bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5878: Trichostatin A overcomes chemotherapeutic resistance of urothelial carcinoma cells through the inactivation of c-Raf/ERK pathway
Authors: Kuo Kuan-Lin , shi Chung-Sheng , Hsieh Ju-Tong , Chen Shiang-Peng , Lin Wei-Chou , Liao Shih-Ming , Liu Shing-Hwa , Huang Kuo-How
Published: July 2017

Trichostatin A (TSA), a potent histone deacetylase (HDAC) inhibitor, has been reported to elicit anti-proliferative response in various tumors. Here, we investigated antitumor effect of TSA alone or in combination with conventional chemotherapeutic agents on urothelial carcinoma (UC) cells. We use one high-grade UC cell line (T24) and another UC cells (NTUUC) obtained by primary culture from the surgical specimen of a women with high-grade and metastatic bladder UC. The cytotoxicity and apoptosis induced by TSA alone , chemotherapeutic agents (cisplatin, gemcitabine and doxorubicin) and combined treatment were assessed by MTT assay and fluorescence-activated cell sorting and flow cytometry. The expression of phosphor c-Raf, phosphor-MEK1/2, and phosphor-ERK1/2 were measured by Western blot. urther elucidation on the role of Raf/MEK/ERK pathoway on the TSA-enhanced cytoxicity were exmained by using ERK1 siRNA knockdown and the specific MEK inhibitor (PD98059). Our results showed TSA markedly enhances the cytotoxicity and apoptosis of three chemotherapeutic agents in UC cells with concurrent suppression of Raf/MEK/ERK signaling pathway. Consistently, inhibition of Down-regulation of ERK by MEK inhibitor or by ERK 1 siRNA knockdown potentiated the chemotherapeutic agnet-induced cytotoxicity of TSA in UC cells. We concluded that TSA potentiates the therapeutic efficacy of cisplatin, gemcitabine and doxorubicin in human UC cells through Raf/MEK/ERK signaling pathway. These findings provide a new treatment strategy against UC.

Note: This abstract was not presented at the meeting.

Citation Format: Kuan-Lin Kuo, Chung-Sheng shi, Ju-Tong Hsieh, Shiang-Peng Chen, Wei-Chou Lin, Shih-Ming Liao, Shing-Hwa Liu, Kuo-How Huang. Trichostatin A overcomes chemotherapeutic resistance of urothelial carcinoma cells through the inactivation of c-Raf/ERK pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5878. doi:10.1158/1538-7445.AM2017-5878
Abstract 5885: Sialyl Lewis X-P-selectin connection between ovarian tumor-mesothelium in early stage metastasis
Authors: Li Shan-Shan , Ip Carman K. M. , Hassan Ayon A. , Tang Matthew Y. H. , Yung Susan , Chan Tak-Mao , Shum Ho Cheung , Wong Alice S. T.
Published: July 2017

Metastasis is one of the major challenges for the treatment of ovarian cancer. Unlike other solid tumors, ovarian cancer primarily disseminates within the peritoneal cavity. A crucial step in metastasis formation is the adhesion of ovarian cancer cells onto the peritoneal mesothelium under ascitic shear flow. However, the adhesion mechanisms engaged in this tumor-mesothelium interaction remain elusive due to a lack of physiologically relevant model to manipulate and investigate this dynamic process. In this study, using a 3D microfluidic platform, we found that the metastatic population of cancer stem cells (M-CSCs) exhibited slower rolling velocity and higher binding ability to the peritoneal mesothelium than non-metastatic (NM)-CSCs under flow condition. This adhesion cascade was mediated by P-selectin which expressed on the peritoneal mesothelium. The key carbohydrate determinant on M-CSCs was a glycoprotein, but not a glycolipid, with its recognition as sialyl-Lewis X (sLeX) in a sialic acid- and fucose-dependent manner. Moreover, several glycosyltransferase genes including B4GALT4, ST3GAL3, ST3GAL4 and FUT-5 involved the synthesis of sLeX were upregulated in M-CSCs. Knocking down FUT-5 significantly inhibited ovarian tumor cell adhesion on the mesothelium in vitro and reduced the metastatic potential in vivo. Taken together, our findings revealed that a distinct sLeX-P-selectin axis of ovarian tumor-mesothelium interaction in early metastasis and may offer the possibility of new therapeutic targets. (This work is supported by RGC grant 17122014)

Citation Format: Shan-Shan Li, Carman K. M. Ip, Ayon A. Hassan, Matthew Y. H. Tang, Susan Yung, Tak-Mao Chan, Ho Cheung Shum, Alice S. T. Wong. Sialyl Lewis X-P-selectin connection between ovarian tumor-mesothelium in early stage metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5885. doi:10.1158/1538-7445.AM2017-5885
Abstract 5899: Compartment-resolved proteomics reveal NNMT as a master metabolic regulator of cancer associated fibroblasts
Authors: Eckert Mark A. , Coscia Fabian , Pan Shawn , Tienda Samantha M. , Chryplewicz Agnieszka A. , Chiang Chun-Yi , Montag Anthony , Yamada S. Diane , Mann Matthias , Lengyel Ernst R.
Published: July 2017

The poor outcome of ovarian cancer patients is due to late detection and the ability of ovarian cancer cells to metastasize quickly throughout the abdominal cavity. Recent research, including histopathological studies and experiments with mouse models, suggest that ovarian cancer may actually arise in the fallopian tube as serous tubal intraepithelial carcinomas (STIC). Lesions of the ovary would, therefore, represent metastases from the fallopian tube. To better understand the molecular events that occur in both the tumor and stromal compartment during ovarian cancer progression, we performed shotgun proteomics on laser-microdissected tumor and stromal compartments from anatomic sites representing the hypothetical progression series for ovarian cancer (STIC → fallopian tube → ovary → omentum). With an optimized sample preparation technique, we successfully quantified 4-5000 proteins per anatomic site with high reproducibility and with as few as several thousand microdissected cells derived from formalin-fixed and paraffin-embedded (FFPE) biobank specimens. Proteomics of the tumor compartments revealed high inter-patient heterogeneity, with no conserved protein signatures associated with invasion or metastasis. In strong contrast, a highly conserved molecular signature of stromal proteins associated with metastasis to the omentum was identified. In particular, nicotinamide N-methyl transferase (NNMT) was highly upregulated in the stroma of omental and peritoneal metastases. Functionally, NNMT was necessary and sufficient for multiple aspects of the cancer associated fibroblast (CAF) phenotype, including expression of CAF markers and secretion of cytokines and oncogenic extracellular matrix. NNMT expression was necessary for the ability of CAFs to support ovarian cancer cell migration, proliferation, adhesion, and in vivo growth. Mechanistically, high expression of NNMT in CAFs led to a depletion of S-adenosyl methionine (SAM) and a reduction in histone methylation associated with gene expression changes in the tumor stroma. This work supports the use of ultra-low input proteomics for compartment-resolved identification of candidate drivers of disease phenotypes and identifies NNMT as a central, metabolic regulator of CAF differentiation.

Citation Format: Mark A. Eckert, Fabian Coscia, Shawn Pan, Samantha M. Tienda, Agnieszka A. Chryplewicz, Chun-Yi Chiang, Anthony Montag, S. Diane Yamada, Matthias Mann, Ernst R. Lengyel. Compartment-resolved proteomics reveal NNMT as a master metabolic regulator of cancer associated fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5899. doi:10.1158/1538-7445.AM2017-5899
Abstract 5921: Microenvironmental changes in the progression from adenocarcinoma in situ to early-invasive adenocarcinoma of the lung
Authors: NAITO MASAHITO , Satoh Yukitoshi , Ishii Genichiro
Published: July 2017

Introduction:

Lepidic predominant adenocarcinoma of the lung was classified into adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA) and invasive lepidic predominant adenocarcinoma (LPA) in current WHO classification, 4th edition. Furthermore, LPA is thought to progress in a stepwise fashion from AIS and MIA. However, those classifications were diagnosed by observation of pathologists based on morphology only. The aim of this study was to investigate the microenvironmental changes in the process of development from AIS, MIA to LPA, and subjective marker of stepwise progression. We have published a part of data in this study in Lung Cancer (100:53-62, 2016), and we believe that this study makes a contribution to elucidate the mechanism of development of LPA of the lung.

Materials and Methods:

Clinicopathological characteristics of AIS (n=51), MIA (n=59), LPA smaller than 3 cm (S-LPA, n=113), and LPA larger than 3 cm (L-LPA, n=47) were analyzed. We then immunohistochemically evaluated the expression levels of epithelial-mesenchymal transition (EMT)-related

molecules (E-cadherin, S100A4), invasion-related molecules (laminin-5, ezrin), and growth factor receptors (EGFR) in cancer cells of each group (n=20). The number of tumor-promoting stromal cells, including podoplanin-positive cancer-associated fibroblasts (PDPN+ CAFs), CD204-positive tumor-associated macrophages (CD204+ TAMs), and CD34+ microvessel cells, were also analyzed.

Results:

LPA-S showed significantly frequent vascular and pleural invasion than MIA (p<0.05); however, no significant difference in those factors was found between S-LPA and L-LPA. Laminin-5 expression in the non-invasive component of MIA was significantly higher than that of AIS (p<0.001). During the progression from MIA to S-LPA, the expression level of laminin-5 in the invasive component was significantly elevated (p<0.01). Moreover, tumor-promoting stromal cells were more frequently recruited in the invasive area of LPA-S (PDPN+ CAFs; p<0.05, CD204+ TAMs; p<0.001, CD34+ microvessel; p<0.05). Ezrin expression in the invasive component of L-LPA was significantly increased (p<0.05) compared to S-LPA; however, the number of tumor-

promoting stromal cells were not different between these two groups.

Conclusion:

Our current study indicated that microenvironmental molecular changes occur during the progression from MIA to S-LPA. Thus, it was suggested that this process may play an important role in disease progression from AIS to LPA of the lung.

Citation Format: MASAHITO NAITO, Yukitoshi Satoh, Genichiro Ishii. Microenvironmental changes in the progression from adenocarcinoma in situ to early-invasive adenocarcinoma of the lung [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5921. doi:10.1158/1538-7445.AM2017-5921
Abstract 606: Targeting multiple immune checkpoints and their ligands using a humanized mouse model of ovarian cancer
Authors: Huang Ruea-Yea , Odunsi Kunle
Published: July 2017

We have recently reported that multiple immune checkpoints are expressed in the TILs (tumor infiltrating T lymphocytes) and TALs (tumor associated T lymphocytes) from a murine ovarian cancer model and that blockade of one checkpoint pathway induces an upregulation of the other unblocked checkpoints. We hypothesized that resistance to single agent blockade (e.g. anti-PD1) is resulted from the compensatory upregulation of the other pathways (e.g. CTLA4 or LAG-3). To test this hypothesis in human ovarian cancer, we first examined the level of several immune checkpoints and their ligands in TILs or TALs from ovarian cancer patients. In addition to the highly expressed PD-1 and CTLA4, other checkpoint molecules such as TIM3, LAG3, VISTA, and TIGIT are also elevated in TALs as compared with control PBMC. The levels of multiple ligands for the immune receptors in the tumor samples were also analyzed. To translate the results from mice to humans, we developed several aggressive human ovarian cancer cell lines from patient derived xenografts to test the combinatorial checkpoint blockade strategies. Using a humanized mouse model of ovarian cancer we observed that adaptive transfer of autologous T cells in combination with checkpoint blockade of PD1 mildly delays ovarian tumor growth. The effect of blocking PD-1 and CTLA-4 pathways on tumor growth and the upregulation of other checkpoints are currently underway.

Citation Format: Ruea-Yea Huang, Kunle Odunsi. Targeting multiple immune checkpoints and their ligands using a humanized mouse model of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 606. doi:10.1158/1538-7445.AM2017-606
Abstract 612: Methylation changes in DNA of CD8 T cells following CD137 costimulation
Authors: Aznar M. Angela , Labiano Sara , Diaz-Lagares Angel , Esteller Manel , Sandoval Juan , Melero Ignacio
Published: July 2017

Introduction: CD137 ligation imprints long term changes in the behavior of costimulated T-cells (1). There is not a satisfactory mechanistic explanation yet.

Experimental procedures: to determine the specific DNA methylation changes occurring upon CD137 costimulation, purified human CD8+ T cells from three healthy donors were activated in vitro for 5 days with anti-CD3 monoclonal antibody and either with an anti-CD137 monoclonal antibody or its corresponding isotype (huIgG4). Activated lymphocytes were left 5 days in culture with huIL-7 without further stimulation. Such back-to-resting CD8+ lymphocytes were reestimulated with anti-CD3 for 12, 24 and 36h to validate the expression of the genes differentially methylated upon primary stimulation at mRNA and protein levels.

DNA methylation profiles of both activated and resting cell subsets were characterized with Infinium 450K DNA methylation array (Illumina). To further confirm our observations, identical experimental procedure was performed with a different anti-CD137 agonist antibody (mIgG1 isotype) in a CD8+ T-cell donor.

Differentially methylated genes between anti-CD3+anti-CD137 versus their corresponding control counterparts were validated by pyrosequencing on activated and resting CD8+ from independent group of healthy donors (n=8 for hIgG anti-CD137 and n= 11 for mIgG anti-CD137). Expression changes were confirmed by qRT-PCR and flow cytometry in activated, rested and reestimulated CD8+ lymphocytes.

Results: 853 genes were differentially methylated in hIgG4 anti-CD137-treated CD8+ T cells as compared with their controls, 52 of which were shared with mIgG1 anti-CD137-costimulated CD8+ T lymphocytes. A number of differentially methylated genes are involved in i) T cell migration, ii) T cell activation, survival and homeostasis and iii) regulation of gene expression including key T-cell transcription factors.

Conclusions: CD137 costimulation induces CD8+ T lymphocytes that are poised to respond more effectively to a second antigen exposure. These acquired functions are imprinted in the genomic DNA of the CD8+ T cells by DNA methylation changes upon CD137 co-stimulation, and involve key genes for CD8+ T cells.

References: 1.

Hendriks, J., Y. Xiao, J.W. Rossen, K.F. van der Sluijs, K. Sugamura, N. Ishii, and J. Borst, During viral infection of the respiratory tract, CD27, 4-1BB, and OX40 collectively determine formation of CD8+ memory T cells and their capacity for secondary expansion. J Immunol 2005, 175: p. 1665-76.

Citation Format: M. Angela Aznar, Sara Labiano, Angel Diaz-Lagares, Manel Esteller, Juan Sandoval, Ignacio Melero. Methylation changes in DNA of CD8 T cells following CD137 costimulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 612. doi:10.1158/1538-7445.AM2017-612
Abstract 614: VB4-845 tumor cell killing in a combination study with the anti-PD-1, Nivolumab
Authors: Dillon Rachelle Lee , Chooniedass Shilpa , Premsukh Arjune , MacDonald Glen , Cizeau Jeannick , Adams Gregory A.
Published: July 2017

VB4-845 is a Targeted Protein Therapeutic (TPT) comprised of a scFv fragment specific for the Epithelial Cell Adhesion Molecule (EpCAM) genetically fused to a truncated form of Pseudomonas exotoxin A (ETA), via a flexible linker. Due to the inherent immunogenicity of the ETA moiety, VB4-845 is only used for the treatment of loco-regionally accessible tumors and is currently in a Phase III clinical trial for the treatment of high grade, non-muscle invasive bladder cancer. In a Phase I study in late stage squamous cell carcinoma of the head and neck, direct injection of VB4-845 led to shrinkage of the principal injected tumor, as well as non-targeted tumors in some patients, suggesting the activation of a T cell-mediated anti-tumor response through cross-priming. Only cells undergoing immunogenic cell death, or ICD, are capable of stimulating cross-priming. ICD is characterized by distinct “eat-me” signals such as the release of ATP and HMGB1, and cell surface translocation of calreticulin, an endoplasmic reticulum chaperone protein. To evaluate whether VB4-845-mediated cell killing results in ICD events, treated tumor cell lines were assessed for these distinct signaling molecules. In vitro studies using flow cytometry and ELISA showed both translocation of calreticulin and release of ATP and HMGB1 by VB4-845-treated tumor cells. Immune T cell activation via an ICD pathway suggests a complimentary outcome when combined with checkpoint inhibitors. A study was performed in PDX tumor-bearing NOG mice reconstituted with a human immune system to assess the combination of intratumoral injection of VB4-845 with an anti-PD1 antibody, Nivolumab, given i.p. VB4-845 showed growth suppression of the injected tumor whereas the growth delay of the contralateral, non-injected tumor was more pronounced with the addition of Nivolumab. In summary, the data presented suggests that VB4-845 mediates tumor cell killing by an ICD pathway and that the resulting cross-priming effect can enhance the anti-tumoral activity of immune checkpoint inhibitors.

Citation Format: Rachelle Lee Dillon, Shilpa Chooniedass, Arjune Premsukh, Glen MacDonald, Jeannick Cizeau, Gregory A. Adams. VB4-845 tumor cell killing in a combination study with the anti-PD-1, Nivolumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 614. doi:10.1158/1538-7445.AM2017-614
Abstract 625: Eradication of cancer cells by T-cell receptor-engineered T cells targeting neoantigens/oncoantigens
Authors: Matsuda Tatsuo , Kato Taigo , Ikeda Yuji , Leisegang Matthias , Yoshimura Sachiko , Hikichi Tetsuro , Harada Makiko , Zewde Makda , Park Jae-Hyun , Schreiber Hans , Kiyotani Kazuma , Nakamura Yusuke
Published: July 2017

Cytotoxic T lymphocytes (CTLs) play critical roles in cancer-immune responses, and functional characterization of CTLs and their cancer-specific antigens will facilitate cancer immunotherapies. Immunogenic peptides, which can be derived from oncogenic proteins specifically expressed in cancer cells but not expressed in normal organs except testis (oncoantigens), or from peptides with somatic nonsynonymous mutations (neoantigens), are known as good targets for CTLs to eradicate cancer cells. In this study, we aimed to establish a method to efficiently identify oncoantigen/neoantigen-specific CTLs. Firstly, we screened candidate HLA-A2402-restricted oncoantigen/neoantigen peptides by in silico prediction of their binding affinity to MHC class I molecules. We conducted an in-vitro stimulation of CD8 lymphocytes carrying HLA-A24:02 allele by each peptide, and then confirmed clonal expansion of the peptide-specific CTLs by TCR repertoire sequencing analysis, interferon-γ enzyme-linked immunospot (ELISPOT) and/or peptide-HLA multimer assays. After identification of TCR alpha-beta pairs, we conducted retroviral transduction and prepared the TCR-engineered T cells to evaluate their cytotoxic activities against cancer cells. As oncoantigens, we isolated the CTLs for FOXM1 and UBE2T from healthy donors, and found these CTLs showed strong cytotoxicity against HLA-A2402-positive cancer cells expressing target proteins, but not against HLA-unmatched cancer cells. Similarly, the TCR-engineered T cells for FOXM1 and UBE2T showed killing effects for only HLA-A2402-positive cancer cells. Neoantigen-specific TCR-engineered CTLs also exhibited the mutated peptide-specific response, but did not cross-react to the nonmutated peptide. In addition, neoantigen-specific cytotoxicity was observed against HLA-A2402-positive cancer cells expressing the proteins with target somatic mutations. In conclusion, we developed the pipeline to screen possible onconatigens/neoantigens and establish antigen-specific TCR-engineered CTLs from peripheral blood lymphocytes. Our approach provides a promising strategy to develop personalized immunotherapies using onconatigen/neoantigen-reactive TCR-engineered T cells to treat cancer.

Citation Format: Tatsuo Matsuda, Taigo Kato, Yuji Ikeda, Matthias Leisegang, Sachiko Yoshimura, Tetsuro Hikichi, Makiko Harada, Makda Zewde, Jae-Hyun Park, Hans Schreiber, Kazuma Kiyotani, Yusuke Nakamura. Eradication of cancer cells by T-cell receptor-engineered T cells targeting neoantigens/oncoantigens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 625. doi:10.1158/1538-7445.AM2017-625
Abstract 645: Immunological landscape of non-small cell lung cancer (NSCLC): PD-1 expression in lung TILs correlate with co-expression of other checkpoint inhibitor molecules
Authors: Jeong Se W. , Bullock Timothy
Published: July 2017

Lung cancer is the leading cause of annual cancer related mortality in the United States. More than 150,000 people in the U.S. will die from lung cancer this year which will lead to roughly as many deaths as breast, prostate, colon, and pancreatic cancers combined. Lung cancer is also of global concern as tobacco and cigarette use, a significant known risk factor, in developing countries have risen significantly. Stage II and III non-small cell lung cancer (NSCLC) patients often have a 5-year disease free survival of less than 50% even after surgical resection and post-operative chemotherapy and a dismal 5% overall survival for patients with stage IV metastatic disease. Even with the advent of PD-1/PDL-1 checkpoint blockade immunotherapies, only about 20% of patients are responsive to the treatment. This limited scope of success may be due to our lack of understanding the immune landscape of NSCLCs. Therefore, we set out to characterize the immune composition from surgical lung resections via flow cytometry. We found that NSCLCs have a diverse set of checkpoint inhibitor and co-stimulatory molecules expressed by T cells, including PD-1, TIM-3, and TIGIT. Interestingly, even though PD-1 was elevated compared to a normal donor, the expression level was lower than either breast or melanoma tumor samples. We also discovered that PD-1 heavily correlates with co-expression of other checkpoint inhibitor molecules. In our regulatory T cell analysis, FoxP3+ populations have a profound presence in these lung tumors suggesting there is a highly suppressive environment for tumor infiltrating lymphocytes (TILs). Our lung resection screenings furthermore suggests a dynamic range of both frequency and proportion of CD3-CD56+ natural killer (NK) cells. Finally, our data indicates there is a large population of CD11b+CD33+ myeloid-derived suppressor cells (MDSCs) in the tumors, with varying degrees of arginase-1 expression, a functional marker for MDSCs. Altogether, these findings in concert suggest there are potential goldmine of opportunities to target molecules on different immune cell populations. This multi-modal approach, directing combination of immunotherapies that influences multiple immune compartments yet complement each other, may have significant implications for future clinical studies.

Citation Format: Se W. Jeong, Timothy Bullock. Immunological landscape of non-small cell lung cancer (NSCLC): PD-1 expression in lung TILs correlate with co-expression of other checkpoint inhibitor molecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 645. doi:10.1158/1538-7445.AM2017-645
Abstract 647: Bortezomib enhances CD8+ T Lymphocyte antitumor effector function: Potential mechanism(s) via notch regulation
Authors: Renrick Ariana N. , Thounaojam Menaka C. , Thomas Portia , Pellom Samuel T. , Shanker Anil
Published: July 2017

The immunosuppressive tumor microenvironment disturbs host antitumor immunity by multiple mechanisms including interference with the Notch system, which is important for various metazoan cell fate decisions and hematopoietic cell differentiation and function. We observed that treatment with the proteasome inhibitor bortezomib in mice bearing various solid tumors resulted in an upregulated expression of various Notch signaling components in lymphoid tissues, thereby increasing CD8+ T lymphocyte IFNγ secretion and expression of effector molecules, perforin and granzyme B, as well as the T-box transcription factor eomesodermin. Of note, bortezomib reversed tumor-induced downregulation of Notch receptors, Notch1 and Notch2, as well as increased the levels of cleaved Notch intracellular domain (NICD) and downstream targets Hes1 and Hey1 in tumor-draining CD8+ T cells. Moreover, bortezomib promoted CD8+ T cell nuclear factor-κB (NFκB) activity by increasing the total and phosphorylated levels of the IκB kinase and IκBα as well as the cytoplasmic and nuclear levels of phosphorylated p65. Even when we blocked NFκB activity by Bay-11-7082, or NICD cleavage by γ-secretase inhibitor, bortezomib significantly increased expression of Notch Hes1 and Hey1 genes as well as perforin, granzyme B and eomesodermin in activated CD8+ T cells. Data suggest that bortezomib can rescue tumor-induced dysfunction of CD8+ T cells by its intrinsic stimulatory effects promoting NICD-NFκB crosstalk. We are also elucidating components of microRNA regulation affecting NICD-NFκB crosstalk. Our preliminary data suggest that bortezomib is also able to positively regulate miR-155 expression in CD8+ T cells from mice bearing tumor. As well as, miR-155 suppression downregulates bortezomib-induced increase in Notch target genes in T cells. We are currently investigating alternative proteasome inhibitors in order to understand whether bortezomib’s effect on miR-155 expression in CD8+ T cells is specific to bortezomib or primarily conducive to a proteasome inhibition effect. These findings provide novel insights on using bortezomib not only as an agent to sensitize tumors to cell death, but also to provide lymphocyte-stimulatory effects, thereby overcoming immunosuppressive actions of tumor on antitumor T cell functions.

Citation Format: Ariana N. Renrick, Menaka C. Thounaojam, Portia Thomas, Samuel T. Pellom, Anil Shanker. Bortezomib enhances CD8+ T Lymphocyte antitumor effector function: Potential mechanism(s) via notch regulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 647. doi:10.1158/1538-7445.AM2017-647
Abstract 671: Prognostic importance of EGFR expression and Kras gene mutations in gallbladder carcinoma
Authors: Singh Anjali , Najmi Abul Kalam , Mishra Pramod , Talikoti Majid
Published: July 2017

Gallbladder carcinomas involve multiple cascades of oncogenes like EGFR and Kras. Activation of Kras downstream pathways predict the sensitivity to anti EGFR treatment. In 2012, for the first time it was established that GBC and cholangiocarcinomas have their unique somatic genomic landscape and thus be studied independently. This study evaluates whether EGFR is a prognostic factor in GBC and identifies the frequency of codon 12 and codon 13 Kras mutations along with its association with subject survival. Seventy two GBC curative resections from North India were immunostained with anti EGFR monoclonal antibody and assessed for codon 12 and 13 KRAS mutation by real time PCR. Strong EGFR expression was observed in 25.4percent cases. About 28/72; G12C, 17/72; G12R, 10/72; G12D and 18/72; G13D cases harboured point mutations in Kras gene, which was significantly higher as compared to control tissues. Multivariate analysis revealed that EGFR expression (p=0.01; HR=3.14; 95percent CI=1.28-7.71) and codon-13 mutation (p=0.001; HR=38.34; 95percent CI=4.90-300.01) had a significant impact on survival and were independent prognostic factors in GBC. Our study suggests that EGFR expression and codon 13 mutations are independent prognostic factors in the selected GBC cohort and showed a high frequency of Kras codon 12 mutations as well.

Citation Format: Anjali Singh, Abul Kalam Najmi, Pramod Mishra, Majid Talikoti. Prognostic importance of EGFR expression and Kras gene mutations in gallbladder carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 671. doi:10.1158/1538-7445.AM2017-671
Abstract 673: Exploration of tissue morphologies in breast cancer samples using unsupervised machine learning
Authors: Turkki Riku , Bychkov Dmitrii , Linder Nina , Isola Jorma , Joensuu Heikki , Lundin Johan
Published: July 2017

We applied a machine learning approach for exploration of tissue morphology in hematoxylin and eosin (H&E) stained breast cancer tissue microarray (TMA) samples. We then investigated whether the morphological categories produced were associated with clinically relevant molecular biomarkers and 10-year overall survival.

The data set comprises digitized (0.22 µm/pixel) and H&E stained TMA spots from tumor samples of 490 women who were diagnosed with primary breast cancer within a Finnish breast cancer database (FinProg) collected in 1991 and 1992. In order to quantitatively describe the tissue morphologies of the TMA spots, we divided the tissue images into rectangular sub-images (224x224 pixels), and extracted features with a pre-trained convolutional neural network. We then clustered the sub-images (n=147,266) with a non-linear data embedding algorithm that creates a two-dimensional mapping of the tissue morphologies. Lastly, we defined a quantitative profile for each tumor, describing the morphologies within the tissue spot image by dividing the two-dimensional map of morphologies into 128 separate clusters with k-nearest neighbor clustering.

Visual inspection of the two-dimensional embedding of tissue spot images verified that the morphologies clustered coherently, i.e. similar looking sub-images formed distinct clusters in the map. Interestingly, some morphological patterns were strongly associated with tumor estrogen receptor content, progesterone receptor content, human epidermal growth factor receptor 2 status, and the proliferation marker Ki-67 status (p<0.0001 for each comparison). In exploratory analyses we identified one morphological category that was associated with a favorable 10-year overall survival with a risk ratio of 0.68 (CI95% 0.53-0.89, p=0.002, power = 0.87).

Our work demonstrates that unsupervised machine learning can be applied to explore and better understand the role of morphological patterns in breast cancer. Methods that quantitatively assess the morphology of cancer tissue may complement molecular biomarkers and potentially reveal novel prognostic and predictive factors.

Citation Format: Riku Turkki, Dmitrii Bychkov, Nina Linder, Jorma Isola, Heikki Joensuu, Johan Lundin. Exploration of tissue morphologies in breast cancer samples using unsupervised machine learning [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 673. doi:10.1158/1538-7445.AM2017-673
Abstract 74: Discovery of new therapeutic monoclonal antibodies to challenging GPCRs, ion channels and transporters
Authors: Stafford Lewis J. , Chambers Ross , Willis Sharon H. , Hall Moniquetta , Screnci Brad , Mabila Manu , Tucker David , Barnes Trevor , Fong Rachel , Ettenger Andrew , Pfaff Jennifer , Sulli Chidananda , Molino Nicholas , Hudacek Andrew , Doranz Benjamin J. , Rucker Joseph
Published: July 2017

The objective of this work was to evaluate the ability to generate panels of monoclonal antibodies against a set of highly challenging targets including GPCRs (CB1, C5AR, CXCR5 and CGRPR), transporters (GLUT4), and ion channels (P2X3). Integral membrane proteins are important drug targets and monoclonal antibodies (MAbs) directed against them are highly sought for therapeutic purposes. However, the complex structure of multispan membrane protein targets makes the discovery of these MAbs especially challenging. To address this need, Integral Molecular has developed the MPS Discovery Engine® to enable the isolation, characterization, and engineering of monoclonal antibodies for GPCRs, ion channels, and transporters. MPS utilizes a collection of technologies to address each of the barriers to monoclonal antibody development against the native extracellular epitopes of multispan membrane proteins. These include, antigen engineering to attain high levels of surface expression, DNA and Lipoparticle immunization to present native epitopes to the immune system, diverse immunization host species to deal with highly conserved proteins, Lipoparticles (high concentration native membrane proteins) to enable phage display and microfludic B-cell isolation, and shotgun mutagenesis (comprehensive Alanine scanning) for epitope mapping. Using the MPS Discovery Engine® we were able to successfully generate large panels of antibodies to the targets that were able to bind to the native extracellular epitopes on cells by flow cytometry. A subset of the antibodies had antagonist activity. With this technology we have the ability to target intact, conformation specific, and functional antibodies to complex membrane proteins.

Citation Format: Lewis J. Stafford, Ross Chambers, Sharon H. Willis, Moniquetta Hall, Brad Screnci, Manu Mabila, David Tucker, Trevor Barnes, Rachel Fong, Andrew Ettenger, Jennifer Pfaff, Chidananda Sulli, Nicholas Molino, Andrew Hudacek, Benjamin J. Doranz, Joseph Rucker. Discovery of new therapeutic monoclonal antibodies to challenging GPCRs, ion channels and transporters [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 74. doi:10.1158/1538-7445.AM2017-74
Abstract 815: Using CRISPR/Cas9 to generate isogenic cell lines and reference standards for applications in cancer diagnostics
Authors: Jin Huanyu , Cheng Xin , Nguyen Nga , Zheng Qi , Shi Mi , Hilmer Andrew , Yuan Yuan , Chen-Tsai Ruby Yanru , Kong Ling-Jie
Published: July 2017

We are entering an era of precision medicine, in which there are significant initiatives to employ companion diagnostics and molecular profiling to inform clinical decisions in therapeutic treatment. In order for these initiatives to work as effectively as possible, there is a need for molecular reference materials that can help facilitate precision assay development. Cell-line based reference standards are ideal for such applications because they represent a reproducible and renewable source of control materials that can be employed for assay development, quality control, or lead compound discovery. With this in mind, we have used the CRISPR/Cas9 platform to engineer series of isogenic cell lines that feature 51 recurrent mutations in the MAPK signaling pathway. These cell lines contain footprint-free, homozygous mutations in the kinase domains of the EGFR, KRAS, and BRAF genes in HCT116 and RKO cancer cell lines, with 32 mutations in EGFR, 12 mutations in KRAS, and 7 mutations in BRAF. The mutations include precise deletions, point mutations and small fragment insertions, and these engineered cell lines have been further employed for the development of derivative reference materials, including genomic DNA (gDNA) and FFPE cell line blocks. Such materials can be employed as positive and negative controls in diverse assay platforms, including next generation sequencing (NGS), quantitative PCR (qPCR), in situ hybridization (ISH), and others. We present quality control data for these reference materials, which demonstrate excellent consistency and uniformity, as well as examples of the application of these materials in molecular assay validation. We conclude that using CRISPR/Cas9 technology to generate molecular reference standards is a very efficient and precise method in generating footprint-free, biorelevant mutations for companion diagnostic assays.

Citation Format: Huanyu Jin, Xin Cheng, Nga Nguyen, Qi Zheng, Mi Shi, Andrew Hilmer, Yuan Yuan, Ruby Yanru Chen-Tsai, Ling-Jie Kong. Using CRISPR/Cas9 to generate isogenic cell lines and reference standards for applications in cancer diagnostics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 815. doi:10.1158/1538-7445.AM2017-815
Abstract 893: Systematic assessment of spliceosome components as drivers of breast cancer progression
Authors: Koedoot Esmee , Dévédec Sylvia Le , Water Bob van de
Published: July 2017

Accumulating evidence indicates that RNA splicing is involved in different steps of carcinogenesis. Although the critical role of a limited number of splicing factors has been demonstrated in several cancer studies, the function of many spliceosome components is still unknown. In breast cancer samples higher expression levels of a subset of spliceosome components consistently correlated with more aggressive tumor types in patient microarray as well as RNA sequencing data from The Cancer Genome Atlas (TCGA). We further systematically evaluated the effect of siRNA-mediated knockdown of 244 individual splicing factors on both cell migration and proliferation in two aggressive breast cancer cell lines MDA-MB-231 and Hs578T using high throughput microscopy. Primary candidates were validated with four single siRNAs and classified based on their effects on migration and proliferation. Knockdown of validated spliceosome factors such as SNRPG and SNRPA1 affected the levels of small nuclear RNAs, the main catalyzers of the spliceosome. Moreover, high expression of these key candidate hits is associated with poor breast cancer metastasis free survival. To investigate the downstream mechanisms and splicing patterns controlled by our candidate genes we performed RNA sequencing on both control and siRNA mediated knockdown cells. Interestingly, knockdown of our hits affect extracellular matrix pathways that are essential in tumor cell migration and invasion. Altogether our data indicate a key role for spliceosome factors in the control of breast cancer progression which provide new leads for future therapeutic intervention.

Citation Format: Esmee Koedoot, Sylvia Le Dévédec, Bob van de Water. Systematic assessment of spliceosome components as drivers of breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 893. doi:10.1158/1538-7445.AM2017-893
Abstract 927: Cancer stem cell proliferation in human prostate cancer cells utilizing a new defined 3D spheroid culture system
Authors: Asbrock Nick , Chu Vi , Saito Kan
Published: July 2017

Solid tumors grow in a three-dimensional (3D) spatial conformation, resulting in a heterogeneous exposure to oxygen and nutrients as well as to other physical and chemical stresses. To mimic the 3D spatial conformation, 3D in vitro culture models have been used in cancer research since the diffusion-limited distribution of oxygen (hypoxia), nutrients, metabolites, and signaling molecules is not mimicked in conventional two-dimensional (2D) monolayer cultures. One of the 3D in vitro culture models, the tumorsphere culture is an emerging model for studying and expanding the cancer stem cell (CSC) population. E006AA cell line is a spontaneously immortalized cell line derived from a Gleason 6 localized prostate cancer in a hormone-naive prostate cancer patient of African American descent. This cell line expresses androgen receptor and was repeatedly reported to be non-tumorigenic in nude mice. However this cell line forms continuously growing tumor in NOG-SCID triple-deficient mice (i.e., NOG-SCID mice not having NK, B, and T-cells) and an establishment of highly tumorgenic subline E006AA-hT was reported suggesting this cell line has a potential for CSC proliferation by 3D sphere cultures. Here we report tumorsphere cultures of E006AA cells by a new 3D spheroid culture media system. The cells showed continuous proliferation supported during serial passage of 3D tumorsphere cultures. In addition to the stable proliferation, increases of Aldehyde Dehydrogenase (ALDH) expressing CSC population were observed with the increase of passages of 3D tumorsphere cultures.

Citation Format: Nick Asbrock, Vi Chu, Kan Saito. Cancer stem cell proliferation in human prostate cancer cells utilizing a new defined 3D spheroid culture system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 927. doi:10.1158/1538-7445.AM2017-927
Abstract 934: EGFR induces ILF3 and G9a expressions to maintain Oct4-mediated stemness property in lung cancer
Authors: Cheng Chun-Chia , Chang Jungshan , Lin Huan-Chau , Ho Ai-Sheng , Lim Ken-Hong , Chang Chun-Chao , Chang Yi-Fang , Wu Cheng-Wen
Published: July 2017

Cancer stem cells survive and lead to tumor recurrence in tumor therapeutic treatments. Lung cancer cells highly express epidermal growth factor receptor (EGFR), which is critical for tumor progression. We hypothesized that EGFR may play a pivotal role in the induction of cancer stemness cells (CSCs). The aim of this study attempted to investigate the molecular mechanism of EGFR-mediated cancer stemness, and to discover the potent therapeutic agents. A high expression level of cancer stemness markers, ALDH1, CD133, Oct4, and Nanog was observed in tumorspheres derived from HCC827. External EGF further elevated the expression of Oct4 in HCC827 cells. Moreover, chemical inhibition and genetic knockdown of EGFR reduced the Oct4 expression and also tumorsphere formation. We found that inhibitors targeting to EGFR (afatinib), ILF3 (YM155), and G9a (UNC0642) are potential to reduce the formations of tumorspheres. The results revealed that EGFR triggered the formation of tumorspheres through elevating the activations of G9a-mediated stemness genes. In addition, YM155 effectively inhibited tumor growth and formation of tumorspheres as an anti-lung cancer agent blocking the EGFR-G9a cell initiating program pathway. This study demonstrated that EGFR participated in maintain of cancer stemness property through expressing ILF3 and G9a expressions. YM155 inhibited the EGFR-G9a stemness pathway, and reduced the formation of tumorspheres as a potent anti-lung cancer agent.

Citation Format: Chun-Chia Cheng, Jungshan Chang, Huan-Chau Lin, Ai-Sheng Ho, Ken-Hong Lim, Chun-Chao Chang, Yi-Fang Chang, Cheng-Wen Wu. EGFR induces ILF3 and G9a expressions to maintain Oct4-mediated stemness property in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 934. doi:10.1158/1538-7445.AM2017-934
Abstract 967: Development of fluorescent-cell based high throughput assay to identify novel therapeutics of bladder cancer cells through upregulation of SPARC expression
Authors: Patel Chirayu M.
Published: July 2017

Bladder cancer is the most common malignancy affecting the urinary system with an estimated 76,960 new cases and projected 16,390 deaths in 2016 in the United States. The role of SPARC in various cancers is contextual and differs based on the tumor classification, malignancy and progression. We have recently reported the tumor suppressor effect of SPARC in urinary bladder cancer. We have shown that the loss of SPARC in bladder cancer resulted accelerated bladder carcinogenesis and metastasis multiple pathways involving cell cycle deregulation, inflammation and angiogenesis. We have also reported that SPARC protein expression significantly decreased in the cancerous compartment in advanced human bladder cancer as well as in carcinogen-induced murine urothelial cancer. However, the mechanism of the downregulation of SPARC expression in cancer in general and in bladder cancer in particular is still unraveled. The purpose of our study is to develop a robust, high throughput assay to identify therapeutic candidates as regulators of SPARC expression in bladder cancer. We cloned SPARC promoter in a third generation lentiviral vector coupled with fluorescent GFP or mCherry fluorescent protein to quantitatively measure SPARC expression. We generated stable bladder cancer cell lines expressing SPARC expression in bladder cancer cell lines. UMUC3 bladder cancer cells were transduced with lentiviral vector to generate SPARC-promoter reporter system to screen regulators of SPARC expression. Reporter cells were plated in 96 well plates and treated with 10uM of FDA-approved and natural product drug libraries. Real time monitoring of changes in SPARC expression was done by measuring GFP fluorescence intensity using IncuCyte® ZOOM Live Imaging Analysis System. We identified drugs that consistently exhibited dosage-dependent augmentation or down-regulation of SPARC expression in UMUC3 cells. Our screens identified novel up-regulators of SPARC expression that are both standard of care and novel bladder cancer therapeutics. Our efforts produced an efficient robust assay to identify novel therapeutics and regulators of the tumor suppressor SPARC in the bladder cancer ecosystem that can be used in the adjuvant and neoadjuvant settings.

Citation Format: Chirayu M. Patel. Development of fluorescent-cell based high throughput assay to identify novel therapeutics of bladder cancer cells through upregulation of SPARC expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 967. doi:10.1158/1538-7445.AM2017-967