bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract LB-006: Receptor tyrosine kinase EphA5 is a functional molecular target in human lung cancer
Authors: Staquicini Fernanda , Dobroff Andrey , Ferrara Fortunato , D'Angelo Sara , Bradbury Andrew RM , Arap Wadih , Pasqualini Renata
Published: August 2015

The concept of targeted cancer therapy is predicated on the assumption that tumors have unique and sustained genetic abnormalities, and that direct targeting of such distinctive biological features can only be accomplished through the identification and validation of certain specific molecular markers. These principles are particularly relevant to lung cancer, the leading cause of cancer-related death worldwide. For patients with early-stage disease, surgical resection or primary radiotherapy are generally the standard treatments, whereas combined-modality therapy (radiation plus chemotherapy, with or without surgery) is preferred for locally advanced disease. However, most patients with lung cancer develop metastatic tumors and resistance to therapy, and ultimately die of their disease. Here, we identify EphA5 as a molecular target of lung cancer, and as a novel regulator of IR-induced cell cycle checkpoint and DNA damage repair, with unexpected roles in the resistance of lung cancer to radiotherapy. In the absence of EphA5, lung cancer cells displayed a defective G1/S cell cycle checkpoint, were unable to resolve DNA damage, and became radiosensitive. Upon irradiation, EphA5 was transported into the nucleus where it interacted with activated ataxia-telangiectasia mutated (ATM) at sites of DNA repair. In addition, we produced a new monoclonal antibody against EphA5 that sensitizes lung cancer cells to IR in vitro and improves the overall survival of mice bearing human lung cancer xenografts in combination with radiation therapy. These findings present a readily available and potentially effective strategy for the detection and treatment of lung cancer.

Citation Format: Fernanda Staquicini, Andrey Dobroff, Fortunato Ferrara, Sara D'Angelo, Andrew RM Bradbury, Wadih Arap, Renata Pasqualini. Receptor tyrosine kinase EphA5 is a functional molecular target in human lung cancer. [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 LB-006. doi:10.1158/1538-7445.AM2015-LB-006
Abstract LB-007: Synergistic inhibition of human gastric and colorectal cancers by Bromelain and N-acetylcysteine: An in vivo study
Authors: Amini Afshin , Masoumi-Moghaddam Samar , Ehteda Anahid , Liauw Winston , Akhter Javed , Pilai Krishna , Morris David L.
Published: August 2015

Mucin is well understood to be an adverse prognostic factor in some cancers. We previously reported that a combination of Bromelain (BR) and N-acetylcysteine (NAC) were synergistic in dissolving pseudomyxoma peritonei mucin, had direct in vitro cytotoxic effects on some gastrointestinal cancer cells and sensitized them to some chemotherapy agents. In the present study, we aimed to evaluate the growth-inhibitory effect of BR and NAC as single agent or combination therapy in nude mice models of gastrointestinal cancer.

Nude mice received 2 million and 1 million cells of MKN45 (gastric) and LS174T (colorectal) by intraperitoneal injection. At day 14 and 7 post inoculation for MKN45 and LS174T cells, respectively, animals were intraperitoneally administrated BR (3, 6 mg/kg), NAC (300, 500 mg/kg) or their combination every other day over 12 days for MKN45 and 17 days for LS174T models. At the end of the study, the animals were euthanized, the number of peritoneal nodules and their weight were collected, and the peritoneal tumors were subjected to immunohistochemistry for evaluation of MUC2.

No toxicity was observed during the experiment. At necropsy, highly significant reductions in the number of tumor nodules and tumor burden were observed, in particular in the combination group, where almost complete inhibition in LS174T group was found. There was more than 95% and 70% decreases in tumor burden and tumor nodules, respectively, in LS174T group after combination treatment. For MKN45 model, the reductions in tumor burden and tumor nodules in combination groups were more than 60% and 70%, respectively. Performing immunohistochemistry on tumor samples, MUC2 staining of the LS174T xenografts showed a greater than 60% reduction of cytoplasmic staining.

To the best of our knowledge, this is the first report of the in vivo use of this combination with synergistic inhibition of human gastric and colorectal cancers. The combination of BR and NAC in mucin secreting gastrointestinal tumors is interesting and could be of potential value in peritoneal cancer.

Citation Format: Afshin Amini, Samar Masoumi-Moghaddam, Anahid Ehteda, Winston Liauw, Javed Akhter, Krishna Pilai, David L. Morris. Synergistic inhibition of human gastric and colorectal cancers by Bromelain and N-acetylcysteine: An in vivo study. [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 LB-007. doi:10.1158/1538-7445.AM2015-LB-007
Abstract LB-029: Signal transduction in EGFR-amplified and NFKBIA-deleted human gliomas
Authors: Mishra Alok , Kong Jun , Brat Daniel J.
Published: August 2015

Nuclear factor Kappa B (NF-κB)- a nuclear transcription factor that is retained in the cytoplasm by IKBα (encoded by NFKBIA)- is implicated as a mediator of aggressive behavior in all glioblastoma (GBM) subclasses. We are interested in elucidating molecular mechanisms that explain the relative mutual exclusivity of EGFR amplification and NFKBIA deletion in GBMs. Bioinformatics analysis of TCGA GBM data showed that NFKBIA hemizygous deletions were significantly depleted in samples with EGFR amplification (one-tailed p value of Fisher Exact test, P = 5.97e-4). Moreover, we showed by immunohistochemistry that IKBα was more highly expressed in EGFR-amplified than non-amplified GBMs. We use Ingenuity Pathway Analysis (IPA) to identify gene sets common to both EGFR amplification and NFKBIA hemizygous deletion to identity shared pathways that might explain mutual exclusivity and identified TNF, p53, PI3K, and NF-κB proliferation and apoptosis signaling pathways. We next genetically modeled glioma cell lines and GBM neurosphere cultures by transient transfection/stable viral transduction to recapitulate EGFR/NFKBIA combinations. qPCR of DNA and mRNA for EGFR and NFKBIA verified genotypes. Among the top hits predicted by our in silico studies, we identified sparcl1 and hgpx4 by RT-qPCR and immunoblots as concordantly upregulated in the EGFRamp/NFKBIA+/+ and EGFRnon-amp/NFKBIA+/- cell lines/neurospheres. We also showed differential DNA binding of NF-κB (by electro-mobility shift assay); target gene expression (eg. bcl-2, Il-6); and expression of upstream regulators of NF-κB pathways (viz. pAKT (S436) and IKKβ (Y466)) in GBM neurospheres with distinct EGFR/NFKBIA status. Moreover in U87MG cells (NFKBIA+/−), transiently silencing and overexpressing NFKBIA in different EGFR backgrounds (non-amplified and amplified) led to activation of PARP-dependent but caspase-3-independent apoptotic pathways. Interestingly, in U87MG cells and HPV E6/E7 transformed astrocytes, Annexin V/PI based FACS experiments indicated that NFKBIA overexpression suppressed necrosis. These data demonstrate a complex interplay of EGFR and IKBα/NF-κB in GBMs and identify shared pathways that might explain the relative mutual exclusivity of EGFR amplification and NFKBIA deletion.

Citation Format: Alok Mishra, Jun Kong, Daniel J. Brat. Signal transduction in EGFR-amplified and NFKBIA-deleted human gliomas. [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 LB-029. doi:10.1158/1538-7445.AM2015-LB-029
Abstract LB-036: Neuronal activity-regulated secretion of neuroligin-3 promotes glioma growth
Authors: Venkatesh Humsa , Johung Tessa , Caretti Viola , Mallick Parag , Monje Michelle
Published: August 2015

Active neurons exert a mitogenic effect on normal neural precursor and oligodendroglial precursor cells, the putative cellular origins of high-grade glioma (HGG). We demonstrate that active neurons similarly promote HGG proliferation and growth in vivo using optogenetic control of cortical neuronal activity in a patient-derived pediatric glioblastoma orthotopic xenograft model. Activity-regulated mitogen(s) are secreted, as the conditioned medium from optogenetically stimulated cortical slices promoted proliferation of pediatric and adult patient-derived HGG cultures. The synaptic protein neuroligin-3 (NLGN3) was identified as the leading candidate mitogen; soluble NLGN3 was sufficient and necessary to promote robust HGG cell proliferation. NLGN3 induced PI3K-mTOR pathway activity and feed-forward expression of NLGN3 in glioma cells, providing mechanistic insight into its surprising role as a mitogen. NLGN3 expression levels in human HGG negatively correlated with patient overall survival. These findings indicate the important role of active neurons in the brain tumor microenvironment and identify secreted NLGN3 as an unexpected mechanism promoting neuronal activity-regulated cancer growth.

Citation Format: Humsa Venkatesh, Tessa Johung, Viola Caretti, Parag Mallick, Michelle Monje. Neuronal activity-regulated secretion of neuroligin-3 promotes glioma growth. [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 LB-036. doi:10.1158/1538-7445.AM2015-LB-036
Abstract LB-052: Kinase identification of proximal substrates (KIPS): A novel chemical genetics approach for kinase substrate identification
Authors: roffey jon , Turnbull Andrew , Dillon Christian , Boyd Susan , Riou Philippe , Linch Mark , Parker Peter , Kjaer Sven , McDonald Neil
Published: August 2015

Protein kinases are attractive targets for pharmacological intervention through their frequent disease associated aberration of cellular signalling networks. In this context an understanding of the myriad of substrates on which a protein kinase operates can help to reveal potential pharmacodynamic and patient stratification biomarkers. Kinase Identification of Proximal Substrates (KIPS) utilises an innovative chemical genetics approach to identify novel proximal biomarkers of kinase target response. This approach relies on the generation of a desensitized mutant protein that retains its kinase function, but loses sensitivity to small-molecule mediated inhibition. Comparative phospho-proteomics is performed on immuno-complexes of tagged wild type and mutant protein kinases in the presence and absence of inhibitor to isolate specific phospho-proteomic changes in proximal substrates. To exemplify the KIPS approach we have focused on the identification of novel substrates of the atypical protein kinase C isoform, PKCι. Through structure based design we identified two acidic amino acid residues in the PKCι nucleotide binding site that are crucial for efficacy of a previously characterized PKCι inhibitor, CRT0066854. Upon mutation to non-acidic alanine residues the PKCι protein is rendered insensitive to compound-mediated inhibition, allowing a comparison of wild type and mutant responses to PKCι inhibition to be made. Using HCT116 cells, we identified Myosin X as a putative substrate for PKCι, and demonstrated its specificity to the PKCι signalling axis through alanine-scanning peptide arrays and generation of phospho-specific antibodies. We show that the key acidic amino acids utilised in KIPS are highly conserved across the AGC and CAMK kinase super families. Furthermore, extensive compound SAR, diverse kinome profiling and structural biology have identified a tool box of inhibitor compounds with a broad range of activities across these kinase families leading to broader applicability of KIPS to enable the identification of individual kinase specific substrates and response across approximately 20% of the human kinome.

Citation Format: jon roffey, Andrew Turnbull, Christian Dillon, Susan Boyd, Philippe Riou, Mark Linch, Peter Parker, Sven Kjaer, Neil McDonald. Kinase identification of proximal substrates (KIPS): A novel chemical genetics approach for kinase substrate identification. [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 LB-052. doi:10.1158/1538-7445.AM2015-LB-052
Abstract LB-110: Expression of multiple androgen receptor splice variants in late stage prostate cancer
Authors: Ghazoui Zara , Jones Emma , Veldman-Jones Margaret , Jacobs Vivien N. , Smith Neil R. , Johnstone Michele , Barrett J. Carl , Harrington Elizabeth A. , Elvin Paul
Published: August 2015

The androgen receptor (AR) remains a key driver of disease progression even with the advent of new therapeutic agents such as abiraterone (A) and enzalutamide (E). Relapse after treatment with A or E has been associated with the emergence of tumour cells expressing AR splice variants, and at least one mutation in AR has been associated with resistance to E. We have investigated the expression of AR splice variants in late stage prostate cancers using Nanostring (nCounter®) to quantify the levels of AR transcripts and by IHC using antibodies targeting the N- and C-terminal AR domains. We also examined the expression of putative AR regulated genes found to be expressed in prostate cancer reported in publically available expression data.

Formalin fixed paraffin embedded tissue from 38 commercially sourced (TriStar Technology Group; Asterand; Avaden Biosciences) late stage prostate cancers including 10 castrate resistant prostate cancers (CRPC), were confirmed by pathology review. Total mRNA was isolated from a single 5μm section and gene expression from nCounter® analysis was expressed as normalised Log2 values. Immunohistochemical analysis of AR expression was determined by a pathologist using a H score.

Our analysis of AR splice variant expression, relative to a Nanostring detection threshold based on housekeeping genes, has shown the presence of more than one constitutively active AR splice variant in individual tumours. ARV7 was detected in 4/10 CRPC and always found together with ARV1, AR45, and the constitutively active ARV12. All of the CRPC that were negative for ARV7 expressed AR45 and ARV12. Unsupervised clustering of 507 putative AR regulated genes revealed three molecular subgroups among the 38 prostate tumours. One of these subgroups was characterised by the presence of 12/14 AR splice variants including ARV7. Of the other two subgroups, one contained AR45, the other ARV12. Pathway analysis of genes that were expressed to a higher level (>1.5 Log2 fold) in the ARV7 positive compared to ARV7 negative tumours were predominantly associated with cell cycle and proliferation functions.

Our data suggests complexity in the molecular landscape of prostate cancer associated with AR splice variants, and that multiple AR splice variants may be of importance to identify patients likely to relapse on treatment with emerging therapy.

Citation Format: Zara Ghazoui, Emma Jones, Margaret Veldman-Jones, Vivien N. Jacobs, Neil R. Smith, Michele Johnstone, J. Carl Barrett, Elizabeth A. Harrington, Paul Elvin. Expression of multiple androgen receptor splice variants in late stage prostate cancer. [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 LB-110. doi:10.1158/1538-7445.AM2015-LB-110
Abstract LB-121: Identification of Carboxylesterase-2 as a determinant of response to irinotecan and neoadjuvant FOLFIRINOX therapy in pancreatic ductal adenocarcinoma
Authors: Capello Michela , Lee Minhee , Wang Hong , Babel Ingrid , Katz Matthew H. , Fleming Jason B. , Maitra Anirban , Wang Huamin , Tian Weihua , Taguchi Ayumu , Hanash Samir M.
Published: August 2015

Background: Serine hydrolases (SHs) are among the largest classes of enzymes in human and play crucial role in many pathophysiological processes of cancer. We have undertaken a comprehensive proteomic analysis to assess the differential expression and cellular localization of SHs, which uncovered distinctive expression of Carboxylesterase 2 (CES2), the most efficient carboxyl esterase in activating the pro-drug irinotecan into SN-38, in pancreatic ductal adenocarcinoma (PDAC). We therefore assessed the extent of heterogeneity in CES2 expression in PDAC and its potential relevance to irinotecan based therapy. Methods: CES2 expression in PDAC and paired non-tumor tissues was evaluated by immunohistochemistry. CES2 activity was assessed by monitoring the hydrolysis of the substrate p-NPA. Kaplan-Meier and Cox regression analyses were applied to assess the association between overall survival and CES2 expression in patients who underwent neoadjuvant FOLFIRINOX treatment. Results: Significant overexpression of CES2, both at the mRNA and protein levels, was observed in PDAC compared to paired non-tumor tissue (P < .0001), with 48/118 (40.7%) tumors exhibiting high CES2 expression. CES2 activity in PDAC cell lines was inversely correlated with irinotecan IC50 values (P = .021), while no molecule involved in irinotecan metabolism yielded a significant correlation between its expression and sensitivity to the drug. Remarkably, we recently found that high CES2 expression in tumor tissue was associated with longer overall survival in resectable and borderline resectable patients who underwent neoadjuvant FOLFIRINOX treatment (P = .024). Conclusion: Our findings suggest that CES2 expression and activity, by mediating the intra-tumoral activation of irinotecan, is a contributor to FOLFIRINOX sensitivity in pancreatic cancer and CES2 assessment may define a subset of patients likely to respond to irinotecan based therapy.

Citation Format: Michela Capello, Minhee Lee, Hong Wang, Ingrid Babel, Matthew H. Katz, Jason B. Fleming, Anirban Maitra, Huamin Wang, Weihua Tian, Ayumu Taguchi, Samir M. Hanash. Identification of Carboxylesterase-2 as a determinant of response to irinotecan and neoadjuvant FOLFIRINOX therapy in pancreatic ductal adenocarcinoma. [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 LB-121. doi:10.1158/1538-7445.AM2015-LB-121
Abstract LB-126: Declining T levels provide increased opportunity for mutagenesis in prostate stem cells
Authors: Zhou Ye , Jones Jeremy
Published: August 2015

Because both normal prostate development and the growth of prostate cancers depend on androgen-mediated activation of androgen receptor (AR) signaling, it is reasonable to hypothesize that androgens and AR signaling play a role in prostate tumor initiation as well; however, little is known about androgens and AR in this process. We hypothesize that the decline in androgen production that occurs naturally with age contributes directly to tumorigenesis. Previously, we showed that low serum testosterone (T) causes changes in the expression of genes in the androgen/AR signaling axis which allows the prostate tissue to sustain functional androgen levels in rodents. These changes may contribute to prostate tumorigenesis. There is ongoing debate concerning the cell of origin for prostate cancer, but most evidence would suggest that it can arise from stem cells or cells with stem-like features. Here, we used a murine model to manipulate serum androgen levels to assess the changes in prostate stem-like cells in response to low serum T and to investigate the potential mechanisms by which androgens and AR contribute to prostate tumorigenesis. Using flow cytometry, we observed that chronic exposure to low T causes a consistent expansion of prostate stem-like cell populations, including Lin-/CD133+/CD117+ cells (from 0.23% to 0.33%), Lin-/CD49fHigh/Trop2+ cells (from 6.47% to 8.42%) and intermediate CK5+/CK8+ cells (from 0.43% to 1.26%). Using an in vitro sphere forming assay, we found that stem-like cells isolated from low T treated animals had an increased ability to form spheres, suggesting that low T increases self-renewal capacity. We are currently testing the ability of low T to increase self-renewal capacity in rodent models. Using qPCR, we found that low T causes changes in the expression of genes in the androgen/AR signaling pathway as well as in the oxidative stress response in stem-like cells. We are currently using RNA-seq to understand on a global scale the changes that chronic low T exposure causes in these stem-like cells. Our studies suggest that chronic exposure to low T, as is the case in older men, could provide a greater opportunity for damage to cells that can become cancer initiating cells.

Citation Format: Ye Zhou, Jeremy Jones. Declining T levels provide increased opportunity for mutagenesis in prostate stem 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 LB-126. doi:10.1158/1538-7445.AM2015-LB-126
Abstract LB-142: Functional role of DNA methyltransferase1 (DNMT1) in regulation of mammary stem/progenitor and cancer stem cells
Authors: Pathania Rajneesh , Ramachandran Sabarish , Prasad Puttur , Ganapathy Vadivel , Thangaraju Muthusamy
Published: August 2015

Tumor propagation is the hallmark feature of the cancer stem/tumor propagating cells. Several genetic and epigenetic components are involved in regulation of this process; however, DNA methylation provides a potential epigenetic mechanism for the cellular memory, which needed to preserve the tumorigenic potential through repeated cell divisions. Further, DNA methylation plays a critical role in stem/progenitor cell maintenance wherein the DNMT proteins get enriched in undifferentiated cells and thereby it retains the regenerative capacity while suppressing differentiation. However, the precise role of DNMTs in maintaining stem/progenitor and tumorigenic phenotype in constantly replenished organ, like mammary glands and mammary tumor is not yet known. Here we show that Dnmt1 is required for mammary gland outgrowth and terminal end bud development and that mammary-gland specific Dnmt1 deletion in mice leads to significant reduction in mammary stem/progenitor cell formation. Interestingly, Dnmt1 deletion almost completely abolishes Neu-Tg- and C3(1)-SV40-Tg- driven mammary tumor formation and metastasis. This phenomenon is associated with significant reduction in cancer stem cell (CSC) formation. Similar observations were also recapitulated using pharmacological inhibitors of Dnmts in Neu-Tg mice. To unravel the cause of tumorigenicity of tumor propagating cells, we used genome-wide methylation and RNA sequence approach and find that DNA methylation plays a vital role in regulation of abnormal self-renewal by hypermethylating genes that are involved in development and cell commitment pathways; thereby leading to immortality and autonomous growth to the tumor propagating cells. Overall, our studies provide the first in vivo evidence that DNMT1 is indispensable for mammary stem, progenitor and cancer stem cell formation and that functional inactivation of this gene drastically reduces mammary tumor formation even in the aggressive triple-negative breast cancer subtype. Furthermore, we identified ISL1 as a functional target of DNMT1 in tumor progenitor cells, and stable expression of ISL1 induces apoptosis in cancer stem cells. Thus, DNMT1 specific inhibitors could have a great impact on eradication of cancer stem cells and associated disease recurrence, and ISL1 hypermethylation status could be used as a prognostic marker for early breast cancer diagnosis.

Citation Format: Rajneesh Pathania, Sabarish Ramachandran, Puttur Prasad, Vadivel Ganapathy, Muthusamy Thangaraju. Functional role of DNA methyltransferase1 (DNMT1) in regulation of mammary stem/progenitor and cancer stem 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 LB-142. doi:10.1158/1538-7445.AM2015-LB-142
Abstract LB-146: TGF-β-induced tumor heterogeneity and drug resistance of cancer stem cells
Authors: Oshimori Naoki , Oristian Daniel , Fuchs Elaine
Published: August 2015

Among the most common and life-threatening cancers world-wide, squamous cell carcinoma (SCC) exhibit high rates of tumor recurrence following anti-cancer therapy. Subsets of cancer stem cells (CSCs) often escape anti-cancer therapeutics and promote recurrence. However, its sources and mechanisms that generate tumor heterogeneity and therapy-resistant cell population are largely unknown. Tumor microenvironment may drive intratumor heterogeneity by transmitting signaling factors, oxygen and metabolites to tumor cells depending on their proximity to the local sources. While the hypothesis is attractive, experimental evidence is lacking, and non-genetic mechanisms that drive functional heterogeneity remain largely unknown. As a potential non-genetic factor, we focused on TGF-β because of its multiple roles in tumor progression.

Here we devise a functional reporter system to monitor, track and modify TGF-β signaling in mouse skin SCC in vivo. Using this approach, we found that perivascular TGF-β in the tumor microenvironment generates heterogeneity in TGF-β signaling in neighboring CSCs. This heterogeneity is functionally important: small subsets of TGF-β-responding CSCs proliferate more slowly than their non-responding counterparts. They also exhibit invasive morphology and a malignant differentiation program compared to their non-responding neighbors. By lineage tracing, we show that although TGF-β-responding CSCs clonally expand more slowly they gain a growth advantage in a remarkable ability to escape cisplatin-induced apoptosis. We show that indeed it is their progenies that make a substantial contribution in tumor recurrence. Surprisingly, the slower proliferating state of this subset of CSCs within the cancer correlated with but did not confer the survival advantage to anti-cancer drugs. Using transcriptomic, biochemical and genetic analyses, we unravel a novel mechanism by which heterogeneity in the tumor microenvironment allows a subset of CSCs to respond to TGF-β, and evade anti-cancer drugs.

Our findings also show that TGF-β established ability to suppress proliferation and promote invasion and metastasis do not happen sequentially, but rather simultaneously. This new work build upon the roles of this factor in tumor progression, and sets an important paradigm for a non-genetic factor that produces tumor heterogeneity.

Citation Format: Naoki Oshimori, Daniel Oristian, Elaine Fuchs. TGF-β-induced tumor heterogeneity and drug resistance of cancer stem 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 LB-146. doi:10.1158/1538-7445.AM2015-LB-146
Abstract LB-154: Genome-wide analysis identified differentially methylated regions in head and neck squamous cell carcinoma anatomic subsites
Authors: Rivera Bianca L. , Folawiyo Oluwasina , Turaga Nitesh , Pirini Francesca , Lopez Ricardo , Vazquez Roger , Guerrero Rafael , Baez Adriana
Published: August 2015

Introduction

Several groups have identified hundreds of genes differentially methylated in head and neck squamous cell carcinoma (HNSCC) using genome-wide technologies. However, there is a paucity of studies examining differential methylation in HNSCC anatomic subsites.

Objective

Our principal objective is to determine DNA methylation differences between Head and Neck Squamous Cell Carcinoma (HNSCC) anatomic subsite: larynx, oral cavity and pharynx.

Methods

DNA was extracted from 24 HNSCC samples and 10 normal oral epithelium tissue samples of Puerto Rican Hispanic patients. To unveil the methylome of HNSCC anatomic subsites genomic DNA was enriched with Methylated DNA immunoprecipitation prior to labeling and hybridization to genome-wide oligonucleotide tilting arrays (3 × 720K Human CpG Island-Plus-Promoter Array, Roche/NimbleGen). Differentially methylated regions were identified with CHARM using the bumphunter algorithm. Downstream analyses were performed to identify differences in the methylation landscape of the three anatomic subsites: larynx, oral cavity and pharynx.

Results

When datasets were analyzed by anatomic site using DAVID v6.7 we identified 2565 differentially methylated genes common to the three subsites. We also identified 738 differentially methylated CpG loci in laryngeal cancer, 889 differentially methylated CpG loci in oral cavity and 363 differentially methylated CpG loci in pharyngeal cancer. We identified 11 key genes which were uniquely methylated in the larynx (CDH1, PAX1, BID, SMAD4, FGF19, BMP2, ITGA2, RXRA, IGFBP1, CDKN2D and RBL2). BCL2, AKT2, TRAF3, STAT5B, PTCH1, WNT9B, AXIN2, DVBL2, and APC were differentially methylated in oral cavity. In the pharynx, a comparatively lower number of differentially methylated genes were detected including B-catenin, RAF, BRAF, ATR, CTNNA1, FOXO1 and MED15.

Conclusion

We found evidence that HNSCC subsites (larynx, oral cavity and pharynx) manifest different methylation patterns that may be consistent with the clinical expression of the disease. A panel of differentially methylated genes identified will be further tested in a large cohort of HNSCC samples to determine their utility as prognostic biomarkers of HNSCC according to subsite.

Citation Format: Bianca L. Rivera, Oluwasina Folawiyo, Nitesh Turaga, Francesca Pirini, Ricardo Lopez, Roger Vazquez, Rafael Guerrero, Adriana Baez. Genome-wide analysis identified differentially methylated regions in head and neck squamous cell carcinoma anatomic subsites. [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 LB-154. doi:10.1158/1538-7445.AM2015-LB-154
Abstract LB-165: Antiproliferative effects of AZD6738 and the inhibition of DDR activity
Authors: Kim Hee Jun , Min Ahrum , Im Seock-Ah , Jang Hyemin , Lee Miso , Kim Seongyeong , Kim Jungen , Lee Kyung-Hun , Han Sae-Won , Kim Tae-Yong , Oh Do-Youn , Kim Tae-You , Bang Yung-Jue
Published: August 2015

Background

Anti-tumor effect of DNA damaging agents was reduced by the activation of DNA damage repair (DDR), which was led to resistance. Therefore, the inhibition of DNA repair could lead to induce the accumulation of errors which is becoming an attractive strategy. The ataxia telangiectasia and Rad3-related (ATR) proteins play a role of sensor for DNA damage, which induces homologous recombination-dependent repair. ATR is a master regulator of DDR, signaling to control DNA replication, DNA repair, and apoptosis. Therefore, the ATR pathway might be useful target for new drug development and it is important that the effects of many current cancer treatments are modulated by DDR.

Materials and Methods

The growth inhibitory effects of ATR inhibitor, AZD6738 were studied on human breast cancer cell lines using MTT assay. Cell cycle analysis and western blotting were also performed to determine molecular changes. Immunofluorescence assay and comet assay were conducted to understand the action mechanisms of AZD6738.

Results

This research identified the anti-proliferative effects and the inhibition of DDR activity by AZD6738 on human breast cancer cell lines. AZD6738 induces cell cycle arrest and apoptosis, which impaired DDR function and promoted cell death by damage accumulation. Results of MTT showed that the heterogeneous response and two cell lines were chosen for the focus on the HER2-positive breast cell lines: SKBR-3 and BT-474. In sensitive cell line, SKBR-3 cell, the expression of phosphorylated CHK1 was reduced with the other repair markers; RAD51, MRE11 and ERCC1 as opposed to less sensitive breast cancer cell BT-474. The decreased functional CHK1 leads to the accumulation of DNA damage due to homologous recombination inactivation. And it was also identified that ATR inhibitor played a role of sensitizer to increase the efficacy of cytotoxic chemotherapeutic agents, cisplatin and paclitaxel in breast cancer cell lines.

Conclusion

Understanding the antitumor efficacy and the mechanisms of ATR inhibitor in the breast cancer cell lines open up the possibility of future clinical trial targeting DNA damage repair in breast cancer

Keywords: DNA damage response, ATR inhibitor, Breast cancer, Homologous recombination

Citation Format: Hee Jun Kim, Ahrum Min, Seock-Ah Im, Hyemin Jang, Miso Lee, Seongyeong Kim, Jungen Kim, Kyung-Hun Lee, Sae-Won Han, Tae-Yong Kim, Do-Youn Oh, Tae-You Kim, Yung-Jue Bang. Antiproliferative effects of AZD6738 and the inhibition of DDR activity. [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 LB-165. doi:10.1158/1538-7445.AM2015-LB-165
Abstract LB-198: Molecular drivers of metabolic reprogramming during EMT
Authors: Teo Zi Qiang , Sng Ming Keat , CHAN Jeremy , Tan Nguan Soon
Published: August 2015

The reprogrammed cancer metabolism is an emerging cancer hallmark pervasive in many cancers and is fundamental to neoplastic progression. Many key cancer-promoting pathways are known to converge on the regulation of cellular metabolism. Besides providing basic sustenance to fuel cancer cells limitless replicative potential, emerging data reveals critical roles for the reprogammed metabolism during maglignant progression. Metastasis is often regarded as the end-stage of malignancy and majority of cancer mortality is attributed to the metastatic dissemination of the malignant cancer cells. The initiation of metastasis has been pinpointed as pivotal event, without which, the malignant cancer cells loses their motility and invasive capabilities for distal dissemination. This initiation event highly resembles epithelial-mesenchymal transition (EMT) process that occurs during embryogenesis and wound healing. Microenvironmental signals such as hypoxia and the inflammatory milleu that initiate EMT, interestingly, are also shown to be critical in rewiring cancer metabolism. Thus, it is conceivable that the manipulation of cancer metabolism can impact EMT. Furthermore, whether such metabolic reprogramming is a functional pre-requisite for the initiation of EMT remains unclear.

Using three distinct in vitro EMT models, we show that EMT is an energy-demanding process. Comparative microarray analysis of the EMT models reveals that genes involved in fatty acid, glucose and ROS metabolism are altered. We went further to show that cancer cells undergoing EMT, derives much of the ATP/energy from glucose and lipid metabolism. Importantly, we identified several key molecular drivers that coordinate EMT and cellular bioenergetics, which is validated by our in vivo EMT model.

Citation Format: Zi Qiang Teo, Ming Keat Sng, Jeremy CHAN, Nguan Soon Tan. Molecular drivers of metabolic reprogramming during EMT. [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 LB-198. doi:10.1158/1538-7445.AM2015-LB-198
Abstract LB-203: Obesity promotes resistance to anti-VEGF therapy in breast cancer via pro-inflammatory and angiogenic pathways
Authors: Incio Joao , McManus Daniel , Suboj Priya , Rahbari Nuh , Chin Shan M. , Babycutty Suboj , Vardan-Kaur Trupti , Huang Yuhui , Jung Keehoon , Duda Dan , Soares Raquel , Fukumura Dai , Jain Rakesh K.
Published: August 2015

Background: Most breast cancer (BC) patients are overweight or obese at the time of diagnosis. Obesity is associated with increased risk, recurrence, and worse prognosis of BC. It has been shown that obesity associates with worse outcome in metastatic kidney or colon cancer treated with bevacizumab. If and how excess body weight contributes to the failure of anti-VEGF therapy in BC is unknown.

Results: Here we found that diet-induced obesity promoted resistance to anti-VEGF therapy in two syngeneic mouse breast cancer models. The effects of anti-VEGF therapy on tumor growth and metastasis, VEGF downstream signaling pathways and vessel density were significantly attenuated in obese mice. Under obesity condition, intra-tumor adipocytes increased. These adipocyte-rich regions in breast cancers were hypoxic and overexpress IL-6 or FGF-2 by adipocytes, fibroblasts, and myeloid cells. In IL-6 overexpressing obese breast cancer model (E0771), neutralization of IL-6, either genetically or pharmacologically, abrogated the obesity-induced resistance to anti-VEGF therapy seen in both primary and metastasis sites. This occurred due to a reversion of the obesity-augmented STAT3 signaling and cell proliferation, of hypoxia via vessel normalization, and of immunosuppression. In another breast cancer model (MCaIV), which overexpress FGF-2 under obesity, anti-FGF receptor antibody restored tumor sensitivity to anti-VEGF treatment in obesity.

Conclusion: Our findings indicate that obesity promotes resistance to anti-VEGF therapy in breast cancer via the production of pro-inflammatory and angiogenic factors that circumvent the loss of VEGF signaling.

Citation Format: Joao Incio, Daniel McManus, Priya Suboj, Nuh Rahbari, Shan M. Chin, Suboj Babycutty, Trupti Vardan-Kaur, Yuhui Huang, Keehoon Jung, Dan Duda, Raquel Soares, Dai Fukumura, Rakesh K. Jain. Obesity promotes resistance to anti-VEGF therapy in breast cancer via pro-inflammatory and angiogenic pathways. [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 LB-203. doi:10.1158/1538-7445.AM2015-LB-203
Abstract LB-242: PTEN regulates the CD20 antigen expression and affects rituximab-based therapy of lymphoma malignancies
Authors: Pyrzynska Beata , Bojarczuk Kamil , Siernicka Marta , Dwojak Michal , Bobrowicz Malgorzata , Miazek Nina , Zapala Piotr , Zagozdzon Agnieszka , Golab Jakub , Winiarska Magdalena
Published: August 2015

The therapeutic strategies currently used in B cell malignancies include the treatment with monoclonal antibodies (rituximab and ofatumumab) directed against CD20 antigen. These antibodies specifically eliminate B cells by triggering indirect effector mechanisms of the immune system, like complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), or immunophagocytosis. In many patients, the reduced level of CD20 antigen on the surface of tumor B cells leads to the resistance to anti-CD20 therapy. The aim of this study was to explore the molecular mechanisms governing the regulation of CD20 expression in lymphoma cells as a potential explanation of the resistance to rituximab/ofatumumab therapy.

We previously observed that CD20 mRNA expression is significantly affected by the SRC family inhibitors. Here, we report that also PTEN tumor suppressor is a negative regulator of CD20 expression. To uncover the transcriptional mechanisms governing the CD20 expression we employed the construct encoding the promoter region of CD20 cloned upstream of the firefly luciferase gene. Overexpression of wild-type PTEN (but not the phosphatase-deficient mutant) strongly affected the promoter activity and the expression of CD20, leading to decreased binding of rituximab and ofatumumab and increased resistance of tumor cells to complement-dependent cytotoxicity. Using the truncated versions of the CD20 promoter we identified a particular region (-313/-198) as the major region sensitive to PTEN overexpression. We found that the negative regulation of CD20 promoter activity by PTEN was mediated by inhibition of AKT signaling. We observed that the overexpression of constitutively active AKT1 (CA-AKT1) overcame the negative effect of PTEN and sensitized cells to rituximab/ofatumumab treatment. The results of our studies indicate that PTEN status in tumor cells should therefore be considered when analyzing the mechanisms of resistance of B cell malignancies to anti-CD20 therapies.

Citation Format: Beata Pyrzynska, Kamil Bojarczuk, Marta Siernicka, Michal Dwojak, Malgorzata Bobrowicz, Nina Miazek, Piotr Zapala, Agnieszka Zagozdzon, Jakub Golab, Magdalena Winiarska. PTEN regulates the CD20 antigen expression and affects rituximab-based therapy of lymphoma malignancies. [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 LB-242. doi:10.1158/1538-7445.AM2015-LB-242
Abstract LB-244: Amino acid deprivation selectively targets multidrug-resistant breast cancer cells
Authors: Del Vecchio Catherine A. , Feng Yuxiong , Sokol Ethan S. , Tillman Erik J. , Sanduja Sandhya , Reinhardt Ferenc , Gupta Piyush B.
Published: August 2015

Tumor recurrence and metastasis underlie the majority of cancer-related deaths. Cancer cells that recur or metastasize are often both de-differentiated and multidrug resistant, but the mechanistic basis for this has been poorly understood. We have recently shown that de-differentiation promotes multidrug resistance by activating Nrf2, which stimulates transcription of drug efflux pumps and enzymes that scavenge reactive oxygen species (ROS). De-differentiation activates Nrf2 by a non-canonical mechanism involving its phosphorylation by the ER membrane kinase PERK. PERK-Nrf2 signaling protects de-differentiated cells from chemotherapy, and inhibiting this signaling axis re-sensitizes de-differentiated cancer cells to treatment. To further explore this pathway we profiled the effects of PERK inhibition on global gene expression in both differentiated and de-differentiated cells upon treatment with chemotherapy. This analysis showed that PERK inhibition results in an amino acid deprivation phenotype, and suggested that de-differentiated cells may be sensitive to perturbations in amino acid availability. Consistent with this, we found that the aminopeptidase inhibitor Tosedostat was selectively toxic to de-differentiated breast cancer cells when given in combination with chemotherapy. Our findings identify a novel vulnerability of therapy-resistant breast cancer cells, and suggest that targeting amino acid availability in combination with chemotherapy could be an effective treatment for aggressive breast cancers that are multidrug resistant.

Citation Format: Catherine A. Del Vecchio, Yuxiong Feng, Ethan S. Sokol, Erik J. Tillman, Sandhya Sanduja, Ferenc Reinhardt, Piyush B. Gupta. Amino acid deprivation selectively targets multidrug-resistant breast cancer 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 LB-244. doi:10.1158/1538-7445.AM2015-LB-244
Abstract LB-250: Induction of c-Cbl is a novel mechanism for the anti-cancer effect of HDAC inhibitor in lung cancer
Authors: Ching-Chow Chen , Wei Tzu-Tang , Lin Yu-Chin , Lin Pei-Hua , Shih Jin-Yuan , Chou Chia-Wei
Published: August 2015

Here we found loss of c-Cbl, an E3 ligase, expression in Non-small cell lung cancer (NSCLC) compared with its adjacent normal tissue in patient specimens. HDAC inhibition by WJ or knockdown of HDAC 1, HDAC2, HDAC3 or HDAC6 all induced c-Cbl. Ectopic expression of c-Cbl induced decreased EGFR, inhibited growth in NSCLC cells. Knockdown of EGFR inhibited NSCLC growth. Mutation of EGFR at Y1045 decreased WJ-induced growth inhibition as well as in vivo anti-cancer effect and EGFR degradation mediated by WJ. Time-lapse confocal analysis showed co-localization of c-Cbl and EGFR after WJ treatment. Furthermore, WJ inhibited lung tumor growth through c-Cbl induction in orthotopic and tail vein injected models. C-Cbl up-regulation induced by HDACi is a potential strategy for NSCLC treatment.

Citation Format: Chen Ching-Chow, Tzu-Tang Wei, Yu-Chin Lin, Pei-Hua Lin, Jin-Yuan Shih, Chia-Wei Chou. Induction of c-Cbl is a novel mechanism for the anti-cancer effect of HDAC inhibitor in lung cancer. [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 LB-250. doi:10.1158/1538-7445.AM2015-LB-250
Abstract LB-260: A small molecule Myc inhibitor has therapeutic effects on mouse pancreatic and other cancers
Authors: Stellas Dimitris , Szabolcs Matthias , Koul Sanjay , Li Zhe , Polyzos Alexander , Anagnostopoulos Constantinos , Cournia Zoe , Tamvakopoulos Constantin , Klinakis Apostolos , Efstratiadis Argiris
Published: August 2015

Mouse models faithfully simulating human cancer are valuable for genetic identification of potential drug-targets but, among them, the most advantageous for practical use in subsequent preclinical testing of candidate therapeutic regimes are those exhibiting rapid tumor development. Considering that pancreatic ductal adenocarcinoma (PDA) is the deadliest of the major malignancies and an unresolved clinical problem, and that a KRAS mutation (predominantly in codon 12, such as KRASG12D; KRAS*) occurs with high frequency (∼90%) in PDA cases, we sought to develop a mouse PDA model that would exhibit high tumor incidence and short latency by ectopic overexpression of Kras*. This transgenic modification caused by two weeks postpartum the appearance of pancreatic intraepithelial neoplasia (PanIN), which evolved into invasive PDA within a week later, and resulted in a moribund condition at one month of age. Interestingly, however, this aggressive form of pancreatic tumorigenesis was effectively prevented by genetic ablation of Myc specifically in the pancreas On the basis of this observation, causally demonstrating that Kras* oncogenicity fully depends on unperturbed Myc activity, we tested with positive results the effective treatment of our mouse PDA model by using orally-administered Mycro3, a small-molecule inhibitor of Myc-Max dimerization. PET/CT image analysis demonstrated marked shrinkage of PDA, while immunohistochemical analyses showed an increase in cancer cell apoptosis and reduction in cell proliferation. Tumor growth was also drastically attenuated in Mycro3-treated NOD/SCID mice carrying orthotopic or heterotopic xenografts of human pancreatic cancer cells. In addition, Mycro3 was efficacious in treating xenografts generated either heterotopically or orthotopically (by tail-injection) using a human lung carcinoma cell line (KRASG12D) and also in treating KRAS-driven mouse mammary tumors. These results provide strong justification for eventual clinical evaluation of anti-Myc drugs as potential chemotherapeutic agents for the treatment of PDA and other Myc-dependent cancers.

Citation Format: Dimitris Stellas, Matthias Szabolcs, Sanjay Koul, Zhe Li, Alexander Polyzos, Constantinos Anagnostopoulos, Zoe Cournia, Constantin Tamvakopoulos, Apostolos Klinakis, Argiris Efstratiadis. A small molecule Myc inhibitor has therapeutic effects on mouse pancreatic and other cancers. [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 LB-260. doi:10.1158/1538-7445.AM2015-LB-260