of the pro-metastatic effects of MenaINV involve its ability to bind α5 and regulate bidirectional signaling with FN and growth factors in the tumor microenvironment.

Citation Format: Madeleine J. Oudin, Liliane C. Broye, Alisha Lussiez, Sreeja B. Asokan, Miles A. Miller, Douglas A. Lauffenburger, James E. Bear, Frank B. Gertler. MenaINV interaction with α5β1 promotes tumor cell invasion in response to gradients of growth factors and fibronectin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1988. doi:10.1158/1538-7445.AM2014-1988
Abstract 20: Adiponectin increases VEGF expression and promotes angiogenesis in human chondrosarcoma
Authors: Shih Jhao-Sheng , Tang Chih-Hsin
Published: May 2015

Vascular endothelial growth factor (VEGF) is an angiogenic mediator in tumors and has been implicated in the pathogenesis and progression of cancer. Adiponectin is a protein hormone secreted by adipose tissue. We recently found that adiponectin increased metastasis in human chondrosarcoma. However, the effect of adiponectin on VEGF expression and angiogenesis in human chondrosarcoma cells is mostly unknown. Here we found that adiponectin promoted VEGF expression in human chondrosarcoma and subsequently increased migration and tube formation in endothelial progenitor cells (EPCs). Pretreatment of cells for 30 min with PI3K inhibitors (LY294002 and wortmannine), Akt inhibitor, mTOR inhibitor (Rapamycin) or HIF-1α inhibitor abolished adiponectin-induced VEGF expression, EPC migration and tube formation. In addition, transfection of cells with PI3K, Akt, mTOR, or HIF-1α siRNA also reduced adiponectin-induced VEGF expression, EPC migration and tube formation. Knockdown adiponectin decreased VEGF expression in vitro as well as angiogenesis effects the chick chorioallantoic membrane and matrigel-plug nude mice model in vivo. In addition, using xenograft tumor angiogenesis model, knockdown adiponectin significantly reduced tumor growth and tumor angiogenesis. Finally, we analyzed from chondrosarcoma patients by immunohistochemical staining and found that the expression of adiponectin and VEGF was significantly higher in chondrosarcoma tissue than normal cartilage. Taken together this study showed that adiponectin mediates VEGF expression and angiogenesis of human chondrosarcoma cells. One of the mechanisms underlying adiponectin induced VEGF expression and angiogenesis was activation of PI3K, Akt, mTOR and HIF1-α pathways.

Citation Format: Jhao-Sheng Shih, Chih-Hsin Tang. Adiponectin increases VEGF expression and promotes angiogenesis in human chondrosarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 20. doi:10.1158/1538-7445.AM2014-20
Abstract 2017: Development of a novel 3D tri-culture system in an in vitro non-small cell lung cancer (NSCLC) model
Authors: Amann Arno , Zwierzina Marit , Huber Julia M. , Gamerith Gabriele , Bitsche Mario , Pechriggl Elisabeth J. , Hilbe Wolfgang , Zwierzina Heinz
Published: May 2015

Introduction: Different molecular processes lead to metastatic spread and the occurrence of tumor cell resistance to therapeutic interventions. Among them, the stromal compartment of tumors plays a key role. We describe the development of our 3D co-culture to a novel tri-culture model, using non-small cell lung cancer (NSCLC) cell lines in combination with a lung fibroblast and two different endothelial cell lines in a hanging drop technology.

Methods: 96-well hanging drop microtiter plates (InSphero AG, Zürich, Switzerland) were applied for the production of 3D mono-, co- and tri-cultures including the human lung cancer cell lines A549 or Colo699 alone or in combination with a human lung fibroblast cell line (SV-80) and either the human umbilical vein endothelial cell line (HUVEC) or the human lung microvascular endothelial cell line (HMVEC-L). In addition, to conventional histology (H&E, PAS) tumor fibroblast spheroid aggregation was displayed immunohistochemically (IHC) by protein expression of e-cadherin, vimentin, Ki67, CD31 and α-smooth muscle actin (α-SMA). Viability of each cell compartment of the microtissue was determined by a modified AnnexinV/Propidium Iodide staining for flow cytometry.

Results: Endothelial cells aggregated either in small colonies with Colo699 or as single cells mainly in the stromal compartment of A540 microtissues. Simultaneously an up-regulation of vimentin and a downregulation of E-Cadherin was observed in co- and tri-cultures compared to monocultures. Furthermore, Ki67 expression increased significantly from mono- to co- and tri-cultures in both cancer cell lines, indicating a high metabolic activity. In addition, a morphological alteration of A549 tumor cells resembling “signet ring cells” was observed for the first time in both endothelial tri-cultures with a significantly increased glycoprotein expression demonstrated by a Periodic acid-Schiff (PAS) stain.

Conclusion: We demonstrate that our method is a promising tool for the generation of multicellular tumor microtissues and reflects in vivo conditions closer than traditional 2D cell culture. Furthermore, it represents an appropriate alternative method to investigate endothelial cell interactions with tumor and stromal cells.

Citation Format: Arno Amann, Marit Zwierzina, Julia M. Huber, Gabriele Gamerith, Mario Bitsche, Elisabeth J. Pechriggl, Wolfgang Hilbe, Heinz Zwierzina. Development of a novel 3D tri-culture system in an in vitro non-small cell lung cancer (NSCLC) model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2017. doi:10.1158/1538-7445.AM2014-2017
Abstract 2033: A comprehensive 3D triple coculture model for evaluating breast cancer progression
Authors: Benton Gabriel J. , George Jay , DeGray Gerald , Arnaoutova Irina , Kleinman Hynda K.
Published: May 2015

Current models for evaluating breast cancer progression lack a comprehensive, physiological approach to modeling the complex tumor microenvironment. There has been much evidence supporting the use of tumor spheroids to mimic tumor physiology; they exhibit cell-cell bond formation, comparable morphology, elevated cell survival and proliferation in the outer cell layers, reduced proliferation rates in the intermediate layers, and a hypoxic core. While the tumor spheroid provides a physiological tumor model, other cell types within the surrounding tumor microenvironment are essential for tumor behavior and subsequent cancer progression. The tissue vasculature provides a critical component for tumor progression given the metabolic requirements of a growing tumor, and by incorporating endothelial tubules with the tumor spheroid, we are able to model interactions between vascular networks and growing tumors. At the same time, there is also an important stromal component involved in cancer progression where stromal cells have been shown to promote cell proliferation, dissemination, and drug resistance during cancer development. By using extracellular matrix proteins, we are able to promote the proper physiology for each of these cell types in triple cell coculture and compartmentalize their activities. To evaluate interactions between each cell type, they are fluorescently labeled with fluorophores with different excitation and emission spectra; MCF7 and MDA-MB-231 human breast cancer cell lines express a red fluorescence protein, while human umbilical vein endothelial cells (HUVECs) and human adipose-derived mesenchymal stem cells (hMSCs) are labeled with stable lipophilic membrane dyes. Tumor spheroids are formed using low adhesion microwells and deposited into fully formed HUVEC tubular networks, and the hMSCs are then added within a hydrogel overlay matrix. Cellular interactions and dissemination are monitored via fluorescence microscopy and cell proliferation of the breast cancer cells is monitored using a fluorescence plate reader. The breast cancer spheroids exhibit cell-cell interactions with endothelial tubules and stromal cells, and there is an increase in cell proliferation and invasion over standard spheroid monoculture model.

Citation Format: Gabriel J. Benton, Jay George, Gerald DeGray, Irina Arnaoutova, Hynda K. Kleinman. A comprehensive 3D triple coculture model for evaluating breast cancer progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2033. doi:10.1158/1538-7445.AM2014-2033
Abstract 2041: Development and analysis of mouse brain tumor models derived from neural stem cells expressing activated ALK
Authors: Onishi Nobuyuki , Sampetrean Oltea , Sugihara Eiji , Saya Hideyuki
Published: May 2015

Neural Stem Cells (NSCs), having self-renewal and multipotent ability, are considered one of the cells-of-origin of glioblastoma maltifome (GBM). Although NSCs can be enriched in neurosphere floating culture with serum-free media containing EGF/FGF as a classical method, neurosphere is composed of not only NSCs but also differentiated and apoptotic cells. Therefore, we utilize the method for efficient derivation of NSCs with long-term self-renewal and differentiate capacity in adherent culture. Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase first identified in a chromosomal translocation associated with anaplastic large cell lymphomas. Subsequently, new ALK translocations were found in a fraction of non-small-cell lung cancers and in other solid tumors. The function of full-length ALK is involved in neuronal cell differentiation, regeneration and synapse formation. Recently, gene amplification and mutations of full-length ALK were identified in neuroblastoma. Furthermore, it is reported that ALK and PTN, ligand of ALK, are required for maintenance of the stem cell population in GBM. Although constitutive activation of ALK signaling results in cell transformation, little is known about the tumorigenic mechanisms induced by activated ALK. We reasoned that ALK activity has a crucial role in GBM. To verify this hypothesis, human GBM stem cell lines were treated with crizotinib, ALK inhibitor. Crizotinib could suppress the proliferation of human GBM stem cell lines in dose-dependent-manner. Next, we have established a stable mouse model of brain tumor transplanting the genetically modified NSCs. Active mutant of H-RAS could transform the Ink4a/Arf KO NSCs but not WT NSCs. However, transplanting of WT NSCs transduced activated ALK could rapidly formed highly proliferative and invasive brain tumors. Histological characteristics of these tumors resembled human GBM phenotype demonstrating necrosis, perivascular cuffing and giant cell formation. Although the activated H-RAS increased expression of Ink4a in WT NSCs, but the activated ALK never changed. On the basis of these findings, we propose a specific regulation against tumor suppressor genes by activated ALK.

Citation Format: Nobuyuki Onishi, Oltea Sampetrean, Eiji Sugihara, Hideyuki Saya. Development and analysis of mouse brain tumor models derived from neural stem cells expressing activated ALK. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2041. doi:10.1158/1538-7445.AM2014-2041
Abstract 2084: RhoGDI2 is associated with HGF-mediated tumor invasion through VEGF in stomach cancer
Authors: Koh Sung A. , Lee Kyung Hee , Choi Eun Young , Kim Min Kyoung , Jang Byung Ik , Lee Si Hyung , Kim Kyeong Ok , Kim Kook Hyun , Kim Jae Ryong , Kim Sang Woon , Kim Se Won
Published: May 2015

RhoGDP dissociation inhibitor 2(RhoGDI2) has been identified as a regulator of tumor metastasis; however, its role in cancer remains controversial. The aims of this study were to analyze RhoGDI2 in gastric cancer growth and metastasis, and to determine its possible signaling pathway.The level of expression of RhoGDI2 was further confirmed by real time RT-RCR and Western blot analysis. Transfection of cells with RhoGDI2 shRNA resulted in no effects of cell proliferation, as determined with MTT assays. In an in vitro invasion assay, significantly fewer cells transfected with RhoGDI2 shRNA,compared with control cells, were able to invade across a Matrigel membrane barrier. The role of RhoGDI2 in the level of HGF-induced up-regulation of VEGF was measured by knockdown of RhoGDI2 with RhoGDI2 shRNA and a chromatic immuno-precipitation assay.The levels of RhoGDI2 and VEGF were up-regulated in cells treated with HGF in a dose-dependent manner. HGF-induced up-regulation of VEGF was repressed by RhoGDI2 knockdown. HGF-induced upregulation of phosphorylated ERK and P38 levels was inhibited in RhoGDI2 knockdown cells. HGF enhanced the binding activity of RhoGDI2 to the VEGF promoter in control cells, but not in RhoGDI2-shRNA cells. Findings of this study also showed a statistically significant difference in the mean RhoGDI2 level before and after surgery (p<0.01) and the mean level of RhoGDI2 before surgery showed a statistically significant difference depending on lymphatic, neural invasion and stage (p<0.05).

In conclusion, RhoGDI2 might play an important role in up-regulation of VEGF induced by HGF and contributes to HGF-mediated tumor invasion and metastasis, which may serve as a promising target for gastric cancer therapy.

Note: This abstract was not presented at the meeting.

Citation Format: Sung Ae Koh, Kyung Hee Lee, Eun Young Choi, Min Kyoung Kim, Byung Ik Jang, Si Hyung Lee, Kyeong Ok Kim, Kook Hyun Kim, Jae Ryong Kim, Sang Woon Kim, Se Won Kim. RhoGDI2 is associated with HGF-mediated tumor invasion through VEGF in stomach cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2084. doi:10.1158/1538-7445.AM2014-2084