bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4965: Multiple ubiquitin-conjugating enzymes modulate the ubiquitination and downregulation of the EGFR by the Cbl RING finger ubiquitin ligase
Authors: Ma Ke , Ryan Philip , Klevit Rachel , Lipkowitz Stanley
Published: August 2015

Receptor tyrosine kinases (RTKs) are known drivers of malignant transformation. Many RTKs (e.g., EGFR, MET) are negatively regulated by ubiquitination and degradation mediated by Cbl proteins, a family of RING finger (RF) ubiquitin ligases (E3s). Loss of Cbl protein function is associated with malignant transformation driven by increased RTK activity. E3s such as the Cbl proteins confer specificity to the ubiquitination process and direct the conjugation of ubiquitin to one or more lysines on the target proteins in collaboration with ubiquitin-conjugating enzymes (E2s). We used enzymatic and yeast two-hybrid assays to characterize the E2s that can interact with Cbl proteins. Using an in vitro E3 assay, we found that only the Ube2d family of E2s mediates autoubiquitination of the Cbl proteins. Subsequently, using the yeast two-hybrid system, we found that Ube2e1, Ube2e2, Ube2e3, Ube2l3, Ube2u, Ube2w and Ube2z can interact with Cbl even though they do not support autoubiquitination of Cbl in the in vitro E3 assay. Among these E2s, three Ube2e family members and Ube2w bind to the RF domain of Cbl as demonstrated by the loss of interaction when the RF domain is disrupted. This suggests that Ube2e and Ube2w are relevant to the ubiquitination and degradation of substrates by Cbl. Knockdown of Ube2w decreases downregulation of EGFR in Hela cells. In the in vitro E3 assay we found that Ube2w can increase autoubiquitination of Cbl mediated by ube2d2. Surprisingly, we found that knockdown of Ube2e increases downregulation and ubiquitination of EGFR in HeLa cells. Mechanistically we found that three Ube2e members inhibit autoubiquitination of Cbl mediated by Ube2d2 in vitro. Further, we showed that Ube2e does not affect ubiquitin charging of Ube2d2 by the ubiquitin-activating enzyme (E1) in vitro. This suggests that Ube2e does not compete with Ube2d2 for the E1 under these conditions. Thus, our data suggest that Ube2e acts as a positive modulator of EGFR signaling by competing for Cbl with Ube2d2 and thus prevents ubiquitination and downregulation of the EGFR by Cbl in combination with Ube2d2. Together these data demonstrate that there is an E2 network which modulates the ubiquitination and downregulation of the EGFR by Cbl.

Citation Format: Ke Ma, Philip Ryan, Rachel Klevit, Stanley Lipkowitz. Multiple ubiquitin-conjugating enzymes modulate the ubiquitination and downregulation of the EGFR by the Cbl RING finger ubiquitin ligase. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4965. doi:10.1158/1538-7445.AM2015-4965
Abstract 4982: Increased core fucosylation, a glycan alteration associated with cancer, is the result of hepatocyte dedifferentiation
Authors: Mehta Anand S. , Comunale Mary Ann , Rawat Siddhartha , Garner Jessica , Betesh Lucy , Wang Mengjun , Steel Laura , Bouchard Michael
Published: August 2015

Alterations in N-linked glycosylation have been associated with the development of cancer. One such modification, core fucosylation has been observed in many cancers and is actually used clinically in the diagnosis of hepatocellular carcinoma (HCC). However, the reason why core fucosylation is increased in cancer is unclear. In this study, we show that the activation of specific cancer-associated cellular signaling pathways can increase core fucosylation and induce additional glycoform alterations on hepatocyte proteins. Specifically, we were able to determine that increased levels of protein sialylation and alpha-1,6-linked core fucosylation were observed following activation of the beta catenin pathway. Activation of the AKT pathway or induction of hypoxia also resulted in increased levels of fucosylation and sialylation. Furthermore, we show that increased core fucosylation was directly associated with de-differentiation of hepatocytes and with the transition of cells from an epithelial to a mesenchymal state. In conclusion, we show that increased core fucosylation, a modification of N-linked glycoproteins associated with HCC, occurs as a result of the process of cellular de-differentiation. We believe that this knowledge may help in the use of this glycan modification in the management of those with HCC.

Citation Format: Anand S. Mehta, Mary Ann Comunale, Siddhartha Rawat, Jessica Garner, Lucy Betesh, Mengjun Wang, Laura Steel, Michael Bouchard. Increased core fucosylation, a glycan alteration associated with cancer, is the result of hepatocyte dedifferentiation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4982. doi:10.1158/1538-7445.AM2015-4982
Abstract 4984: EMT and breast cancer metastasis driven by plasma membrane fluidity
Authors: Chang Jeffrey T.
Published: August 2015

New therapeutic targets that can inhibit cancer metastasis are desperately needed. To address this issue, we have leveraged a computational approach and performed an unbiased screen for therapies that can inhibit an epithelial-to-mesenchymal transition (EMT) gene expression profile, a phenotype that is believed to promote metastases. This yielded predictions of drugs that can both promote and inhibit an EMT transcriptional profile. Following up on these predictions, we confirmed that these drugs can induce or repress an EMT in a range of breast cancer cell lines. Furthermore, we discovered that they do so by altering the fluidity of the plasma membrane. An increase in plasma membrane fluidity is necessary and sufficient to induce and maintain an EMT state, increased motility, and increased mammosphere forming ability. Therapies that decrease fluidity can inhibit metastasis in xenograft mouse models. Finally, we found that human cancers can regulate their membrane fluidity through cholesterol homeostasis pathways. 41% of human breast cancers have alterations in cholesterol regulatory pathways, and are associated with a 9 year loss in time to distant metastasis. These data demonstrate that plasma membrane fluidity is a cellular attribute that is leveraged by human tumors to increase metastatic capacity, and that it can, in principle, be targeted by therapeutics to prevent metastasis.

Citation Format: Jeffrey T. Chang. EMT and breast cancer metastasis driven by plasma membrane fluidity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4984. doi:10.1158/1538-7445.AM2015-4984
Abstract 5011: generation of rejuvenated murine antigen-specific T cells by reprogramming to pluripotency and redifferentiation
Authors: Ito Fumito , Fusaki Noemi , Saito Hidehito
Published: August 2015

Background: Adoptive cell therapy with autologous antigen-specific T cells is a promising approach for treating patients with chronic viral infections and various cancers. A major limitation of this approach is the difficulty of obtaining sufficient numbers of T cells with the characteristics necessary for in vivo effectiveness. Less-differentiated T cells with long telomeres would be an ideal T-cell subset for adoptive T cell therapy; however, generating large numbers of these “young” T cells is problematic. This limitation of adoptive T cell therapy can be overcome by using induced pluripotent stem cells (iPSCs) that self-renew, maintain pluripotency, and provide an unlimited source of autologous T cells for immunotherapy. However, in vivo antitumor reactivity of iPSC-rejuvenated T cells remains unknown due to the lack of animal model.

Methods: To establish preclinical model of evaluating iPSC-rejuvenated antigen-specific T cells, we reprogrammed OT-1 T cell receptor (TCR) transgenic CD8 T cells using Sendai virus vectors encoding Oct3/4, Sox2, Klf4 and c-Myc transcription factors. Pluripotency and differentiation capacity of generated iPSCs were evaluated.

Results: Twenty days after infection with Sendai virus vectors, we obtained murine embryonic stem cell-like colonies from OT-1 CD8 T cells. Immunofluorescence staining and RT-PCR analyses revealed pluripotency of established colonies. The iPSCs formed embryoid bodies in vitro, and upregulation of marker genes for all three germ layers was detected. The iPSCs differentiated into teratomas in vivo comprising all three embryonic germ layers in immune-deficient NOD/SCID mice. The iPSCs retained the same rearranged configuration of TCR chain genes as the original OT-1 T cells. Furthermore, OT-1 T cell-derived iPSCs differentiated into T cells on delta-like ligand 1 expressing OP9 (OP9/DL1) murine bone marrow stromal cells.

Conclusions: Generation of rejuvenated T cells by reprogramming to pluripotency and redifferentiation is feasible. This type of approach may be useful for adoptive T cell therapy.

We acknowledge DNAVEC Corporation for kindly providing Sendai virus vectors. This study was supported by the University of Michigan Start-up fund.

Citation Format: Fumito Ito, Noemi Fusaki, Hidehito Saito. generation of rejuvenated murine antigen-specific T cells by reprogramming to pluripotency and redifferentiation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5011. doi:10.1158/1538-7445.AM2015-5011
Abstract 502: Copy number alterations identify targeted therapies in preclinical models of osteosarcoma
Authors: Sayles Leanne C. , Breese Marcus , Sweet-Cordero Alejandro
Published: August 2015

Osteosarcoma (OS) is an aggressive pediatric bone malignancy that affects 400 children per year in the United States. It has one of the lowest 5 yr. survival rates of any pediatric cancer. OS is characterized by genomic instability and numerous copy number alterations (CNA). We hypothesized that CNA may be drivers of OS and that targeting cancer genes with CNAs may be a novel approach for treatment of OS. We have established a robust approach for generation of patient-derived xenografts (PDX) from patients with OS. To date, 12 PDX have been established from OS patients. Using whole-genome sequencing (WGS), we have identified possible target CNAs for individual patients and are currently testing them in their corresponding PDX. In one patient, an amplification in c-myc suggested possible susceptibility to the bromodomain inhibitor JQ1. In a second patient sample, amplification of Cyclin D1 in the setting of p16 loss and wild-type Rb suggested responsiveness to a CDK4/6 specific inhibitor. This patient sample also has an Aurora kinase B amplification, suggesting susceptibility to inhibition of this kinase as a therapeutic approach. We treated the corresponding PDX with the drug predicted to target the patient sample based on CNA. In one case, JQ1 treatment inhibited tumor growth in the PDX generated from the patient sample with c-myc amplification. In the second case, Aurora kinase B inhibition arrested tumor growth in the PDX harboring the Aurora kinase B amplification. We are currently testing whether CDK4/6 inhibition is sufficient to inhibit tumor growth as predicted by WGS. As a follow up we are currently submitting several more patient samples and paired PDX for WGS as well as RNAseq to determine other potential targeted therapeutics based on CNA. In summary, our work suggests that WGS can be used to identify potential novel therapies for patients with OS. Further work will be needed to determine whether response in PDX models correlates with clinical response in patients.

Citation Format: Leanne C. Sayles, Marcus Breese, Alejandro Sweet-Cordero. Copy number alterations identify targeted therapies in preclinical models of osteosarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 502. doi:10.1158/1538-7445.AM2015-502
Abstract 5051: Androgen receptor-mediated regulation of p14ARF transcription in prostate tumor cells
Authors: Mudryj Maria , Siddiqui Salma , Libertini Stephen J. , Lombard Alan P. , Mooso Benjamin , D'Abronzo Leandro , Melgoza Frank , Borowsky Alexander , Drake Christiana , Qi LiHong , Ghosh Paramita M.
Published: August 2015

The p14ARF tumor suppressor is often deleted or silenced in malignancies. Prostate tumors are an exception, where p14 expression is elevated. To understand this phenomenon, we assessed the expression of p53 pathway members, which are most effected by p14ARF. The expression of androgen receptor (AR), a pivotal prostate cancer regulator, which is also affected by p14arf and MDM2 was analyzed as well. The studies used archival prostate tumor tissues obtained from prostatectomies performed at the Veterans Affairs-Northern California Health Care System in Mather California between 1996 and 2002 to better define the relationship between these interrelated networks. A prostate tumor tissue array consisting of 78 tumors of differing stages and grades was constructed to evaluate correlations between multiple parameters. Immunohistochemical studies assessed expression of the proliferation marker Ki67, p53, MDM2, MDM4, p14ARF, and the AR in the nuclear and cytoplasmic compartments of tumor and adjacent cells. p53, MDM4, p14ARF and AR were detected in nuclear and cytoplasmic compartments of tumor and non-tumor cells, but were predominantly nuclear. MDM2 expression was primarily cytoplasmic in tumor cells. Multivariate analysis of the immunohistochemical markers identified a strong correlation between expression of p14ARF and AR. Studies utilizing the prostate CWR22 xenograft and LNCaP cell line models revealed that castration or androgen deprivation resulted in reduced p14arf levels and that this effect correlated with a precipitous decline in E2F1-3a levels. In a reciprocal analysis, RB ablation enhanced p14ARF transcription, arguing that the E2F/RB pathway mediates AR-dependent p14ARF expression. However, we also identified an AR binding site located ∼40 KB upstream of the p14ARF gene. Chromatin immunoprecipitation (ChIP) studies showed that in prostate cells this site was bound by AR. ChIP studies also revealed E2F1 and E2F3 were present at the p14ARF promoter. Together, the studies argue p14ARF is a direct transcriptional target of AR and that AR and E2F collaborate to promote p14ARF expression.

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Citation Format: Maria Mudryj, Salma Siddiqui, Stephen J. Libertini, Alan P. Lombard, Benjamin Mooso, Leandro D'Abronzo, Frank Melgoza, Alexander Borowsky, Christiana Drake, LiHong Qi, Paramita M. Ghosh. Androgen receptor-mediated regulation of p14ARF transcription in prostate tumor cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5051. doi:10.1158/1538-7445.AM2015-5051
Abstract 506: TROY-EGFR signaling complex mediates glioblastoma cells invasion and survival
Authors: Roos Alison , Mayo Zachary , Kloss Jean , Tuncali Serdar , Dhruv Harshil , Berens Michael E. , Loftus Joseph C. , Tran Nhan L.
Published: August 2015

Glioblastoma (GBM) is a highly lethal CNS malignancy in adults with a median survival of about 15 months. Effective therapeutic control of GBM is thwarted by the invasive nature of the tumor, which prevents complete surgical removal and results in tumor recurrence. Thus, there is a dire need to develop innovative approaches to target the invasive tumor cells for improved treatment of this disease. Expression of TROY, a member of the TNFR family, increases with glial tumor grade and inversely correlates with patient survival. Increased expression of TROY stimulates glioblastoma cell invasion and increases resistance to temozolomide (TMZ) and radiation treatment. We have demonstrated that TROY forms a novel complex with EGFR and modulates EGFR survival signaling. IHC analysis of GBM specimens showed that tumors with elevated TROY expression had a statistically positive correlation with increased EGFR expression. TROY expression enhanced EGFR phosphorylation, stabilized EGFR surface expression in the presence of ligand, and significantly increased EGF-stimulated GBM cell invasion. Consistent with invasion, co-expression of TROY with EGFR increased cell survival to TMZ. Moreover, we have shown that in cells treated with EGF, the TROY and ErbB4 receptor are recruited to the EGFR complex. Although the role of ErbB4 in GBM is not well defined, inhibition of ErbB4 expression suppressed tumor growth and invasion in multiple tumor subtypes, decreased EGFR surface retention, and limited EGF-induced cell migration. These data suggest that the TROY-EGFR-ErbB4 complex may represent an unappreciated therapeutic target to inhibit glioma invasion and decrease therapeutic resistance.

Citation Format: Alison Roos, Zachary Mayo, Jean Kloss, Serdar Tuncali, Harshil Dhruv, Michael E. Berens, Joseph C. Loftus, Nhan L. Tran. TROY-EGFR signaling complex mediates glioblastoma cells invasion and survival. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 506. doi:10.1158/1538-7445.AM2015-506
Abstract 5063: Lysyl oxidase-like 2 (LOXL2) from cancer-associated fibroblasts stimulates the progression of gastric carcinoma
Authors: Kasashima Hiroaki , Yashiro Masakazu , Kitayama Kisyu , Masuda Go , Kinoshita Haruhito , Sakurai Katsunobu , Toyokawa Takahiro , Kubo Naoshi , Tanaka Hiroaki , Muguruma Kazuya , Ohira Masaichi , Hirakawa Kosei
Published: August 2015

Purpose: Lysyl oxidase families are involved in a variety of pathological processes, including cancer progression. Lysyl oxidase-like 2 (LOXL2) in the tumor stroma was mainly expressed in fibroblasts, which are a major stromal compartment of gastric cancer. Significance of LOXL2 remains unclear in gastric carcinoma. The aim of this study was to clarify the role of fibroblast-derived LOXL2 in the development of gastric cancer.

Methods: Two gastric cancer cell lines, OCUM-12 and NUGC-3, and cancer-associated fibroblasts (CAF) were used in this in vitro study. The effect of fibroblast-derived LOXL2 on the motility of gastric cancer cells was analyzed by wound-healing assay, double-chamber invasion assay, and western blot. The correlation between LOXL2 expression and the clinicopathological features of 548 primary gastric carcinomas was examined by immunohistochemistry.

Results: LOXL2 mRNA expression of CAF, was significantly higher than that of normal fibroblasts. CAF significantly stimulated the migration and invasion of OCUM-12 and NUGC-3 cells. This motility-stimulating ability of CAF was inhibited by LOXL2 siRNA. Western blot analysis indicated that phosphorylation of focal adhesion kinase (FAK) in cancer cells was increased by the conditioned medium from CAF, and was not affected by the conditioned medium from LOXL2 siRNA-treated CAF. Immunohistochemical study indicated that LOXL2 expression in stromal cells was significantly associated with tumor invasion depth, lymph node metastasis, lymphatic invasion, venous invasion, and peritoneal dissemination. The overall survival of all patients with LOXL2-positive stromal cells was significantly poorer than those with LOXL2-negative stromal cells. Multivariable logistic regression analysis revealed that LOXL2 expression in stromal cells was an independent predictive parameter for the overall survival of patients with gastric cancer.

Conclusion: LOXL2 from CAF may stimulate the motility of gastric cancer cells. LOXL2 expression in stromal cells may be a useful prognostic factor for patients with gastric cancer.

Citation Format: Hiroaki Kasashima, Masakazu Yashiro, Kisyu Kitayama, Go Masuda, Haruhito Kinoshita, Katsunobu Sakurai, Takahiro Toyokawa, Naoshi Kubo, Hiroaki Tanaka, Kazuya Muguruma, Masaichi Ohira, Kosei Hirakawa. Lysyl oxidase-like 2 (LOXL2) from cancer-associated fibroblasts stimulates the progression of gastric carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5063. doi:10.1158/1538-7445.AM2015-5063
Abstract 5064: EGFR and HER3 are important in the interaction between lung cancer cells and fibroblasts
Authors: Demuth Christina , Jakobsen Kristine Raaby , Meldgaard Peter , Nielsen Anders Lade , Sorensen Boe Sandahl
Published: August 2015

Introduction: The cancer microenvironment has been recognised as a major factor in the development and treatment of lung cancer. Fibroblasts have gained attention owing to their ability to stimulate the growth of cancer cells and to change the response to chemotherapy. Though, not much is known about the underlying mechanisms.

Methods: Lung cancer cell lines (H1568, Calu-3 and HCC827) and a lung fibroblast cell line (WI-38) were grown in co-culture using a Boyden chamber assay.

Results: The presence of lung fibroblasts markedly increased growth of the lung cancer cells during co-culture. We investigated the activation of 49 different receptor tyrosine kinases and found that H1568 activated only EGFR and HER3. Activation of EGFR and HER3 was confirmed in Calu-3 and HCC827 after co-culture with fibroblasts. To account for this activation, we measured the expression of all EGFR ligands in the cell lines. We found that all lung cancer cell lines had increased levels of amphiregulin mRNA after co-culture with fibroblasts. ELISA confirmed increased protein production. We also found that the amount of available amphiregulin in the media of the co-culture is increased compared to controls.

Conclusion: Lung fibroblasts stimulate the growth of lung cancer cells lines. We suggest that this effect is obtained by activation of EGFR and HER3. Furthermore we show that co-culture induce increased amphiregulin production in the cancer cells. This study identifies EGFR and HER3 as important components in the interaction between lung cancer cells and fibroblasts.

Citation Format: Christina Demuth, Kristine Raaby Jakobsen, Peter Meldgaard, Anders Lade Nielsen, Boe Sandahl Sorensen. EGFR and HER3 are important in the interaction between lung cancer cells and fibroblasts. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5064. doi:10.1158/1538-7445.AM2015-5064
Abstract 5070: Inhibition of collagen receptor discoidin domain receptor-1 (DDR1) reduces colon cancer cell migration and metastasis
Authors: Yuge Ryo , Kitadai Yasuhiko , Shinagawa Kei , Onoyama Mieko , Tanaka Shinji , Yasui Wataru , Chayama Kazuaki
Published: August 2015

Accumulating evidence suggests that a unique set of receptor tyrosine kinases, known as discoidin domain receptors (DDRs), plays a role in cancer progression by regulating the interactions of tumor cells with their surrounding collagen matrix. We investigated the role of DDR1 in the tumor progression of colorectal cancer.

We examined the DDR1 mRNA and protein expression levels in a series of human colorectal cancer cell lines by RT-PCR and Western blotting. We also analyzed the DDR1 protein expression in 27 surgical specimens of colorectal cancer by immunohistochemistry. Migration, invasion, and proliferation assays were conducted with a high-content time-lapse assay system in a series of colon cancer cell lines. Nilotinib, a well-known DDR1 inhibitor, and DDR1-silenced colon cancer cells were used for inhibition of DDR1 tyrosine kinase activity. In an in vivo study, we orthotopically implanted KM12SM human colon cancer cells into the cecum of nude mice and administered nilotinib (100 mg/kg/day) by daily oral gavage. After 4 weeks of treatment, primary tumors and metastatic lesions were resected, and specimens were analyzed histologically.

DDR1 mRNA and protein expression was observed in the human colon cancer cell lines and in cancer cells of most of the surgical specimens. Colon cancer cell lines KM12SM, KM12c, LoVo, and HT29 exposed to nilotinib (20 μM) showed reduced migration. When incubated with type I collagen, however, these cells showed increased migratory activity. Nilotinib treatment significantly inhibited this effect. The migratory activity of DDR1-silenced cells was not affected by nilotinib treatment. The orthotopic xenografts grew invasively as aggressive tumors with a stromal reaction, but under nilotinib treatment, the stromal reaction was reduced, and tumors grew expansively. In addition, the numbers of lymph node metastases were significantly reduced.

Disruption of DDR1 attenuated colon cancer cell migration and led to altered primary tumor morphology and to impaired lymph node metastasis. DDR1 may be a promising therapeutic target in patients with colon cancer.

Citation Format: Ryo Yuge, Yasuhiko Kitadai, Kei Shinagawa, Mieko Onoyama, Shinji Tanaka, Wataru Yasui, Kazuaki Chayama. Inhibition of collagen receptor discoidin domain receptor-1 (DDR1) reduces colon cancer cell migration and metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5070. doi:10.1158/1538-7445.AM2015-5070
Abstract 508: Identification of FRA-1 as a potential driver of pro-invasive properties in pancreatic cancer in conjunction with MUC1
Authors: Hanson Ryan L. , Hollingsworth Michael A.
Published: August 2015

The transmembrane mucin, MUC1, contributes to the progression of pancreatic cancer, in part by conducting signals from the cell surface that regulate expression of molecules associated with aggressive tumor behavior. We investigated transcriptional co-regulatory effects mediated by interactions of the MUC1 cytoplasmic tail with activator protein 1 (AP-1). Previous results suggest that MUC1 regulates the capacity of AP-1 to bind specific promoter elements and regulate genes that contribute to tumor progression. The DNA binding activity of AP-1 is controlled in several ways including composition of the protein dimer. The precise mechanism by which MUC1 alters transcriptional complexes remains unknown. We sought to determine how MUC1 influences the formation of AP-1 complexes and potential downstream effects.

We found MUC1 increased c-Jun protein levels, the major component of AP-1, in a manner largely independent of transcriptional upregulation (mRNA upregulation was observed in only one cell line of several analyzed). Formation of the AP-1 dimer was assessed by quantitative and semi-quantitative assays. MUC1 promoted the association of c-Jun with the Fos protein, FRA-1. This association was promoted by enhanced ERK2 signaling in MUC1 expressing cell lines. In breast cancer, FRA-1 drives an invasive and metastatic phenotype. Overexpression of FRA-1 in pancreatic cancer cell lines promoted migratory and invasive behaviors in vitro. Blocking FRA-1 activity by shRNA knockdown or ERK inhibition decreased migration and invasion. Analysis of gene expression in pancreatic tumor samples and normal, uninvolved tissue [mined from the Gene Expression Omnibus (GEO)] revealed that tumor samples had significantly increased levels of FRA-1 mRNA. A subset of these samples exhibited gene expression patterns consistent with a FRA-1:EMT phenotype previously observed in colorectal cancer cells. We evaluated expression of FRA-1 protein in primary pancreatic tumors, liver metastases, and uninvolved non-malignant pancreas. Robust nuclear localization of FRA-1 was observed in primary and metastatic tumor cells whereas uninvolved pancreas showed lower levels of diffuse cytoplasmic staining.

Based on these results, we hypothesize that FRA-1 plays an important role in a subset of pancreatic cancers by altering the binding profile of AP-1. Due to a dependence on ERK activity for FRA-1 function, we propose that cancers with activating mutations in the RAS-RAF-MEK-ERK cascade are the most likely candidates for utilization of FRA-1.

Citation Format: Ryan L. Hanson, Michael A. Hollingsworth. Identification of FRA-1 as a potential driver of pro-invasive properties in pancreatic cancer in conjunction with MUC1. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 508. doi:10.1158/1538-7445.AM2015-508
Abstract 5092: Midkine as a potential target for combating drug resistance and invasion in melanoma
Authors: Kaur Amanpreet , O'Connell Michael , Webster Marie , Behera Reeti , Dang Vanessa , Ndoye Abibatou , Weeraratna Ashani
Published: August 2015

Melanoma is an aggressive disease. Although about 13% of melanoma cases are diagnosed at metastatic stage, the 5-year survival rate for these patients is 16%. We have previously shown that metastatic melanoma cells express high levels of Wnt5a. Wnt5a is a member of the Wnt family of proteins, which play an important role during development and tumorigenesis. In melanoma, Wnt5a signaling is mediated by an autocrine loop that also modulates the activity of midkine. Midkine is a 16kDa protein that is elevated in many different cancers. Overexpression of midkine promotes the growth, migration, survival and angiogenic capability of the tumors. However, the molecular mechanisms that regulate the activity of midkine are unknown, especially in metastatic melanoma. We asked whether Wnt5a can positively regulate the functions of midkine to promote the progression of metastatic melanoma. To address this, we analyzed melanoma cells for midkine expression and observed that Wnt5a high melanoma cells also express high levels of midkine. Further, an increase in Wnt5a signaling by treatment with recombinant Wnt5a led to an increased midkine signaling. By performing 3D spheroid assays, we observed that higher expression of midkine is correlated with an increased metastatic ability of the cells. The expression of midkine also correlated with an increased resistance to BRAF inhibitors in BRAF V600E mutated melanoma. This indicates that midkine may signal through MAPK pathway to affect invasion and drug resistance in metastatic melanoma. Developing adjuvant therapies targeting midkine can prolong the overall survival of patients with metastatic melanoma.

Citation Format: Amanpreet Kaur, Michael O'Connell, Marie Webster, Reeti Behera, Vanessa Dang, Abibatou Ndoye, Ashani Weeraratna. Midkine as a potential target for combating drug resistance and invasion in melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5092. doi:10.1158/1538-7445.AM2015-5092
Abstract 5098: Participation of Crk-associated substrate (CAS) in human pancreatic cancer cell migration, invasion and metastatic processes
Authors: Okamoto Hirotaka , Fujii Hideki
Published: August 2015

Introduction; Crk-associated substrate (CAS, p130Cas) is a major tyrosine phosphorylated protein in cells transformed by v-crk and v-src oncogenes. In non-transformed cells, CAS localizes to focal adhesions and undergoes tyrosine phosphorylation in response to integrin-mediated signaling. In the current study, we evaluate the role of CAS in cell migration, invasion and metastatic processes in human pancreatic cancer cells. Materials and Methods; We investigated that CAS tyrosine kinase activity and tumor cell migration, invasion to endothelial cell monolayer in human pancreatic cancer cell lines, in vitro. CAS tyrosine phosphorylation were measured by immunoblotting and immunoprecipitation using anti-CAS monoclonal antibody and anti-phosphotyrosine antibody. Tumor cell migration was measured with Matrigel-coated modified-Boyden's chamber assay. Tumor cell invasion to endothelial cell monolayer was assayed with a co-culture invasion bioassay. Results; Tyrosine phosphorylation of CAS in human pancreatic cancer cells were induced during tumor cell migration and invasion. Phosphorylated tyrosine level of CAS was coincident with invasion capability in human pancreatic cancer cell lines, PSN-1, MiaPacaII, Panc1, despite of similar protein expression level. Protein tyrosine kinase inhibitor, Herbimycin A, had an inhibitory effect on migration and invasion as well as CAS tyrosine phosphorylation in tumor cells. Conclusion; These results indicated that CAS tyrosine phosphorylation in pancreatic tumor cells was correlated with invasive capability, suggesting a pivotal role of CAS in migration, invasion and metastatic processes.

Citation Format: Hirotaka Okamoto, Hideki Fujii. Participation of Crk-associated substrate (CAS) in human pancreatic cancer cell migration, invasion and metastatic processes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5098. doi:10.1158/1538-7445.AM2015-5098
Abstract 5099: Abrogation of cancer cell extravasation in renal cell carcinoma PDX lines via invadopodia dysregulation mediated by PAK1 inhibitors
Authors: Williams Karla , Mazzola Clarisse , Brugarolas James , Power Nicholas , Chambers Ann , Leong Hon
Published: August 2015

Cellular remodeling of the extracellular matrix (ECM), facilitating tumour cell invasion, is a key intrinsic quality in metastatic cells.   Invadopodia are actin-driven protease-rich subcellular membrane protrusions formed by tumor cells for localized degradation and remodeling of the ECM.  Invadopodia mediate tumor cell extravasation, a key step in the metastatic cascade in which tumor cells undergo transendothelial migration when arrested at distant sites.  Using several PDX cell lines, we determine that treatment with PAK1 inhibitors leads to significantly decreased rates of cancer cell extravasation in vivo (IPA-3, 1 uM final concentration) with no impacts on cell viability in vitro. Interestingly, in vitro, there was no significant change in the number of cells forming invadopodia; however, there was more gelatin degradation per cell as determined by in vitro gelatin-Alexa594 invadopodia assays.  Time lapse imaging of invadopodia formation in PDX cell lines reveal a decrease in invadopodia disassembly in vitro, resulting in alterations to adhesions to maintain tumor cell anchorage to the CAM vessel wall in vivo.  These results reveal the feasibility of targeting extravasation through other mechanisms whilst impairing invadopodia disassembly prior to cancer cell transportation to the underlying tissue.

Citation Format: Karla Williams, Clarisse Mazzola, James Brugarolas, Nicholas Power, Ann Chambers, Hon Leong. Abrogation of cancer cell extravasation in renal cell carcinoma PDX lines via invadopodia dysregulation mediated by PAK1 inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5099. doi:10.1158/1538-7445.AM2015-5099
Abstract 5100: Aerobic glycolysis is the primary metabolic pathway used for prostate cancer cell motility and invasion processes regardless of androgen sensitivity
Authors: Verdone James E. , Zarif Jelani C. , Pienta Kenneth J.
Published: August 2015

The ability of a cancer cell to detach from a primary tumor and intravasate is a fundamental prerequisite for development of metastatic disease. As metastatic tumors are frequently the principal cause of death in cancer patients with advanced disease, targeting the ability of cancer cells to metastasize may improve patient outcomes. Advanced, aggressive prostate cancers are characterized by abnormal metabolic upregulation of aerobic glycolysis, a phenomenon known as the Warburg effect. We have previously demonstrated that prostate cancer and breast cancer cells use aerobic glycolysis as the primary energy pathway for cellular motility and cytoskeletal remodeling. In clinical prostate cancer patients, the progression of cells to an androgen insensitive state renders treatment difficult and often unsuccessful. In the present study, we investigate the role of aerobic glycolysis in motility and invasion in prostate cancer cells with intact androgen signaling.

To test this, we utilized a Seahorse XF24 metabolic to measure aerobic glycolysis and oxygen consumption rate of prostate cancer cells with and without intact androgen receptor signaling. Cellular motility was measured by tracking cell movement over time while subjecting cells to pharmacological inhibition of aerobic glycolysis, mitochondrial oxidative phosphorylation, or fatty acid beta-oxidation. Cellular invasion was measured by a Boyden chamber invasion assay with similar pharmacological inhibition.

As anticipated, prostate cancer cells with intact androgen receptor signaling displayed a modulated usage of aerobic glycolysis, whereas mitochondrial oxidative phosphorylation was unchanged. Consequently, androgen receptor signaling altered cancer cell invasiveness. Notably, regardless of invasiveness and androgen receptor status, cellular motility and invasion capacity can be attenuated upon pharmacological inhibition of aerobic glycolysis. Taken together, these data suggest that targeting aerobic glycolysis in prostate cancer could severely weaken prostate cancer metastatic capability and limit tumor spread from the primary site. In the future, combining glycolytic inhibitors with conventional therapy may prevent dissemination of metastatic clones to distal sites and thus drastically reduce risk of metastatic recurrence.

Citation Format: James E. Verdone, Jelani C. Zarif, Kenneth J. Pienta. Aerobic glycolysis is the primary metabolic pathway used for prostate cancer cell motility and invasion processes regardless of androgen sensitivity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5100. doi:10.1158/1538-7445.AM2015-5100
Abstract 5107: Divergent behaviors and underlying mechanisms of cell migration and invasion in non-metastatic T24 and its metastatic derivative T24T bladder cancer cell lines
Authors: Jin Honglei , Li Jingxia , Huang Chuanshu
Published: August 2015

Previous studies on cancer cell invasion were primarily focused on its migration because these two events were often considered biologically equivalent. Here we found that T24T cells exhibited higher invasion but lower migration abilities than T24 cells. Expression of Rho-GDPases was much lower and expression of SOD2 was much higher in T24T cells than in T24 cells. Indeed, knockdown of SOD2 in T24T cells can reverse the cell migration but without affecting cell invasion. We also found that SOD2 inhibited the JNK/C-Jun cascade, and the inhibition of c-Jun activation by ectopic expression of TAM67 impaired Rho-GDPases expression and cell migration in T24T shSOD2 cells. Further, we found that Sp1 can upregulate SOD2 transcription in T24T cells. Importantly, matrix metalloproteinase-2 (MMP-2) was overexpressed in T24T and participated in increasing its invasion, and MMP-2 overexpression was mediated by increasing nuclear transport of nucleolin, which enhanced mmp-2 mRNA stability. Taken together, our study unravels an inverse relationship between cell migration and invasion in human bladder cancer T24T cells and suggests a novel mechanism underlying the divergent roles of SOD2 and MMP-2 in regulating metastatic behaviors of human bladder T24T in cell migration and invasion.

Citation Format: Honglei Jin, Jingxia Li, Chuanshu Huang. Divergent behaviors and underlying mechanisms of cell migration and invasion in non-metastatic T24 and its metastatic derivative T24T bladder cancer cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5107. doi:10.1158/1538-7445.AM2015-5107
Abstract 5127: The most common mistakes in cancer cell pharmacology
Authors: Lenz Guido , Silva Andrew O.
Published: August 2015

Background: Cancer cell culture has contributed with pivotal discoveries for cancer biology and pharmacology. However, cancer cells in culture have not provided a good test method for patient´s response to chemotherapy to a similar extent as antibiograms have been useful to predict response to antibiotics. Although the reason for this is multifactorial, some very common mistakes in cancer cell pharmacology are made in most studies that, if corrected, could improve the overall results.

Methods: We systematically tested key conditions such as concentration, time of exposure and analysis, methods of analysis and number of cells analyzed in glioma cell lines.

Results: We found that time of exposure to the drug and time of analysis have a large impact on the outcome, with some drugs producing in vitro “relapse” only after 20 days of analysis. Additionally, the initial number of cells plated is also central for the outcome, with resistance being uncovered only after a certain number of cells is treated. Finally, a direct comparison between the MTT viability assay with population doubling assay clearly indicates that the former is not suited for studying effective anticancer drugs and resistance due to its low sensitivity. By systematically analyzing concentration, time of exposure and analysis, number of initial cells and method of analysis we showed that for more meaningful in culture cancer pharmacology, drug exposure should mimic the patient´s exposure time, (5 days for gliomas), analysis should be for at least one treatment cycle (28 days for gliomas), the number of cells tested should be at least more than 15 thousand and the method for analyzing cell number should be repetitive direct counting instead of viability assays such as MTT.

Conclusion: Despite not addressing other very fundamental issues in cancer cell culture such as medium, adhesion, 3D culture and presence of other cell types, these simple improvements should aid to produce more significant in culture cancer pharmacology.

Financial support: FAPERGS, CNPq e CAPES.

Citation Format: Guido Lenz, Andrew O. Silva. The most common mistakes in cancer cell pharmacology. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5127. doi:10.1158/1538-7445.AM2015-5127
Abstract 5128: Changes in mitochondrial background affect nuclear DNA methylation
Authors: Vivian Carolyn J. , Brinker Amanda E. , Gooden Gerald C. , Legendre Christophe , Salhia Bodour , Welch Danny R.
Published: August 2015

There is emerging evidence demonstrating that mitochondrial DNA (mtDNA) contributes to complex genetic diseases such as cancer. To test directly the role of mtDNA in tumorigenicity and metastasis, a new genetically engineered mouse model was created that contains nuclear DNA from one mouse strain and mtDNA contribution from different mouse strain(s). Taking advantage of maternal inheritance, Mitochondrial Nuclear Exchange (MNX) mice can be bred to mice sharing the same nuclear DNA background. As a result, MNX serves as an important tool to determine if and how mitochondrial genetic background contributes to cancer, negating the concern for mixing maternal and paternal nuclear DNA polymorphisms. MNX mice are healthy, breed normally, show no overt differences in cell cycle or apoptosis and are genetically stable for multiple (>10) generations. Nonetheless, they are distinct strains based upon behaviors and gene expression patterns. The mechanisms controlling gene expression in different MNX strains are not yet known; however, retrograde and anterograde cross-talk exists between the nuclear and mitochondrial genomes. Our hypothesis is that important epigenetic changes occur in the nuclear genome depending upon mitochondrial genetics. To test this hypothesis, we performed Meth-Seq using the Agilent Mouse SureSelect platform and Affymetrix gene expression arrays on four mouse brains (8 wk) that were pooled from different litters and cages of wild-type and MNX to minimize age, developmental, inter-individual and generational heterogeneity. Both differential DNA methylation and gene expression patterns were observed but analyses are ongoing to better define the observed differences. Together these observations provide novel insights into how mtDNA could be altering epigenetic regulation and thereby contribute to cancer pathogenesis. Support: Susan G. Komen for the Cure (SAC11037), Natl Fndn Cancer Res., Kansas Bioscience Authority, Steiner Family Fund.

Citation Format: Carolyn J. Vivian, Amanda E. Brinker, Gerald C. Gooden, Christophe Legendre, Bodour Salhia, Danny R. Welch. Changes in mitochondrial background affect nuclear DNA methylation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5128. doi:10.1158/1538-7445.AM2015-5128