bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 805: KRASG12D and TP53R167H cooperate to induce pancreatic carcinoma in conditional transgenic Sus scrofta
Authors: Principe Daniel , Overgaard Nana , Diaz Andrew , Torres Carolina , McKinney Ronald , Dawson David , Rund Laurie , Schwind Regina , Grippo Paul J. , Schook Lawrence
Published: July 2017

Although survival has improved in recent years, pancreatic ductal adenocarcinoma (PDAC) remains remarkably virulent with a median survival time of only four months. As there is no effective strategy for the management of PDAC, there is a need for the development of new preclinical strategies. The majority of this work is being done in genetically modified mice, which faithfully recapitulate a variety of pancreatic cancer histotypes. While mice have allowed for unprecedented insight into pancreatic cancer etiology, due to fundamental differences in anatomy and physiology, mouse models of PDAC often fail to accurately predict responsiveness to therapy. As the pig pancreas has more similar anatomical orientation and localization to humans than that of rodents, pigs may serve as a more relevant model for PDAC and other malignancies of the pancreas. As KRAS and TP53 mutations are observed in approximately 95% and 70% of PDAC patients, respectively, we generated Cre/lox transgenic Sus scrofta expressing a LSL-KRASG12D-TP53R167H cassette and administered an Adeno-Cre particle to the pancreas gland parenchyma. This resulted in a mixed histoype of metastatic leiomysarcoma and neoplastic disease of the pancreas. To produce a similar pancreas histoype while avoiding the leiomyosarcoma, we next restricted the Adeno-Cre administration to the main pancreatic duct, which resulted in both PDAC and neuroendocrine carcinoma with no abnormality of the smooth muscle. This serves as the first large animal model of pancreatic carcinogenesis, and given the anatomical/physiological similarity of pigs and humans, may allow for insight into new avenues of research not before possible in rodents.

Citation Format: Daniel Principe, Nana Overgaard, Andrew Diaz, Carolina Torres, Ronald McKinney, David Dawson, Laurie Rund, Regina Schwind, Paul J. Grippo, Lawrence Schook. KRASG12D and TP53R167H cooperate to induce pancreatic carcinoma in conditional transgenic Sus scrofta [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 805. doi:10.1158/1538-7445.AM2017-805
Abstract 81: E2F4/p107 complex regulates chemotherapy resistance in human colorectal cancer stem cells
Authors: Matsubara Junichi , Li Yong F. , Dalerba Piero , Sahoo Debashis , Isobe Taichi , Altman Russ B. , Clarke Michael F.
Published: July 2017

Most cancer patients experience disease progression despite one or multiple lines of treatment with cytotoxic chemotherapy. Evidence from human trials and in vivo experiments on patient-derived xenograft (PDX) tumor lines indicates that cancer stem cells (CSCs) can survive chemotherapy treatment. However, the mechanism of chemotherapy resistance in CSCs is not fully elucidated. To identify new mechanisms of the drug-resistance in colorectal cancer, we used gene-expression microarrays to examine the gene expression profile of CSCs and non-CSCs in four human PDX models of colorectal cancer, both before-and-after treatment with cytotoxic chemotherapy. The gene-expression data were analyzed using Independent Component Analysis (ICA) followed by Transcription Factor Target Genes (TFTG) enrichment analysis, which identified the transcription factor E2F4 as a candidate regulator of chemotherapy resistance in colorectal CSCs. E2F4 was predominantly expressed in the CSC-enriched cell population (CD44+/CD166+ human colorectal cancer cells). E2F4 knock-down sensitizes cancer cells to chemotherapy in three different PDX tumors as well as HT29 cells. Co-immunoprecipitation experiments showed that E2F4 forms a complex with p107 and DP-1, which also contributed to the chemotherapy resistance. Simultaneous knock-down of both E2F4 and p107 enhanced chemotherapy sensitivity more than E2F4 knock-down alone. In normal intestinal crypt cells E2F4 was dispensable for chemotherapy resistance. ChIP-seq and RNA-seq experiments revealed that LDHA is a key downstream target of E2F4. LDHA knock-down sensitizes cancer cells to chemotherapy. High LDHA expression in tumor tissues was an independent poor prognostic factor in colorectal cancer patients (n=466), as well as in non-small cell lung cancer (n=1,926) and breast cancer (n=3,554). Thus, the E2F4/p107-LDHA pathway represents a novel therapeutic target to overcome chemotherapy resistance in CSCs.

Citation Format: Junichi Matsubara, Yong F. Li, Piero Dalerba, Debashis Sahoo, Taichi Isobe, Russ B. Altman, Michael F. Clarke. E2F4/p107 complex regulates chemotherapy resistance in human colorectal cancer stem cells [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 81. doi:10.1158/1538-7445.AM2017-81
Abstract 827: Statistical analyses of stable housekeeping gene expression in cancer post-irradiation
Authors: Wang Albert R. , Iyer Gopal , Brennan Sean , Bourgeois Shay , Armstrong Eric , Shah Pari , Harari Paul M.
Published: July 2017

Ionizing radiation (IR) can induce DNA damage in human cells and result in changes in gene expression. The changes could also include some of the commonly used housekeeping genes (HKG), and thus making them unstable over the time period after being exposed to radiation. Normalization of housekeeping genes is critical for understanding gene expression post-irradiation. Toward the validation of stable HKGs, cancer cell lines from head and neck, non-small cell lung and pancreas were irradiated to 2, 4 and 6 Gy IR doses. Statistical analysis of RNA expression of fourteen HKGs measured at 5 min to 48hrs post-irradiation reveal tradition HKGs such as beta-actin (ACTB) and glucose-6-phosphate-dehydrogenase (G6PD) to be unstable, while TATA-Box Binding Protein (TBP) and Importin 8 (IPO8) was identified as stable HKGs in non-small lung and pancreas cell across all IR doses and time. Interestingly in head and neck cancer, TBP is also found stable across all IR doses and time. The statistical framework used for identification and validation of HKGs can serve as a reliable metric for quantifying gene expression post-irradiation.

Citation Format: Albert R. Wang, Gopal Iyer, Sean Brennan, Shay Bourgeois, Eric Armstrong, Pari Shah, Paul M. Harari. Statistical analyses of stable housekeeping gene expression in cancer post-irradiation [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 827. doi:10.1158/1538-7445.AM2017-827
Abstract 831: TGFβ controls the DNA damage response via miR-182 regulation of BRCA1 and ATM
Authors: Liu Qi , Martinez-Ruiz Haydeliz , Murnane John , Powell Simon N. , Barcellos-Hoff Mary Helen
Published: July 2017

Transforming growth factor β (TGFβ) is a well-documented tumor suppressor; a poorly studied aspect of TGFβ biology is its control of genomic stability. Our prior work showed that TGFβ compromises ATM (ataxia telangiectasia mutated) kinase activity, which mediates recognition and repair of DNA damage. Consequently, inhibiting TGFβ following ionizing radiation increases clonogenic death in vitro and tumor control in vivo in breast, brain, and lung cancer preclinical models. As yet unknown is how TGFβ controls ATM kinase. We recently determined that TGFβ regulates mammary lineage commitment by post-transcriptional control of BRCA1 mRNA and protein by its suppression of miR-182 (Martinez-Ruiz et al., Science Signaling, in press). miR-182-mediated downregulation of BRCA1 impacts DNA repair and sensitivity to PARP inhibitors in cancer cell lines (10.1016/j.molcel.2010.12.005) Here we used two cell culture models to investigate the effect of TGFβ regulation of BRCA1 and miR-182 on DNA damage response. Spontaneously immortalized Tgfb1 heterozygote and wild type fibroblasts from primary mammary epithelial preparations were synchronized in S-phase and exposed to UV radiation before fixation to detect unrepaired DNA damage by quantitative image analysis of 53BP1 foci. Consistent with compromised BRCA1 function, more than twice as many cells with 53BP1 foci were evident in Tgfb1 heterozygote fibroblasts compared to wild type cells (p<0.01). The second model consisted of non-malignant human breast epithelial cell line, MCF10A, stably transduced with a scrambled or miR-182 antagomir, grown under serum free conditions and treated with a small molecule inhibitor of TGFβ type I receptor kinase, LY364937. FOXO3 is a target of miR-182 and thus also regulated by TGFβ. and FOXO3 association with ATM is required for its kinase activity (10.1038/ncb1709). As reported, FOXO3 and ATM co-immunoprecipitated, but was eliminated by TGFβ inhibition and this effect was lost in MCF10A cells expressing a miR-182 antagomir. The impaired DNA repair phenotype induced by TGFβ inhibition can be fully rescued by antagonizing miR-182. These studies support TGFβ as stringent regulator of the DNA damage response via suppression miR-182, which directly targets BRCA1 and indirectly affects ATM activity via FOXO3. We predict that cancers that have lost TGFβ signaling capacity will be genomically unstable due to defective DNA damage control, which is consistent with our studies showing that TGFβ inhibition sensitize cancers to radiation therapy.

Citation Format: Qi Liu, Haydeliz Martinez-Ruiz, John Murnane, Simon N. Powell, Mary Helen Barcellos-Hoff. TGFβ controls the DNA damage response via miR-182 regulation of BRCA1 and ATM [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 831. doi:10.1158/1538-7445.AM2017-831
Abstract 844: PPARD downregulation in cancer cells suppresses metastases by inhibiting tumor angiogenesis and EMT
Authors: Zuo Xiangsheng , Xu Weiguo , Xu Min , Tian Rui , Moussalli Micheline J. Moussalli J. , Mao Fei , Gagea Mihai , Wei Daoyan , Sood Anil K. , Shureiqi Imad
Published: July 2017

PPARD is upregulated in many major human cancers, but the role that its expression in cancer cells has in metastasis remains poorly understood. Here, we show that specific PPARD downregulation or genetic deletion of PPARD in cancer cells significantly repressed metastasis in various cancer models in vivo. Mechanistically, PPARD promoted angiogenesis via interleukin 8 in vivo and in vitro. Analysis of transcriptome profiling of HCT116 colon cancer cells with or without genetic deletion of PPARD and gene expression patterns in The Cancer Genome Atlas colorectal adenocarcinoma database identified novel pro-metastatic genes (GJA1, VIM, SPARC, STC1, SNCG) as PPARD targets. PPARD expression in cancer cells drastically affected epithelial-mesenchymal transition, migration, and invasion, further underscoring its necessity to metastasis. Clinically, high PPARD expression in various major human cancers (e.g., colorectal, lung, breast) was associated with significantly reduced metastasis-free survival. Our results demonstrate that PPARD, a druggable protein, is an important molecular target in metastatic cancer.

Citation Format: Xiangsheng Zuo, Weiguo Xu, Min Xu, Rui Tian, Micheline J. Moussalli J. Moussalli, Fei Mao, Mihai Gagea, Daoyan Wei, Anil K. Sood, Imad Shureiqi. PPARD downregulation in cancer cells suppresses metastases by inhibiting tumor angiogenesis and EMT [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 844. doi:10.1158/1538-7445.AM2017-844
Abstract 846: A novel high-throughput 3D screening system for EMT inhibitors
Authors: Arai Kazuya , Rahman M Mamunur , Itoh Manabu , Masuda Norio , Eguchi Takanori , Nakatsura Tetsuya , Calderwood Stuart K.
Published: July 2017

EMT (epithelial-mesenchymal transition) is a significant event in tumor metastasis and malignancy. Therefore, inhibition of EMT is considered to enable controlling of malignant transformation. In this study, we introduce an innovative three-dimensional (3D) high-throughput (HTS) system that leads to an identification of EMT inhibitors. At first, we established 3D-HTS EMT model with NanoCulture Plate (NCP) that provided a gel-free micro-patterned scaffold for cells forming spheroids naturally. In the NCP-based 3D cell culture system, A549 lung cancer cells migrated, gathered, and then formed multiple spheroids within 7 days. Live cell imaging experiments showed that an established EMT-inducer TGF-β promoted peripheral cells around the core of spheroids to acquire mesenchymal spindle shapes, loss of intercellular adhesion, and migration from the spheroids. Along with such morphological change, EMT-related gene expression signatures were altered, particularly alteration of mRNA levels of ECAD/CDH1, NCAD/CDH2, VIM and ZEB1/TCF8. These EMT-related phenotypic changes were blocked by SB431542, a TGF-βreceptor I (TGFβR1) inhibitor. Inside of the spheroids were highly hypoxic; in contrast, spheroid derived peripheral migrating cells were normoxic, revealed by visualization and quantification using Hypoxia Probe. Thus, TGF-β-triggered EMT caused spheroid hypoplasia and loss of hypoxia. Spheroid EMT inhibitory (SEMTIN) activity of SB431542 was calculated from fluorescence intensities of the Hypoxia Probe, and then was utilized in a drug screening of EMT-inhibitory small molecule compounds. In a pilot screening, 9 of 1,330 compounds were above the thresholds of the SEMTIN activity and cell viability. Finally, two compounds SB-525334 and SU9516 showed SEMTIN activities in a dose dependent manner. SB-525334 was a known TGFβR1 inhibitor. SU9516 was a cyclin-dependent kinase 2 (CDK2) inhibitor, which we showed also had an EMT-inhibitory activity. Taken together, it was shown that this 3D NCP-based HTS system was useful for screening of EMT-regulatory drugs.

Citation Format: Kazuya Arai, M Mamunur Rahman, Manabu Itoh, Norio Masuda, Takanori Eguchi, Tetsuya Nakatsura, Stuart K. Calderwood. A novel high-throughput 3D screening system for EMT inhibitors [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 846. doi:10.1158/1538-7445.AM2017-846
Abstract 848: Netropsin blocks EMT of murine BRAF mutated melanoma cells
Authors: Freitas Juliano , Leng Fenfei , Kos Lidia
Published: July 2017

Melanoma is the most aggressive and deadliest type of skin cancer due to its high propensity to metastasize. Despite current progress in the identification of promising drugs for treating melanoma, drug resistance has emerged as a serious issue indicating that novel therapeutic strategies may be required. It is well documented that the process of converting the epithelial tumorigenic cell to a mesenchymal phenotype, the epithelial-mesenchymal transition (EMT), is responsible for conferring an invasive phenotype to cancer cells. An EMT-like process has been shown to occur during melanoma progression. High-mobility-protein AT-hook 2 (HMGA2) is a multi-function transcription factor linked to oncogenesis and EMT in a variety of cancers. HMGA2 is a small DNA-binding protein with three “AT-hook” DNA-binding motifs that specifically recognize the minor groove of AT-rich DNA sequences. HMGA2 is significantly upregulated in human primary melanoma and metastases, including those with BRAF mutations. We accessed the potential application of netropsin, a potent inhibitor of HMGA2-DNA interactions, as an EMT blocking agent of BRAF mutated melanoma cells. Three murine melanoma cells lines that carry mutations in the BRAF gene and show resistance to vemurafenib (D4M, YUMM1.1, and YUMM1.7) were used. Cells were treated with EMT Inducing Media Supplement along with Netropsin and the expression of E-cadherin as well as their migratory behavior were evaluated. Netropsin treatment of EMT induced cells was not cytotoxic, prevented changes in cell morphology, maintained high levels of E-cadherin expression and decreased migratory behavior. All three cell lines express HMGA2 which continues to be expressed in the tumors resulting from subcutaneous injections in mice. YUMM1.1 cells showed the highest levels of expression of HMGA2 amongst the three cell lines. Interestingly, in our preliminary study only YUMM1.1-derived tumors generated overt metastasis in the lung 30 days after the injections. Altogether our data showed that Netropsin is capable of blocking EMT of murine BRAF mutated melanoma cells. The results of this study will contribute to the development of strategies to prevent EMT that may result in the implementation of more effective therapies to treat melanoma.

Citation Format: Juliano Freitas, Fenfei Leng, Lidia Kos. Netropsin blocks EMT of murine BRAF mutated melanoma cells [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 848. doi:10.1158/1538-7445.AM2017-848
Abstract 852: Bromodomain and extraterminal proteins regulate the epithelial-mesenchymal transition in breast cancer
Authors: Andrieu Guillaume , Denis Gerald V.
Published: July 2017

The epithelial-mesenchymal transition (EMT) is a developmental program that cancer cells often activate to acquire a highly plastic phenotype that promotes invasion, metastasis, but also chemoresistance and cancer stem cell generation. As readers of epigenetic marks, bromodomain and extra-terminal (BET) proteins BRD2, BRD3 and BRD4 participate in the regulation of multiple transcriptional programs implicated in cancer progression. We sought to unravel the roles of BET proteins in EMT in breast cancer.

Despite their homology, we report that BET proteins differentially regulate EMT. Based on an EMT PCR array, we identify a BRD2-specific transcriptional profile that promotes EMT, whereas BRD3 and BRD4 signatures repress this program. These individual signatures are unidentifiable upon pan-BET inhibition using JQ1, reinforcing the necessity to target each BET member separately to better understand their functions. Upon BRD2 depletion, basal-like breast cancer cells, which present a mesenchymal phenotype, exhibit a reduced expression of mesenchymal markers (N-cadherin, vimentin) and re-express epithelial markers (E-cadherin, cytokeratins). Moreover, a large panel of EMT master transcription factors is downregulated in BRD2-depleted cells, including the Snail and ZEB families or Twist. Interestingly, we found that BRD3 or BRD4 depletion leads to the opposite phenotype: an increase of mesenchymal marker expression and repression of the epithelial markers. In luminal A breast cancer cells which present an epithelial phenotype, BRD2 overexpression leads to the expression of mesenchymal markers. Similar results were obtained by depleting BRD3 or BRD4 in these cells, confirming the differential roles of BET proteins in EMT regulation.

Taken together, our results establish that BRD2 positively regulates EMT, whereas BRD3 and BRD4 repress this program. BET proteins possess separate and opposite biological functions, reinforcing the relevance of an individual targeting instead of a pan-BET inhibition using JQ1. We hypothesize that BET proteins modulate EMT through the regulation of its master transcription factors. We propose that the balance of BET proteins presence at the promoters of the EMT genes is a novel mechanism of regulation of this program in breast cancer cells.

Citation Format: Guillaume Andrieu, Gerald V. Denis. Bromodomain and extraterminal proteins regulate the epithelial-mesenchymal transition in breast 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 852. doi:10.1158/1538-7445.AM2017-852
Abstract 862: Atypical protein kinase c inhibitors can repress epithelial to mesenchymal transition (type III) in malignant melanoma
Authors: Ratnayake Wishrawana Sarathi , Acevedo-Duncan Mildred
Published: July 2017

Melanoma is a type of cancer occurs in melanocytes. Approximately 90% of melanoma occurs in skin (cutaneous melanoma) but can rarely arise from the mucosal surfaces or areas which neural cells migrate. Examples are eye, intestine and mouth [Eur. J. Cancer, 69: 39-42 (2016)]. 76,380 of new cases and 10,130 number of deaths are expected in 2016 in the USA due to melanoma [http://seer.cancer.gov/statfacts/html/melan.html (11/05/2016)]. Atypical PKCs contains two structurally and functionally distinct isozymes in human which are PKC-ι (iota) and PKC-ζ (zeta). They are believed to be involved in cell cycle progression, tumorigenesis, cell survival and cell migration. We believe that atypical PKCs play an important role in cell motility of melanoma by involving the signaling pathways which induces EMT-type III (Epithelial to Mesenchymal Transition). In normal melanocytes, PKC-ζ was found in low levels and PKC-ι was not detected. But both proteins are detected in very high levels in malignant melanoma [Melenoma Res. 12:201-209 (2002)]. In the current study, we have investigated the effects of novel atypical PKC inhibitors [4-(5-amino-4-carbamoylimidazol-1-yl)-2, 3-dihydroxycyclopentyl] methyl dihydrogen phosphate (ICA-1) which is specific to PKC-ι and 8-hydroxy-1, 3, 6-naphthalenetrisulfonic acid (Compound-50) which is specific to PKC-ζ on the cell proliferation, apoptosis and cell migration of two malignant melanoma cell lines (SK-MEL-2 and MeWo) compared to a normal melanocyte cell line (PCS-200-013). We showed that both inhibitors can decrease the levels of total and phosphorylated levels of PKC-ζ and PKC-ι. Furthermore, both inhibitors increased the levels of E-cadherin and decreased the levels of Vimentin which is a mesenchymal marker associated with EMT. Treatments with inhibitors altered the levels of CD44, a cell-surface glycoprotein involved in cell-cell interactions, cell adhesion, migration and tumor cell homing during metastasis. These results suggest that both PKC-ι and PKC-ζ are involved in signaling pathways which upregulate EMT and which can be effectively suppress using ICA-1 and Compound-50. Furthermore we established that treatment with ICA-1/Compound-50 induced apoptosis as shown by increasing Caspase-3 levels and decreasing Bcl-2 levels.

Citation Format: Wishrawana Sarathi Ratnayake, Mildred Acevedo-Duncan. Atypical protein kinase c inhibitors can repress epithelial to mesenchymal transition (type III) in malignant melanoma [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 862. doi:10.1158/1538-7445.AM2017-862
Abstract 865: Cytoplasmic polyadenylation element binding protein 2 splice variants have opposing functions in the epithelial to mesenchymal transition pathway
Authors: DeLigio James T. , Chalfant Charles E. , Park Margaret A.
Published: July 2017

Alternative splicing of cytoplasmic polyadenylation element binding protein 2 (CPEB2) generates two isoforms, CPEB2B and CPEB2A, through inclusion/exclusion of exon 4, respectively, in mature mRNA. We previously reported that CPEB2B is essential to establish anoikis resistance (AnR), and promotes metastasis using in vivo orthotopic models. In contrast, CPEB2A decreases AnR and metastatic transformation. In the current studies, we examined the function of CPEB2 isoforms using next generation sequencing analysis of downstream signaling pathways. We found that CPEB2B expression upregulated AnR in breast cancer cells and induced key genes/proteins in the epithelial to mesenchymal transition (EMT) and hypoxic response (hypoxia inducible factor 1α (HIF1α)-dependent) pathways. Inhibition of either TWIST1 or HIF1α reduced the CPEB2B-mediated AnR in cells ectopically expressing CPEB2B, confirming a link between CPEB2 alternative splicing and the induction of the EMT/hypoxia pathways. We then extended our findings, and demonstrated that CPEB2 alternative splicing induced de novo translation of both HIF1α and TWIST1, whereas CPEB2A decreased translation of these factors. Furthermore, CPEB2 isoforms were found to modulate differential polyadenylation site selection of the TWIST1 3’UTR and competed for binding to HIF1α and TWIST1 3’UTR cytoplasmic polyadenylation element (CPE) site sequences. These results demonstrate that CPEB2A and CPEB2B modulate the translation of factors in the EMT/hypoxia pathways in an opposing fashion via differential polyadenylation site selection and subsequent translation.

Citation Format: James T. DeLigio, Charles E. Chalfant, Margaret A. Park. Cytoplasmic polyadenylation element binding protein 2 splice variants have opposing functions in the epithelial to mesenchymal transition 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 865. doi:10.1158/1538-7445.AM2017-865
Abstract 866: The epithelial-to-mesenchymal transition regulates E-selectin ligand activities of breast cancer cells
Authors: Carlson Grady E. , Ostermann Alexander O. , Shirure Venktesh S. , Benencia Fabian , Burdick Monica M.
Published: July 2017

The epithelial-to-mesenchymal transition (EMT) leads to specific stem-like and invasive properties in breast cancer cells. However, it remains unclear whether the EMT modifies sialofucosylated ligands that circulating tumor cells use to attach and adhere to vascular endothelial E-selectin during metastasis. To address this issue, immunohistochemistry and E-selectin microsphere dynamic adhesion assays were initially used to assay 110 cases of breast cancer for EMT biomarkers and E-selectin ligand activity. The expression of E-cadherin, but not N-cadherin or vimentin, directly correlated with E-selectin ligand activity. Thus, it was hypothesized that the EMT, and in turn the mesenchymal-to-epithelial transition (MET), regulate expression of functional E-selectin ligands in breast cancer cells. The ectopic expression or shRNA knockdown of transcription factors (i.e., Snail and Twist) in target cell lines were used to induce EMT or MET, respectively. Shear flow adhesion assays and flow cytometry demonstrated that cells with epithelial phenotypes had greater E-selectin ligand activities and greater expression of sialofucosylated carbohydrates compared to cells with mesenchymal phenotypes. Moreover, glycoprotein E-selectin ligand activities of breast cancer cells were more affected by the EMT compared to glycolipid E-selectin ligand activities. Assessing expression of α1,3- and α1,4- fucosyltransferases (FUT) at the RNA level using qRT-PCR revealed that FUT3 and FUT6 gene expression were decreased via the EMT, corresponding to the lowered E-selectin ligand activities observed in mesenchymal phenotype cells. Altogether, the results of this investigation demonstrate that the EMT, as well as the MET, mechanistically regulate the expression of functional E-selectin ligands expressed by breast cancer cells.

Citation Format: Grady E. Carlson, Alexander O. Ostermann, Venktesh S. Shirure, Fabian Benencia, Monica M. Burdick. The epithelial-to-mesenchymal transition regulates E-selectin ligand activities of breast cancer cells [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 866. doi:10.1158/1538-7445.AM2017-866
Abstract 88: The role of RBPMS in cisplatin resistant ovarian cancer
Authors: Báez-Vega Perla M. , Valiyeva Fatma , Santiago Ginette , Vivas-Mejia Pablo
Published: July 2017

Epithelial ovarian cancer is the fifth leading cause of cancer-associated deaths among women in the United States. High-grade serous ovarian cancer (HGSOC) is the most common type of epithelial ovarian cancer and accounts for 70-80% of ovarian cancer patient deaths. RNA-Binding Protein With Multiple Splicing (RBPMS) is a member of a family of proteins that bind to the nascent RNA transcripts and regulate their processing, including the pre-mRNA splicing and the transport, localization, and stability of the RNA molecules. However, little is known about the biological function of RBPMS family in ovarian cancer. We hypothesized that decreasing the expression of RBPMS in high-grade serous ovarian cancer increases ovarian cancer progression and platinum-based drug resistance. Quantitative real-time PCR (qPCR) and Western blot (WB) analysis were conducted to determine the basal mRNA and protein expression of RBPMS in ovarian cancer cells. The invasion ability of the ovarian cancer cells after targeting RBPMS with silencing RNA (siRNA) was determined by invasion assays. The proliferation rate of the ovarian cancer cells overexpressing RBPMS was determine by colony formation assay. RBPMS is highly expressed in A2780 cells when compared to the cisplatin resistance ovarian cancer cells and HGSOC cancer cells. Targeting RBPMS increased the invasion ability of the A2780 cells. Cisplatin resistant, A2780CP20 cells overexpressing RBPMS showed a decreased in cell proliferation when compared to the A2780CP20 empty vector clones. We described here for the first time the mRNA and protein expression profile of RBPMS in HGSOC and epithelial ovarian cancer cells. These studies will help to better understand the role of RBPMS in ovarian cancer and may support the use of RBPMS as an adjuvant treatment to overcome the platinum-based chemotherapy resistance characteristic of the high-grade serous ovarian cancer.

Note: This abstract was not presented at the meeting.

Citation Format: Perla M. Báez-Vega, Fatma Valiyeva, Ginette Santiago, Pablo Vivas-Mejia. The role of RBPMS in cisplatin resistant 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 88. doi:10.1158/1538-7445.AM2017-88
Abstract 886: An integrative platform for three-dimensional quantitative analysis of spatially heterogeneous metastasis landscapes
Authors: Guldner Ian H. , Yang Lin , Liu Fang , Chen Danny Z. , Zhang Siyuan
Published: July 2017

Metastatic microenvironments are spatially and compositionally heterogeneous. This seemingly stochastic heterogeneity provides researchers great challenges in elucidating factors that determine metastatic outgrowth. Herein, we develop and implement an integrative platform that will enable researchers to obtain novel insights from intricate metastatic landscapes. Our two-segment platform begins with whole tissue clearing, staining, and imaging to globally delineate metastatic landscape heterogeneity with spatial and molecular resolution. The second segment of our platform applies our custom-developed SMART 3D (Spatial filtering-based background removal and Multi-chAnnel forest classifiers-based 3D ReconsTruction), a multi-faceted image analysis pipeline, permitting quantitative interrogation of functional implications of heterogeneous metastatic landscape constituents, from subcellular features to multicellular structures, within our large three-dimensional (3D) image datasets. Coupling whole tissue imaging of brain metastasis animal models with SMART 3D, we demonstrate the capability of our integrative pipeline to reveal and quantify volumetric and spatial aspects of brain metastasis landscapes, including diverse tumor morphology, heterogeneous proliferative indices, metastasis-associated astrogliosis, and vasculature spatial distribution. Collectively, our study demonstrates the utility of our novel integrative platform to reveal and quantify the global spatial and volumetric characteristics of the 3D metastatic landscape with unparalleled accuracy, opening new opportunities for unbiased investigation of novel biological phenomena in situ.

Citation Format: Ian H. Guldner, Lin Yang, Fang Liu, Danny Z. Chen, Siyuan Zhang. An integrative platform for three-dimensional quantitative analysis of spatially heterogeneous metastasis landscapes [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 886. doi:10.1158/1538-7445.AM2017-886
Abstract 894: Heparanase-induced shedding of syndecan-1 promotes cancer cell invasion : prevention by inhibitory synstatin peptide
Authors: Jung Oisun , Rapraeger Alan
Published: July 2017

Syndecan-1 (Sdc1, CD138) is highly expressed in multiple myeloma (MM). Heparanase (HPSE), an enzyme that cleaves heparan sulfate (HS) chains on Sdc1, induces shedding of the Sdc1 ectodomain; this is associated with poor prognosis in this disease. However, the link between Sdc1 shedding and poor prognosis remains unclear. We now show that HPSE-induced shed Sdc1 (sSdc1) engages α4β1 integrin (VLA-4) and vascular endothelial cell growth factor receptor-2 (VEGFR2) to form a ternary receptor complex on the cell surface. This coupling of VEGFR2 to clusters of VLA-4 via sSdc1 leads to autoactivation of VEGFR2 and the subsequent activation of protein kinase A (PKA) bound to the VEGFR2 cytoplasmic domain. PKA activation proceeds via VEGFR2-mediated stimulation of the G-protein coupled cytokine receptor CXCR4 and the subsequent generation of cAMP by its activation of adenylate cyclase 7. This mechanism is prevented by SSTNVEGFR2, a peptide that competes for the VEGFR2 docking site in the Sdc1 ectodomain, thereby disrupting VEGFR2 binding to sSdc1. VLA-4 has been shown to mediate the polarized invasion of a variety of cells due to α4 integrin phosphorylation on Ser988 by PKA, followed by Rac GTPase activation, a process that is restricted to the leading edge of the cell. But how this is confined at the leading edge is wholly unknown. We find that HPSE-mediated shedding of Sdc1 promotes an invasive phenotype by re-localizing VLA-4 and VEGFR2 to the leading edge of migrating myeloma cells as well as human T-acute lymphoblastic leukemia, melanoma, and endothelial cells where VEGFR2-dependent PKA activation causes α4 integrin phosphorylation. These results reveal a novel mechanism in which the expression of HPSE, acting by causing Sdc1 shedding, potentially regulates angiogenesis and the extravasation and invasion of cancer cells and identifies SSTNVEGFR2 as a promising cancer therapeutic.

Citation Format: Oisun Jung, Alan Rapraeger. Heparanase-induced shedding of syndecan-1 promotes cancer cell invasion : prevention by inhibitory synstatin peptide [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 894. doi:10.1158/1538-7445.AM2017-894
Abstract 896: Long term exposure to Oroxylin A inhibits cell migration via suppressing CCL2 in oral squamous cell carcinoma cells
Authors: Tung Jiun-Jia , Lee Tony Jer-Fu , Ku Wei-Ting , Lai Kuo-Chu
Published: July 2017

Oral squamous cell carcinoma (OSCC) is the sixth most common cancer in the world. Like other cancers, the major causes of OSCC-related death are cervical node and distant metastasis. Therefore, it is a critical need to identify new potential therapeutic agents against OSCC migration, invasion, and metastasis. Oroxylin A, a main bioactive flavonoids extracted from Scutellaria radix, has been reported to inhibit migration in breast and non-small-cell lung cancer. In this study, we explored the anti-migration effects of oroxylin A on OSCC cells and investigated its underlying mechanisms. A 24-hour exposure (short-term) of OSCC cells to oroxylin A at noncytotoxic concentrations (5-20 μM) significantly suppressed cell migration using the wound healing assay. Furthermore, a 30-days exposure (long-term) to oroxylin A (20 μM), which still had no cytotoxic effect on OSCC cells, but significantly suppressed more cell migration than that of short-term oroxylin A exposed OSCC cells. To uncover the molecular mechanisms involved in the inhibitory effect of oroxylin A on OSCC migration, cDNA microarray technique and Ingenuity software were used. There were 468 differentially expressed genes, including 112 upregulated and 356 downregulated genes identified in long-term oroxylin A exposed cells compared to untreated OSCC cells. Among them, 77 genes have been reported to be associated with cancer cell migration. Consistent with results from cDNA microarray, we revealed that the expressions of several cell migration-related genes, such as CCL2, S100A9, LCN2, and THBS1 were significantly decreased in long-term oroxylin A exposed OSCC cells using QPCR assay. Western blotting and ELISA results also demonstrated that CCL2 expressions at mRNA and protein levels were significantly decreased in long-term oroxylin A exposed OSCC cells compared to untreated OSCC cells. In the meantime, the expressions of downstream targets of CCL2, including p-ERK, NFκB, MMP2, and MMP9 were also decreased in long-term oroxylin A exposed OSCC cells. These results suggest that long-term oroxylin A treatment inhibits migration may via suppressing CCL2 in OSCC cells.

Citation Format: Jiun-Jia Tung, Tony Jer-Fu Lee, Wei-Ting Ku, Kuo-Chu Lai. Long term exposure to Oroxylin A inhibits cell migration via suppressing CCL2 in oral squamous cell carcinoma cells [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 896. doi:10.1158/1538-7445.AM2017-896
Abstract 902: Cancer-stem-cell (CSC) marker, DCLK1-S, enhances invasive potential of cancer cells by phosphorylating/activating NFATc2: role of COL3A1 and SPARC in mediating metastatic effects of DCLK1-S/NFATc2
Authors: Connell Malaney O. , Sarkar Shubhashish , Spratt Heidi , Widen Steven , Wood Thomas G. , Singh Pomila
Published: July 2017

DCLK1 expression is critically required for colon carcinogenesis in mice, and for maintaining tumorogenic potential of human colon cancer cells (hCCCs) (Singh et al, 2016). Down-regulation of DCLK1, combined with chemoprevention, eliminates CSCs, and avoids colon cancer relapse (Kantara et al, 2014). We recently discovered that long (L) and Short (S) isoforms of DCLK1 (DCLK1-L/DCLK1-S) are transcribed by two separate promoters (5’(α)/IntronV(β)) in the hDCLK1-gene (O’Connell et al, 2015). During adenoma-carcinoma sequence of colon-tumorigeneses, L-isoform becomes silenced by DNA-methylation while S-isoform gets upregulated by many fold (O’Connell et al, 2015). S-isoform specifically imparts invasive potential to cancer cells, unlike L-isoform (Singh et al, 2016); others have similarly reported metastatic potential of DCLK1 expressing cancer cells (Ito et al, 2016). Thus the goal of our studies was to evaluate molecular/genetic pathways mediating invasive effects of DCLK1-S in cancer cells. Isogenic clones of HCT116-cells, wild-type or down-regulated for DCLK1-S (HCT-C/HCT-D), were subjected to next generation sequencing and pathways analysis. SPARC and COL3A1 emerged as two candidate genes/proteins, which were decreased/increased by several fold in response to loss/overexpression of DCLK1-S, respectively. We present data confirming a critical role of COL3A1 and SPARC in mediating metastatic effects of DCLK1-S expression in hCCCs. We additionally discovered that DCLK1-S functions as a specific kinase for the transcriptional factor, NFATc2, and phosphorylates 53SPPS56 motif of NFATc2, resulting in activation of NFATc2 and increased expression of COL3A1. Conclusions. Our novel findings, suggest for the first time, that DCLK1-S expression by colonic tumors in humans, mediates invasive potential of colon cancer cells by phosphorylation/activation of NFATc2, resulting in up-regulation of COL3A1/SPARC; the latter proteins re-model extracellular matrix, assisting unhindered invasion of colon cancer cells.

Note: This abstract was not presented at the meeting.

Citation Format: Malaney O. Connell, Shubhashish Sarkar, Heidi Spratt, Steven Widen, Thomas G. Wood, Pomila Singh. Cancer-stem-cell (CSC) marker, DCLK1-S, enhances invasive potential of cancer cells by phosphorylating/activating NFATc2: role of COL3A1 and SPARC in mediating metastatic effects of DCLK1-S/NFATc2 [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 902. doi:10.1158/1538-7445.AM2017-902
Abstract 908: A role for MST4 in organelle organization and breast cancer cell vascular invasion
Authors: Berens Eric B. , Gilardi Mara , Moretti Matteo , Riegel Anna T. , Wellstein Anton
Published: July 2017

There exist 5 Mammalian Sterile20-like (MST) kinases that are broadly distributed into two subgroups. While MST1 and MST2 have established roles in the canonical Hippo signaling pathway, MST3, MST4, and YSK1 are known to regulate the actin cytoskeleton and cell polarity. Here we clarify distinct roles for MST3 vs. MST4 in breast cancer cell lines. Stable knockdown of MST4, but not MST3, enhances the ability of breast cancer cells to disrupt an endothelial monolayer as well as invade from an endothelial network formed within a microfluidic device. In addition, depletion of MST4 in MDA-MB-231 cells alters their morphology, induces abnormalities in nuclear structure, and affects the organization of cellular organelles like the Golgi apparatus. We conclude that, despite their relative homology, MST4 versus MST3 uniquely impacts the intracellular organelle status and vascular invasive behavior of breast cancer cells.

Citation Format: Eric B. Berens, Mara Gilardi, Matteo Moretti, Anna T. Riegel, Anton Wellstein. A role for MST4 in organelle organization and breast cancer cell vascular invasion [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 908. doi:10.1158/1538-7445.AM2017-908
Abstract 910: Phloridzin docosahexaenoate (PZ-DHA) inhibits breast cancer cell invasion and angiogenesis
Authors: Fernando Wasundara , MacLean Emma , Coyle Krysta , Marcato Paola , Hoskin David W. , Rupasinghe H. P. Vasantha
Published: July 2017

In our previous studies, we showed the selective cytotoxicity, in vitro anti-proliferative and anti-metastatic activities of phloridzin docosahexaenoate (PZ-DHA). PZ-DHA combines phloridzin (PZ), a dihydrochalcone found in apple peels, and docosahexaenoic acid (DHA), an ω-3 fatty acid found in fish oil, through an acylation reaction. Significant suppression of tumor growth resulted from the intratumoral administration of PZ-DHA to non-obese diabetic severe combined immunodeficient (NOD-SCID) female mice xenografted with MDA-MB-231 breast cancer cells. Examination of haematoxylin and eosin-stained tumor sections confirmed that PZ-DHA caused tumor cell death. PZ-DHA also suppressed the invasion of MDA-MB-231 cells through membranes coated with extra cellular matrix proteins, fibronectin and gelatin, in a Boyden chamber assay. The effect of PZ-DHA on the mRNA transcription of the gelatinase, matrix metalloproteinase-2 (MMP-2,) was measured in RT-qPCR analysis, which showed that MMP-2 mRNA expression was inhibited at 12 h and 24 h following PZ-DHA treatments. Furthermore, suppression of MMP-2 protein expression was shown by western blot analysis of cell lysates of PZ-DHA-treated MDA-MB-231 cells. Intraperitoneal administration of PZ-DHA suppressed the growth of primary tumors and metastasis of 4T1 and green fluorescence protein (GFP)-tagged-MDA-MB-231 cells to lungs of BALB/c and NOD-SCID female mice, respectively. Anti-angiogenic activity of PZ-DHA was investigated using human umbilical vein endothelial cells (HUVECs). PZ-DHA suppressed the metabolic activity of HUVECs in MTS assays and the cell death was confirmed using flow cytometric analysis of 7-AAD and Annexin-V-FLUOS/propidium iodide-stained HUVECs. Anti-proliferative activity of PZ-DHA was shown using Oregon Green 488 stained-HUVECs by flow cytometry. PZ-DHA treatment arrested the HUVEC cell cycle at S-phase. PZ-DHA also suppressed the migration of HUVECs in a gap closure assay and inhibited HUVEC tubule formation on a matrigel matrix. For comparison purposes, PZ and DHA were included in all experiments, but in all cases were less effective than PZ-DHA. Taken together, these findings reveal the potential of PZ-DHA to inhibit triple-negative breast cancer progression. We are currently working on identifying potential phase I and II metabolites of PZ-DHA and understanding its pharmacokinetic parameters.

Citation Format: Wasundara Fernando, Emma MacLean, Krysta Coyle, Paola Marcato, David W. Hoskin, H. P. Vasantha Rupasinghe. Phloridzin docosahexaenoate (PZ-DHA) inhibits breast cancer cell invasion and angiogenesis [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 910. doi:10.1158/1538-7445.AM2017-910