bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 41: DCA bind to EGFR/EGFRvIII/PDK1 and affect the proliferation and growth in TMZ resistant glioblastoma model
Authors: Velpula Kiran Kumar , Sahu Kamlesh , Tuszynski Jack , Guda Maheedhara R. , Asuthkar Swapna , Martin Sarah E. , Lathia Justin D. , Tsung Andrew J.
Published: July 2016

Glioblastomas are characterized by amplification of epidermal growth factor receptor (EGFR).

Approximately half of the GBM tumors with EGFR over-expression also express a constitutively active ligand independent EGFR variant III (EGFRvIII). This phenotype represents a tumor specific target correlating with a high growth potential. Despite advances in treatment, whether surgery, chemotherapeutics or molecular targeted therapies, very few have shown promise. Temozolomide (TMZ), as standard of care alkylation therapy, demonstrates improved survival when administered with concomitant radiotherapy. Unfortunately, acquired TMZ chemo-resistance is observed in more than 90% of recurrent GBMs. To understand the mechanisms in the context of TMZ resistance, we generated an in vitro TMZ resistant model by continuous exposure of U373 cells constitutively expressing EGFRvIII (U373vIII) to 150uM TMZ for 6 months (U373vIIIR). Dicholoroacetate (DCA) is a known metabolic inhibitor of pyruvate dehydrogenase kinase 1 (PDK1). Its application to cancer has recently been revived in the realm of metabolic oncology, where reversal of the Warburg effect has resulted in decreased tumor growth. Treatment of U373, U373vIII, and U373vIIIR GBM cell lines with DCA was found to induce cytotoxicity and reduced cell survival across all cell lines. Further, micro array studies conducted on U373vIII or U373vIIIR and their subsequent treatment with DCA revealed that PDK1 is the sole target in TMZ resistant tumors expressing EGFRvIII. Additionally, we demonstrated that 1mM DCA induced mitochondrial membrane potential change as evidenced in JC-1 staining and electron microscopic studies confirming mitochondrial apoptosis. Consistent with our previous findings that EGFR interacts with PDK1, our computational analysis revealed that DCA aligns itself to the binding sites of both EGFR and EGFRvIII with strong binding affinity apart from its binding to PDK1. Clinically, expression of EGFRvIII correlated with PDK1 when compared to EGFR in GBM surgical specimens. Collectively, our studies demonstrate that principles of metabolic oncology and DCA are active in GBM treatment despite resistance providing new insight into the development of alternative treatment in TMZ failure.

Citation Format: Kiran Kumar Velpula, Kamlesh Sahu, Jack Tuszynski, Maheedhara R. Guda, Swapna Asuthkar, Sarah E. Martin, Justin D. Lathia, Andrew J. Tsung. DCA bind to EGFR/EGFRvIII/PDK1 and affect the proliferation and growth in TMZ resistant glioblastoma model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 41.
Abstract 4101: Stromal cell-induced alterations in the response of colorectal cancer cell to demethylating agents
Authors: Das Viswanath , Skolekova Svetlana , Agrawal Khushboo , Gursky Jan , Kučerová Lucia , Hajdúch Marián
Published: July 2016

Epigenetic mechanisms such as DNA hypermethylation, that results in gene silencing, is closely associated with resistance to platinum-based chemotherapy for colorectal cancer (CRC). DNA methyltransferase inhibitors (DNMTi) that reactivate and induce reexpression of silenced gene have the potential to improve the outcome of platinum-based and other therapies in CRC. In fact, non-cytotoxic concentrations of azacitidine, a clinically used DNMTi, has been reported to resensitize platinum-resistant ovarian cells to carboplatin. Hypomethylating agents, such as azacitidine and decitabine have shown significant promise in hematologic malignancies; however, studies on their application to CRC and other solid tumors have yielded ambivalent results potentially due to the complexities of the tumor microenvironment that result from the numerous cell type interactions.

In this study, we analyzed the impact of different tumor-stroma ratios on cancer cell response to azacitidine, decitabine, and two lead compounds identified by in vitro screens in two-dimensional (2D) and three-dimensional (3D) co-cultures of colorectal HCT116 carcinoma demethylation reporter cells and adipose-derived mesenchymal stromal cells (MSCs) isolated from healthy individuals undergoing elective lipoaspiration. Each subject provided informed consent. Activity of hypomethylating drugs was examined in 2D and 3D cultures of different tumor-stroma ratios using confocal microscopy, flow cytometry, and MTT analyses.

Our preliminary flow cytometry results show an altered response of hypomethylating agents in cocultures of HCT116 and MSCs compared to monocultures of HCT116 cells alone. There was an increase in demethylation of HCT116 cells in 3D cultures containing a higher ratio of stromal cells following treatment with low concentration of decitabine. Furthermore, confocal analysis 3D co-cultures at various z-plane heights revealed increased demethylation at low concentrations of all drugs in cultures with high stromal cell numbers. These results indicate that intratumoral stroma may alter the sensitivity of cancer cells to demethylating agents and warrant further studies.

Citation Format: Viswanath Das, Svetlana Skolekova, Khushboo Agrawal, Jan Gursky, Lucia Kučerová, Marián Hajdúch. Stromal cell-induced alterations in the response of colorectal cancer cell to demethylating agents. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4101.
Abstract 4103: Cancer cell-stromal cell crosstalk drives fibroblast senescence and tumor progression in large cell carcinoma of the lung in culture and in vivo
Authors: Lugo Roberto , Gabasa Marta , Andriani Francesca , Puig Marta , Facchinetti Federica , Ramírez Josep , Gómez-Caro Abel , Pastorino Ugo , Gascón Pere , Davalos Albert , Reguart Noemí , Roz Luca , Alcaraz Jordi
Published: July 2016

Permanent cell cycle arrest through senescence has been previously regarded as a protective mechanism against tumor progression. In contrast, there is evidence that senescence in tumor-associated fibroblasts (TAFs) enhances tumor growth. Senescence has been reported in tumor associated fibroblasts (TAFs) from a growing list of selected cancer types. However, the presence of senescent TAFs in lung cancer remains undefined. To address this gap of knowledge, we examined common markers of senescence in primary TAFs from the 3 major non-small cell lung cancer (NSCLC) subtypes: adenocarcinoma (ADC), squamous cell carcinoma (SCC) and large cell carcinoma (LCC). Given the difficulties in gathering LCC-TAFs owing to the lower prevalence of LCC compared to the other subtypes, primary fibroblasts from 2 independent cell collections were used. We found an enrichment of the myofibroblast-like phenotype in TAFs regardless their histologic subtype, yet senescence was observed in LCC-TAFs only. Likewise, co-culturing normal lung fibroblasts with LCC (but not ADC or SCC) cancer cells was sufficient to induce senescence, and this induction was prevented in the presence of an antioxidant, indicating that it is mediated through oxidative stress. Of note, senescent fibroblasts provided growth and invasive advantages to LCC cells in culture and in vivo beyond those provided by control (non-senescent) fibroblasts. These results expand recent evidence that challenges the common assumption that lung TAFs are a heterogeneous myofibroblast-like cell population regardless their histologic subtype. Of note, because LCC often distinguishes itself in the clinic by its aggressive nature, our findings support that senescent or senescent-like TAFs may contribute to the selective aggressive behavior of LCC tumors.

Citation Format: Roberto Lugo, Marta Gabasa, Francesca Andriani, Marta Puig, Federica Facchinetti, Josep Ramírez, Abel Gómez-Caro, Ugo Pastorino, Pere Gascón, Albert Davalos, Noemí Reguart, Luca Roz, Jordi Alcaraz. Cancer cell-stromal cell crosstalk drives fibroblast senescence and tumor progression in large cell carcinoma of the lung in culture and in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4103.
Abstract 4104: Characterization and validation of an implantable chronic lung imaging window
Authors: Entenberg David , Harney Allison , Voiculescu Sonia E. , Wang Yarong , Oktay Maja , Condeelis John
Published: July 2016

Metastatic disease is the major cause of cancer mortality and is responsible for over 1/2 million deaths each year in the U.S. alone. Of the three most common sites of metastasis observed clinically (bone, lung and liver), the most difficult to study using intravital microscopy is the lung; a delicate organ in perpetual motion. Recent advances in intravital microscopy have enabled visualization of the live intact lung, but are limited in the duration over which they can be utilized (hours) and require major invasive surgeries. Both of these limitations reduce their usefulness in the study of single cell events in the lung and are susceptible to introducing artefacts. We report the development of a novel, minimally invasive surgical protocol for the implantation of a long-lasting lung imaging window. Once implanted, this window allows for the repeated, non-invasive visualization of living, breathing lung tissue over a period of two weeks without the need for ventilation, vacuum plates, or gated imaging equipment. Here we characterize and validate the use of this window in living mice. The window is well tolerated, does not cause infection or inflammation, and offers a 4mm view of the left lung. We utilize it to visualize, with single cell resolution, the progression of experimental metastasis from the arrival of a single cell to the formation of micro-metastases. The development of this novel optical window will allow visualization of, with single cell resolution, tumor microenvironments and metastatic progression within the lung over extended periods of time. This approach to imaging live lung tissue has the potential to reveal the mechanisms underlying many diseases affecting the lung as well as the means to directly evaluate responses to therapeutics in in real time and within a single animal.

Citation Format: David Entenberg, Allison Harney, Sonia E. Voiculescu, Yarong Wang, Maja Oktay, John Condeelis. Characterization and validation of an implantable chronic lung imaging window. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4104.
Abstract 4110: Color-coded imaging of the tumor microenvironment of malignant lymphoma in a syngeneic mouse model
Authors: Matsumoto Takuro , Suetsugu Atsushi , Hasegawa Kosuke , Nakamura Miki , Aoki Hitomi , Kunisada Takahiro , Tsurumi Hisashi , Shimizu Masahito , Hoffman Robert M.
Published: July 2016

EL4-RFP lymphoma cells were injected in the spleen of C57/BL6 GFP transgenic mice. EL4-RFP metastasis was observed in the lymph nodes of the supra mediastinum and abdomen and in the liver 28 days later. The EL4-RFP tumors in the spleen (primary injection site), liver, supra mediastinum lymph nodes, abdominal lymph nodes, bone marrow, and lung were visualized with the Olympus FV1000 scanning laser confocal microscope. EL4-RFP tumors in the liver, lymph nodes, and bone marrow in C57/BL6 GFP mice were rich in GFP stromal cells such as macrophages, fibroblasts, dendritic cells, and normal lymphocytes derived from the host animal. Phagocytosis of EL4-RFP lymphoma cells by macrophages, as well as dendritic cells and fibroblasts, were observed in culture. Small tumors were observed in the spleen (primary injection site), but the stroma cells in the spleen tumors were abundant. We could observe that in the lung, no mass formation of lymphoma cells occurred, but lymphoma cells circulated in lung peripheral blood vessels.

Citation Format: Takuro Matsumoto, Atsushi Suetsugu, Kosuke Hasegawa, Miki Nakamura, Hitomi Aoki, Takahiro Kunisada, Hisashi Tsurumi, Masahito Shimizu, Robert M. Hoffman. Color-coded imaging of the tumor microenvironment of malignant lymphoma in a syngeneic mouse model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4110.
Abstract 4112: Geminin overexpression-dependent recruitment and crosstalk with mesenchymal stem cells enhances aggressiveness in triple-negative breast cancers
Authors: Bonner Theresa , Ananthula Suryatheja , Nordan Janene , Batth Simran , Marshall Gailen D. , Gardner Lauren H. , Shimizu Yoshiko , ElShamy Wael M.
Published: July 2016

Introduction: Tumor resident mesenchymal stem cells (MSCs) promote cancer progression. Pathways involved in recruitment of MSCs to breast tumor cells remain largely undefined. We aimed at defining the mechanism whereby geminin overexpressing breast tumor cells recruit and interact with MSCs to increase their own aggressiveness.

Experimental Procedures: We used co-culturing assay, immunohistochemistry, in vitro invasion assay with or without cytokines neutralization, western blot analysis, immunoprecipitation, immunofluorescence, ELISA, exposure to hypoxic conditions, cytokines receptor inactivation, in vivo orthotopic tumor formation assay in the presence or absence of inhibitors to the proposed signaling pathways, analysis of publically available data sets for the association of selected factors with overall survival, distant metastasis free survival and recurrent free survival to define the mechanism geminin overexpressing cells employ in vivo to recruit and initiate bi-directional interactions with MSCs in order to enhance their own aggressiveness.

Results: Here we show that geminin-dependent acetylation of chromatin HMGB1 (Ac-HMGB1) leads to its release to the extracellular space. Extracellular Ac-HMGB1 promotes geminin overexpressing (GemOE) cells survival by binding to RAGE receptors. Extracellular Ac-HMGB1 also triggers expression and activation of RAGE in the non-expressing MSCs leading to induction of CXCR4 expression and migration of MSCs towards SDF1/CXCL12-expressing GemOE cells. Inhibiting c-Abl, RAGE or CXCR4 prevented MSCs recruitment to GemOE cells in vitro and in vivo. Reciprocal interactions between GemOE cells and MSCs elevate the TIC, basal and EMT traits in GemOE cells leading to enhanced aggressiveness. Indeed, faster, larger and more aggressive tumors develop when GemOE cells are co-injected with MSCs in orthotropic tumor model. Concurrently, inhibiting c-Abl and thus geminin function or RAGE or CXCR4 activity in the in vivo model not only suppressed MSCs recruitment to the orthotopic breast tumors, but also led to decrease in TIC, basal and EMT phenotypes in these tumors.

Conclusion: We suggest that GemOE generates metastatic tumors in part through recruiting and interacting with MSCs.

Citation Format: Theresa Bonner, Suryatheja Ananthula, Janene Nordan, Simran Batth, Gailen D. Marshall, Lauren H. Gardner, Yoshiko Shimizu, Wael M. ElShamy. Geminin overexpression-dependent recruitment and crosstalk with mesenchymal stem cells enhances aggressiveness in triple-negative breast cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4112.
Abstract 4116: Anti-tumor and anti-metastasis efficacy of a novel Notch inhibitor (PF-03084014) in hepatocellular carcinoma
Authors: Chuanxing Wu , Xiaoqi Wang
Published: July 2016

Hepatocellular Carcinoma (HCC) is well-known for its aggressive growth and highly metastatic potential. Efficacy of current therapy is limited by fast tumor growth and rapid metastasis. Cancer stem cells (CSCs) represent a distinct population that can be prospectively isolated from tumor tissues and have long-term clonal repopulation and self-renewal capacity. They are highly tumorigenic, metastatic, and resistance to conventional radiation and chemotherapy. Therefore, it is essential to identify effective therapy to target CSCs. In our prior study, Notch deregulation was correlated with poor outcome in HCC. In this study, we investigated anti-tumor and anti-metastatic efficacy of a novel Notch inhibitor (PF-03084014) in liver cancer spherical cells in vitro and sphere cell-derived orthotropic model in vivo. PF-03084014 significantly suppressed liver CSCs spheroid formation (derived from 97H HCC cells), with survival rate less than 20% by higher dose of PF-03084014. In accordance, CD90+, EpCAM+ and Oct4+ liver CSC populations significantly decreased, so as down-regulation the stem cell genes expression such as Sox2 and Nanog. These results suggest a suppressive effect of PF-03084014 on liver CSC self-renewal ability. Low dose PF-03084014 induced cancer spheroid cell differentiation leading to chemosensitization. Moreover, Notch inhibitor (PF-03084014) altered liver CSCs stemness properties and blocked spherogenesis of CSCs in vitro, leading to decrease tumorigenicity in vivo. In HCC spherical cell- and non-spherical cell-derived orthotropic model, PF-03084014 effectively inhibited tumor growth, with tumor size was 2570±690 mm3 in vehicle group vs. 800±500 mm3 in Notch inhibitor group (p < 0.001). More importantly, under PF-03084014 treatment, HCC tumor metastasis to lung was significantly reduced (87.5% metastasis rate in vehicle group vs. 18.75% in PF-03084014 group). To explore the potential mechanism, we found PF-03084014 was able to suppress phosphorylation of STAT3 and AKT, the kinases involving in self-renewal of stem cells. PF-03084014 also reversed endothelial mesenchymal transition (EMT) shown by down-regulation of Snail-1 and up-regulation of E-cadherin. Together, PF-03084014 shows a strong anti-cancer and anti-metastasis effect on HCC spherical cell derived in vitro and in vivo model, which provides a preclinical rational of the PF-03084014 to serve as a new novel drug in liver cancer therapy.

Citation Format: Wu Chuanxing, Wang Xiaoqi. Anti-tumor and anti-metastasis efficacy of a novel Notch inhibitor (PF-03084014) in hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4116.
Abstract 4118: Honokiol inhibits lung cancer brain metastasis through inhibition of STAT3 signaling pathway
Authors: Lee Yongik , Pan Jing , Zhang Qi , Wang Yian , You Ming
Published: July 2016

Lung cancer is the leading cause of cancer death in the United States, and metastasis to lymph-node (LN) and distal organs especially brain leads to sever complications and are the major cause of death. Preventing lung cancer development and metastases is important strategy to reduce lung cancer mortality. Honokiol (HNK), a natural compound present in magnolia bark extracts, has a good bioavailability profile and recently has also been shown to readily cross both the blood-brain barrier (BBB). We previously demonstrated that HNK can prevent and inhibit squamous lung cancer cell carcinoma both in-vitro and in-vivo. In the current study, we evaluated the anti-metastatic effects of HNK in both LN and brain murine models of lung tumor metastasis. The H2030-BrM3 (BR-brain seeking) lung tumor cells stably transfected with luciferase and GFP were orthotopically injected into lung for LN metastases, or directly to the left ventricle of mouse heart for brain metastases, LN or brain metastasis was monitored using a non-invasive IVIS bio-luminescent imaging system. To test the efficacy of HNK in preventing tumor cells migrating to LN or brain, mice were given 10 mg/kg per body weight HNK by aerosol (for lung to LN metastasis model) or oral gavage starting the day after tumor cell injection, our results reveal that HNK significantly prevented the metastasis of lung cancer cells to LN and brain. Our results demonstrate that HNK decreased LN metastases incidence to 30% compare to 100% in control mice, and inhibited brain metastasis near 70% compare to control. Furthermore, analysis of HNK's mechanism of action by utilizing both RNA sequencing and receptor tyrosine kinase assay suggests that its effect is primarily mediated by inhibiting STAT3 pathway. HNK specifically inhibits STAT3 phosphorylation, and knock-down STAT3 totally abrogated the anti-metastasis effects of HNK in brain metastatic lung cancer cell lines, PC9-BrM3 and H2030-BrM3. These finding suggest that HNK could provide novel chemopreventive or therapeutic options for preventing both lung tumor progression and lung cancer brain metastasis.

Citation Format: Yongik Lee, Jing Pan, Qi Zhang, Yian Wang, Ming You. Honokiol inhibits lung cancer brain metastasis through inhibition of STAT3 signaling pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4118.
Abstract 4127: Blockade of TGFβ signaling enhances anticancer effect of chemotherapeutics in gynecologic cancers
Authors: Zhu Haiyan , Yang Junhua , Gu Xiang , Sun Luzhe
Published: July 2016

Chemoresistance is a significant clinical challenge in the management of gynecologic malignancies. Previous studies have showed that TGF-β signaling pathway can mediate chemotherapy resistance. However, the underlying mechanisms of how TGF-β pathway is activated and causes chemoresistance are not well understood. Here, we found that ovarian cancer patients displayed an increased RNA transcripts of genes associated with TGF-β signaling after chemotherapy. In vitro, treatment with four commonly used drugs for gynecologic malignancies, including cisplatin, doxorubicin, paclitaxel and camptothecin, activated TGFβ signaling by stimulating phosphorylation of Smad2 and Smad3, two intracellular mediators of TGFβ signaling pathway in ovarian and cervical cancer cell lines. Furthermore, cisplatin and doxorubicin can induced epithelial-mesenchymal transition (EMT) and promote migration. Blockade of TGFβ signaling abrogated chemotherapeutics-stimulated Smad phosphorylation, epithelial-mesenchymal transition, and migration. These observations suggest that chemotherapy-mediated activation of TGFβ signaling may be a mechanism of chemoresistance, and that TGFβ pathway inhibitor may prevent the development of chemoresistant. We are currently testing the anti-tumor efficacy of the combination therapy with a novel pan-TGFβ inhibitor and cisplatin in a mouse xenograft model of ovarian cancer and whether the treatment with TGFβ inhibitor can alleviate symptoms of nephrotoxicity, which is known to be caused by cisplatin. Our studies may reveal new strategies for the treatment of gynecologic malignancies.

Citation Format: Haiyan Zhu, Junhua Yang, Xiang Gu, Luzhe Sun. Blockade of TGFβ signaling enhances anticancer effect of chemotherapeutics in gynecologic cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4127.
Abstract 4131: Blocking endogenous TBX2 abrogates prostate cancer bone metastasis through WNT signaling
Authors: Nandana Srinivas , Tripathi Manisha , Duan Peng , Chu Gina , Zhau Haiyen E. , Matusik Robert J. , Chung Leland W.K.
Published: July 2016

Bone metastasis is a crucial turning point in the progression of PCa and is seen in > 90% of the lethal cases. Identifying factors that mediate the metastatic spread and subsequent remodeling of the bone is highly relevant to successful therapeutic intervention in advanced human prostate cancer (PCa). TBX2, a transcription factor that belongs to the T-box family negatively regulates p21 cell cycle inhibitor and is known to play critical roles during embryonic development and cancer progression. Interestingly, a recent report found TBX2 over-expression in the bone metastases from patients with castrate resistant prostate cancer. We found TBX2 over-expression in human prostate cancer specimens and in aggressive androgen independent human PCa cell lines. Further, we found TBX2 over-expression in bone metastases in multiple xenograft mouse models of human PCa progression. Blocking endogenous TBX2 in PC3 and ARCaPM PCa cells through the use of a dominant negative construct resulted in decreased proliferation, colony formation and invasion in vitro. Utilizing various pre-clinical xenograft mouse models, we found that blocking endogenous TBX2 in PCa cells leads to decreased invasion and abrogation of bone and soft tissue metastasis in vivo. Further, utilizing the intra-tibial mouse model of growth in the bone microenvironment, we found that blocking endogenous TBX2 in PCa cells dramatically reduces bone remodeling. Additionally we found that TBX2 acts upstream of the canonical WNT (WNT3A) signaling in PCa and positively regulates its transcription by binding to its promoter. Further, rescue of WNT3A levels in PCa cells in the context of blocking endogenous TBX2 rescues the metastatic ability of the cells in vivo. Taken together, our findings highlight a novel and critical role of TBX2 in PCa progression, metastasis and the subsequent bone remodeling, and that TBX2 function in PCa is mediated through the canonical WNT signaling.

Citation Format: Srinivas Nandana, Manisha Tripathi, Peng Duan, Gina Chu, Haiyen E. Zhau, Robert J. Matusik, Leland W.K. Chung. Blocking endogenous TBX2 abrogates prostate cancer bone metastasis through WNT signaling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4131.
Abstract 4133: Suppressed B7-H3 expression reduces glycolytic capacity and sensitizes tumor cells to anti-cancer agents
Authors: Nunes-Xavier Caroline E. , Flem Karlsen Karine , Øyjord Tove , Tan Ming , Fodstad Øystein
Published: July 2016

B7 family proteins are important immune response regulators, and can mediate oncogenicity-related signals and support cancer development. We have prevously shown that the B7-H3 protein promotes tumor cell invasion, proliferation and mestastasis, and increases resistance to chemotherapy. Now we have used human breast and melanoma cancer cell lines with different expression levels of B7-H3 to further evaluate the role of B7-H3 on the sensitivity to anticancer compounds. B7-H3 knockdown tumor cells showed enhanced chemosensitivity compared to their B7-H3 expressing counterparts, as well as increased inhibition to treatment with compounds that target proteins in the PI3K/AKT/mTOR- and MAPK-pathways (Nunes-Xavier et al. in revision). Treatment of control cells with an anti-B7-H3 monoclonal antibody resulted in similar sensitization, whereas B7-H3 overexpressing cells and xenografts were less sensitive. Of note, knockdown of B7-H3 decreased and B7-H3 overexpression increased the glycolytic capacity of the breast cancer and malignant melanoma cells. In conclusion, we have demonstrated a previously unknown relationship between B7-H3 expression and glycolysis in tumor cells, and found that B7-H3 confers resistance to also to pathway inhibiting agents. The results provide novel insights into the function of B7-H3 in cancer, and suggest that targeting of B7-H3 expression may be a promising alternative to improve current anticancer therapy.

Citation Format: Caroline E. Nunes-Xavier, Karine Flem Karlsen, Tove Øyjord, Ming Tan, Øystein Fodstad. Suppressed B7-H3 expression reduces glycolytic capacity and sensitizes tumor cells to anti-cancer agents. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4133.
Abstract 4148: Different immune cell responses are associated with glioblastoma subclassification and typical genetic alterations
Authors: Luoto Suvi , Kesseli Juha , Nykter Matti , Granberg Kirsi J.
Published: July 2016

Interactions between various components in the tumor microenvironment and dysregulated immune responses are thought to play important roles in cancer development. To better understand the role of immune cells in tumor pathogenesis and destruction, we computationally model the microenvironment of an aggressive brain tumor glioblastoma multiforme (GBM).

We downloaded GBM patient RNA-seq data from the Cancer Genome Atlas (TCGA). Using cluster analysis, we identified 16 clusters, each containing 10 to 933 genes that show a statistical enrichment of immune response related gene ontology terms. Utilizing a panel of RNA-seq data from normal cell types, we constructed global and cluster specific regression models to characterize the expression profiles of GBM samples in the clusters of interest as linear combinations of normal cell and reference GBM expression profiles. Simulated data was used to validate that the regression model coefficients accurately reflect the contributions of normal cell types to the expression profiles of tumor samples. Based on the regression analysis, we were able to uncover high variability in the composition of microenvironment across the TCGA cohort, suggesting diverse immune responses in tumors.

The results from global regression analysis were then associated with common genetic alterations in GBM and with GBM subclassification. Especially the estimated macrophage, granulocyte, and CD8+ T lymphocyte proportions show significant differences between different GBM subgroups. Accumulation of immune cells was increased in the mesenchymal and neural subtype compared to other subtypes. Furthermore, estimated immune cell proportions were associated with alterations in EGFR and NF1.

Taken together, our analysis provided a characterization of the immunomicroenvironment in GBM and linked immune cell responses to typical GBM alterations and GBM subtypes. More detailed characterization of diverse immune responses will facilitate patient stratification and might provide tools for personalized immunotherapy in the future.

Citation Format: Suvi Luoto, Juha Kesseli, Matti Nykter, Kirsi J. Granberg. Different immune cell responses are associated with glioblastoma subclassification and typical genetic alterations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4148.
Abstract 4149: Direct immune cell contact to basal-like triple negative breast cancer cells evokes downregulation of EGFR and PD-L1
Authors: Yamaguchi Ayane , Suzuki Eiji , Kawaguchi Kosuke , Nishie Mariko , Tsuda Moe , Toi Masakazu
Published: July 2016

BACKGROUND:

We previously reported that direct co-culture of triple negative breast cancer cell line MDA-MB-231 and immune cells results in reduction of EGFR expression on cell surface of MDA-MB-231 (AACR 2015 #2349). However, a role of reduction of EGFR on MDA-MB-231 via co-culture with immune cells still remains unclear. Therefore, we evaluated a role of reduction of EGFR on MDA-MB-231 at the immunological point of view by testing expression of immune related genes including PD-L1.

METHODS:

In order to verify the importance of direct immune cell contact to breast cancer cell, MDA-MB-231 cells were co-cultured directly or indirectly with THP-1 cells (human monocytic cell line) at 1:50 cellular ratios. In order to study indirect co-culture assay, we used cell culture insert to avoid direct cancer cell-immune cell contact. We analyzed gene expression by quantitative real time PCR and membrane protein expression by flow cytometry of EGFR, also other HER family on MDA-MB-231 which is directly or indirectly co-cultured with THP-1. We also evaluated the expression of immune related genes including PD-L1.

RESULTS:

Both mRNA and protein level of EGFR on MDA-MB-231 cells directly co-cultured with THP-1 were significantly decreased as compared to those with indirectly co-cultured MDA-MB-231 cells. There are no significant differences in EGFR expression between indirectly co-cultured MDA-MB-231 cells and control MDA-MB-231 cells. Importantly, PD-L1 expression on MDA-MB-231 cells directly co-cultured with THP-1 was significantly decreased as compared to that with indirectly co-cultured MDA-MB-231 cells.

CONCLUSION:

It has been reported that PD-L1 expression in cancers is regulated by phosphatidylinositol 3-kinase (PI3K) and Akt signaling. Thus, our findings may give a novel insight on regulation of PD-L1 expression on cancer cells in tumor microenvironment that tumor infiltrated immune cell directly contact with cancer cells and EGFR down-regulation leads to reduction of PD-L1 expression on cancer cells.

Further investigation is needed to elucidate the mechanism of reduction of EGFR by direct immune cell contact to cancer cells and its interaction with modulation of PD-L1 expression. This will provide novel aspects for immune therapy of breast cancer patients.

Citation Format: Ayane Yamaguchi, Eiji Suzuki, Kosuke Kawaguchi, Mariko Nishie, Moe Tsuda, Masakazu Toi. Direct immune cell contact to basal-like triple negative breast cancer cells evokes downregulation of EGFR and PD-L1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4149.
Abstract 415: A new, highly sensitive ALK antibody improves the screening of rearranged-ALK by IHC
Authors: Lu Hsiangmin , Gonzalez Rachel , Shen Yi , Jin Mu-lan , Wu Yipan , Zhang Yungang , Yuan Kehu , Chu Boyang , Qi Lili , Liu Huibo , Wang Chenlin , Wang Guangli , Shu Youmin , McDowell Julie , Ma Donghui , He Wei-wu , Chen Jian , Lin Ray
Published: July 2016

All non-small cell lung cancer (NSCLC) patients are recommended to be screened for anaplastic lymphoma kinase (ALK)-rearrangement despite its low occurrence (< 7%). This is due to recent advances in treatment for patients with ALK-positive NSCLC with receptor tyrosine kinase inhibitors Crizotinib and Ceritini. Current rearranged-ALK testing includes fluorescence in situ hybridization (FISH), reverse transcription polymerase chain reaction (RT-PCR), and immunohistochemistry (IHC). Despite the high sensitivities and specificities, the limitations of the first two assays include time-consuming, infeasible, and specialized techniques that make them unsuitable for the routine screening for which IHC has been proposed. However, the biggest obstacle to detecting rearranged-ALK in lung cancer patients by IHC is the lack of highly sensitive ALK antibodies that detect the extremely low abundance of ALK in patients. To overcome this limitation, we have developed an ALK mouse monoclonal antibody (clone OTI1A4) with higher sensitivity in comparison to a rabbit ALK antibody (clone D5F3). Moreover, OTI1A4 identified ALK-positive NSCLC samples (16/16 cases) and showed negative staining for ALK-negative samples (11/11 cases) previously validated by FISH (10/16 ALK-positive cases) or qPCR (10/16 ALK-positive cases). This indicated the potential advantage of using clone OTI1A4 over FISH or qPCR to detect ALK rearrangements. Lastly, there was 100% concordance between OTI1A4 and D5F3 in detecting ALK-rearranged NSCLC samples previously confirmed by FISH (76 ALK-positive and 438 ALK-negative samples). These results support the possibility that ALK clone OTI1A4 could be used for routine screening of patients with ALK-positive NSCLC by IHC.

Citation Format: Hsiangmin Lu, Rachel Gonzalez, Yi Shen, Mu-lan Jin, Yipan Wu, Yungang Zhang, Kehu Yuan, Boyang Chu, Lili Qi, Huibo Liu, Chenlin Wang, Guangli Wang, Youmin Shu, Julie McDowell, Donghui Ma, Wei-wu He, Jian Chen, Ray Lin. A new, highly sensitive ALK antibody improves the screening of rearranged-ALK by IHC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 415.
Abstract 4153: Abnormal expression of MUC1 mucin on colon epithelia stimulates production of pro-inflammatory cytokines promoting colitis-associated colon cancer in a murine model
Authors: Cascio Sandra , Faylo Jacque , Xue Jia , Finn Olivera
Published: July 2016

MUC1 is a transmembrane glycoprotein aberrantly expressed in human adenocarcinoma as well as chronic inflammatory conditions. We have previously demonstrated that the tumor form of MUC1, in association with p65, upregulates the expression of pro-inflammatory cytokines in the tumor microenvironment, including IL-6 and TNF-alpha, by modulating their transcriptional activity in epithelial cancer cells. Here, we explored the mechanism underlying MUC1/p65-induced transcription of IL-6 and TNF-alpha in colon cancer cells and its significance for the microenviroment of colitis that is known to be the precursor to cancer. We used a mouse model of colitis-associated colon cancer where wild type (WT) and human MUC1 transgenic (MUC1.Tg) mice are given carcinogen azoxymethane (AOM), followed by three cycles of 1.2% dextran sodium sulfate (DSS) in drinking water to induce colitis. MUC1.Tg showed higher tumor incidence, decreased survival, increased body weight loss and shorter colon length. These features accelerated the severity of inflammation-induced colon cancer. Consistent with our previous in vitro data, expression of NF-kB family members was higher in MUC1.Tg mice compared to WT. We previously showed that in tumors, MUC1/p65 complex modulated the expression of histone methyltransferase Enhancer of Zeste protein-2 (EZH2) and interaction with pro-inflammatory cytokine promoters. In order to understand the significance of MUC1/p65-modulated cytokines in progressive colitis that gives rise to colon cancer, we analyzed infiltration of inflammatory cells into the inflamed colon tissues. The number of infiltrating macrophages was much higher in AOM/DSS-treated MUC1.Tg mice compared to WT. ELISA assay and gene expression analyses demonstrated that the treatment with AOM/DSS in the presence of human MUC1 on the colonic epithelia resulted in a significant increase in M1 type macrophage-associated genes, including IL-6, TNF-alpha and iNOS, whereas M2 type macrophage markers such as Arginase 1, Ym1, and IL-10 were drastically reduced when compared to colon tissues of treated WT mice. Taken together, our findings reveal a pro-inflammatory role for MUC1 in colitis-related carcinogenesis. Our study also provides a mechanism by which MUC1 accelerates tumor initiation and progression.

Citation Format: Sandra Cascio, Jacque Faylo, Jia Xue, Olivera Finn. Abnormal expression of MUC1 mucin on colon epithelia stimulates production of pro-inflammatory cytokines promoting colitis-associated colon cancer in a murine model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4153.
Abstract 416: Tumor cell isolation using antibody/aptamer-based multivalent binding
Authors: Fan Z. Hugh , Zhang Jinling , Sheng Weian
Published: July 2016

Isolation and enumeration of circulating tumor cells (CTCs) in peripheral blood have been used for cancer diagnosis, prognosis, and theragnosis. The majority of the CTC isolation methods, including the FDA-approved CellSearch®, employ one antibody to capture tumor cells. Using such a monovalent-binding capture agent likely results in lower cell capture efficiency than multivalent-binding methods, which tend to have stronger binding force (or lower dissociation constant).

We have investigated in a multivalent-binding-based tumor cell isolation method by immobilizing a mixture of aptamers and antibodies on the surfaces of microfluidic devices. Since aptamers are much smaller in the size (8-15 kDa) than antibodies (150 kDa), aptamers and antibodies could interact with different receptors on cell surfaces, resulting in a configuration that one cell simultaneously interacts with multiple different types of capture agents. The microfluidic devices we used are in the size of a microscope slide and each of them consists of an inlet, eight parallel channels connected via consecutive bifurcation, and an outlet. There are micromixers inside the channels for enhanced interactions. Biotinylated aptamers and biotinylated antibodies were coated onto microchannel surfaces using avidin-biotin chemistry.

We tested the devices by isolating human leukemia cells (CCRF-CEM) in devices immobilized with a mixture of sgc8 aptamers and protein tyrosine kinase-7 (PTK7) antibodies. Both sgc8 aptamers and anti-PTK7 exhibited strong binding with CCRF-CEM cells according to flow cytometry. An antibody-to-aptamer ratio of 1:300 showed the highest capture efficiency among the range we studied (from 1:30 to 1:3000). We found that the antibody-aptamer surfaces had higher capture efficiency than antibody-alone or aptamer-alone surfaces; the degree of enhancement increased with the flow rate. Control experiments using non-specific aptamer (TD05) and non-specific antibody (anti-EpCAM) were carried out to verify the benefits of multivalent binding. We also found that the multivalent-binding surfaces have higher specificity than monovalent-binding surface, especially at a high flow rate. A calibration curve was obtained by spiking leukemia cells into human whole blood.

In summary, antibody/aptamer-coated multivalent-binding surfaces in microfluidic devices showed higher capture efficiency and specificity than monovalent-binding surfaces, suggesting their potential for the isolation and enumeration of CTCs for clinical applications.

Citation Format: Z. Hugh Fan, Jinling Zhang, Weian Sheng. Tumor cell isolation using antibody/aptamer-based multivalent binding. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 416.
Abstract 4165: Myc vs. Akt therapy in RapidCap, a GEM model for metastatic prostate cancer
Authors: Stahlhut Carlos E. , Ambrico Alexandra J. , Watrud Kaitlin E. , Cho Hyejin , Wang Lily , Qi Jun , Cantley Lewis C. , Bradner James , Trotman Lloyd
Published: July 2016

We describe our efforts to determine the effect of MYC and PI3K inhibition in a genetically engineered mouse model of naïve and castration-resistant metastatic prostate cancer. Metastasis is a major driver of mortality and morbidity in prostate cancer, the most common cancer type in men and the second leading cause of cancer-related deaths in the western world. To recapitulate the metastatic process in genetically engineered mice, we have developed the RapidCaP system, which uses prostate-directed viral infection to trigger focal loss of Pten and Trp53. Using luminescence and fluorescence, the RapidCaP system allows us to track disease progression in real-time and isolate metastases in distant tissues. This system provides a powerful platform for the identification and validation of candidate driver genes and for the efficient testing of novel therapeutics. Using this system, we have recently identified Myc as a critical, spontaneously activated driver in Pten-negative metastatic prostate cancer. Preliminary trials indicate that the Myc-suppressing Brd4 inhibitor JQ1 is ineffective towards primary disease, but induces metastatic regression. This specificity correlates with a switch from AKT-driven primary disease to MYC-driven metastatic disease. Our current efforts seek to determine how, when and why targeting of Myc using JQ1 and the PI 3-Kinase pathway using NVP-BKM120 can be best used to treat naïve and castration-resistant metastatic prostate cancer. In addition, we are examining changes to the transcriptome and epigenetic alterations that characterize Pten-negative metastatic prostate cancer, with the goal of understanding the mechanisms of progression toward a metastatic state and establishing new therapeutic targets.

Citation Format: Carlos E. Stahlhut, Alexandra J. Ambrico, Kaitlin E. Watrud, Hyejin Cho, Lily Wang, Jun Qi, Lewis C. Cantley, James Bradner, Lloyd Trotman. Myc vs. Akt therapy in RapidCap, a GEM model for metastatic prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4165.
Abstract 4169: Systematic in vivo analysis of PI3K pathway aberrations in a mouse model of invasive lobular carcinoma
Authors: Van Miltenburg Martine
Published: July 2016

Invasive lobular carcinoma (ILC) is the second most prevalent breast cancer subtype, which develops in the milk-producing glands (lobules) and metastasizes to distant organs. A key feature of ILC is loss of the CDH1 gene, encoding the E-cadherin cell-cell adhesion receptor. Though E-cadherin is thought to be a tumour suppressor, it is interesting to note that mammary-gland specific deletion of E-cadherin does not lead to tumour development indicating that other factors are necessary for the induction of ILC. Recent data from the cancer genome atlas reveals that besides E-cadherin loss, mutations in PIK3CA, and to a lesser extent PTEN, TBX3, and FOXA1 were found as ILC enriched features. These data implicate PI3K signaling as a driving force in ILC development. Using our in-house established GEMM-ESC strategy, we have developed innovative mouse models for ILC to study novel aspects of the functional interplay between E-cadherin and PI3K in ILC development. We have discovered an intriguing new interplay between these components in which E-cadherin loss suppresses growth of normal mammary epithelium, which appears to involve senescence, whereas it effectively accelerates tumor growth in a PIK3CAmutant background. Therapeutically, PIK3CAE545K and AKTE17K ILCs were sensitive to AZD8055, an mTOR inhibitor, and BKM120, a PI3K 110α inhibitor in vivo, indicating that PI3K signaling is required for their tumor maintenance. Currently, we are dissecting the sensitivity of ILCs displaying PIK3CAE545K or AKTE17K mutations, and PTEN loss, to a large panel of inhibitors, which will likely provide new clinical options.

Citation Format: Martine Van Miltenburg. Systematic in vivo analysis of PI3K pathway aberrations in a mouse model of invasive lobular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4169.