bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 286: Preclinical study: HDAC inhibition combined with gemcitabine for the treatment of leiomyosarcoma
Authors: Lopez Gonzalo , Choy Edwin , Patel Shreyaskumar , Bid Hemant , Braggio Danielle , Lev Dina , Pollock Raphael
Published: July 2016

Objective

Leiomyosarcoma (LMS) is a malignant soft tissue sarcoma (STS) with a dismal prognosis following metastasic disease. Chemotherapeutic intervention has demonstrated to have modest clinical efficacy with no curative potential in LMS patients. Previously, we demonstrated pan-HDAC inhibition to have a superior effect in various complex karyotypic sarcomas. In this study, our goal is to evaluate the therapeutic efficacy of pan-HDACi alone and in combination with gemcitabine in LMS.

Methods

Human leiomyosarcoma (LMS) cell lines were used for in vitro and in vivo studies. Compounds tested included a pan-HDAC inhibitor (HDACi) and nucleoside analog, gemcitabine (Gem). MTS and clonogenic assays were used to evaluate the effect of HDACi on LMS cell growth. Cell cycle FACS and annexin V PI/FACS analysis were used to determine the effects of HDACi on cell cycle and apoptosis, respectively. Compusyn software was used to determine in vitro synergy studies for the combination of HDACi + Gem. A LMS xenograft model in SCID mice was used to test the impact of HDACi alone, Gem alone and the combination of HDACi + Gemcitabine.

Results

Pan-HDAC inhibition abrogated LMS cell growth and clonogenic potential. HDACi induced G2/M cell cycle growth arrest and enhanced apoptosis in LMS cell lines. The combination of HDACi + Gem exhibited a synergistic effect in LMS cells in vitro. Similarly, HDACi combined with Gem resulted in superior anti-LMS effects in vivo.

Conclusion

LMS are aggressive, metastatic tumors with poor prognosis where effective therapeutic interventions are wanting. Our studies demonstrate the potential utility of pan-HDAC inhibition combined with gemcitabine for the treatment of LMS.

Citation Format: Gonzalo Lopez, Edwin Choy, Shreyaskumar Patel, Hemant Bid, Danielle Braggio, Dina Lev, Raphael Pollock. Preclinical study: HDAC inhibition combined with gemcitabine for the treatment of leiomyosarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 286.
Abstract 2863: Mechanisms underlying estrogen-induced inflammation in estrogen-deprived breast cancer cells
Authors: Fan Ping , Agboke Fadeke A. , Kumar Tyagi Amit , Jordan V. Craig
Published: July 2016

Inflammation is critical for estrogen (E2) to undertake apoptosis in E2-deprived breast cancer cells. However, it remains unclear how E2 induces inflammation. Here, we demonstrate that E2 increases lipid metabolism and accumulates unfolded proteins in the endoplasmic reticulum in E2-deprived breast cancer cells. These two major alterations result in different inflammatory responses. E2 rapidly increases lipid metabolism related genes such as fatty acid desaturase 1 (FADS1) and CCAAT/enhancer binding protein beta (CEBPB) to modulate adipose inflammation related factors, e.g. IL6/IL6R after two hours treatment, which regulate the cellular proliferation or differentiation. Simultaneously, accumulation of the unfolded protein activates unfolded protein response (UPR) sensors, protein kinase RNA-like endoplasmic reticulum kinase (PERK) and inositol-requiring protein 1 alpha (IRE1α) within few hours after exposure to E2. Importantly, inhibition of PERK, rather than IRE1α, is able to completely prevent E2 from induction of late event inflammatory genes associated with apoptosis, such as tumor necrosis factor (TNF) superfamily members, BCL2L11 (Bim), and HMOX1,which are activated by E2 after 48 hours. Consistently, nuclear factor-kappa B (NF-κB) is activated when TNFα is maximal after E2 treatment. Further examination revealed that an anti-inflammatory agent, dexamethasone (Dex), activates glucocorticoid receptor (GR) transcriptional activity and blocks E2-induced apoptosis. These effects can be reversed by the knockdown of GR. A notable mechanistic change is that Dex effectively blocks the phosphorylation of PERK and produces equivalent inhibitory effects on the apoptosis-associated inflammatory genes as the PERK inhibitor. However, Dex has an additional potential to activate lipid metabolism and are additive with E2 to elevate FADS1 and adipokine IL6/IL6R. All of these data, for the first time, describe the mechanisms underlying E2-induced inflammation in E2-deprived breast cancer. It provides important information to restrict the clinical use of glucocorticoids, which will undermine the beneficial effects of E2-induced apoptosis in estrogen deprived breast cancer patients.

Citation Format: Ping Fan, Fadeke A. Agboke, Amit Kumar Tyagi, V. Craig Jordan. Mechanisms underlying estrogen-induced inflammation in estrogen-deprived breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2863.
Abstract 2871: Dual role of EpCAM cleavage in adhesion attenuation and transcription enhancement for cell migration
Authors: Hsu Ya-Ting , Osmulski Pawel A. , Wang Yao , Huang Yi-Wen , Liu Lu , Ruan Jianhua , Jin Victor X. , Kirma Nameer B. , Gaczynska Maria E. , Huang Tim H.M.
Published: July 2016

Epithelial cell adhesion molecule (EpCAM), a membrane protein known to modulate cell-cell adhesion, is also a regulatory molecule internalized into the nucleus for transcriptional control of gene expression. Here we demonstrate that activated EGF/EGFR is a signaling factor to drive the cleavage of the extracellular fragment EpEX culminating in removal of cell-surface EpCAM as monitored with recognition atomic force microscopy (AFM). As a result, internalization of the cytoplasmic domain EpICD leads to formation of transcription factor complexes with LEF1 that regulate gene transcription for enhancing mobility functions of cancer cells. Comprehensive probing with AFM further reveals increased elasticity and decreased adhesiveness of these cells, implicating acquisition of an epithelial-mesenchymal transition phenotype. While EpCAM cleavage contributes to the loss of cell-surface adhesiveness, its internalized EpICD additionally regulates targets for promoting cell migration. Thus, this EGF/EGFR-modulated action on structural EpCAM and regulatory EpICD can enhance invasion potential of transformed cells.

Citation Format: Ya-Ting Hsu, Pawel A. Osmulski, Yao Wang, Yi-Wen Huang, Lu Liu, Jianhua Ruan, Victor X. Jin, Nameer B. Kirma, Maria E. Gaczynska, Tim H.M. Huang. Dual role of EpCAM cleavage in adhesion attenuation and transcription enhancement for cell migration. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2871.
Abstract 2878: The tumor-permissive role of KHSRP in colorectal cancer: Mechanistic insights from a gene expression study
Authors: Caiazza Francesco , Power Robert , Killick Kate , Higgins Des , Kolch Walter , Breen Laura , Aherne Sinead , Das Sudipto , O’Connor Darran , Tosetto Miriam , Nolan Blathnaid , Fennelly David , Sheahan Kieran , Doherty Glen , Ryan Elizabeth J.
Published: July 2016

Inflammation plays a key role in the development of Colorectal Cancer (CRC), but the underlying regulatory mechanisms are yet to be fully elucidated. The K-homology splicing regulatory protein (KHSRP) is a multifunctional RNA-binding protein which regulates translation of transcripts containing ARE sequences in their 3’-UTR, affecting RNA fate at different levels. KHSRP has been implicated in different functions associated with cancer cell biology, such as inflammation and cell-fate determination. Importantly, KHSRP regulates expression of pro-tumorigenic and anti-inflammatory cytokines, potentially impacting on the tumor microenvironment. We have previously shown that knockdown of KHSRP expression in CRC cell lines regulates cell proliferation and clonogenic survival. KHSRP is also required for the secretion of the pro-angiogenic cytokines, IL-8 and VEGF, impacting on endothelial tube formation. To understand the global impact of KHSRP in regulation of CRC cell function we used Affimetrix Human Transcriptome array to investigate gene expression upon KHSRP silencing in SW480 CRC cells. A total of 210 genes were differentially expressed, 155 of which were up-regulated (100 coding and 55 non-coding genes) and 55 were down-regulated (34 coding and 21 non-coding genes). Among these were genes involved in RNA biology, inflammation, immunity, cell signaling and tumor-stroma interaction, shedding light on the potential mechanisms of action of KHSRP in epithelial cells. Representative targets have been selected for validation and further mechanistic studies. These data builds upon our previous findings suggesting a role for KHSRP in creating a tumor-permissive microenvironment in CRC.

Citation Format: Francesco Caiazza, Robert Power, Kate Killick, Des Higgins, Walter Kolch, Laura Breen, Sinead Aherne, Sudipto Das, Darran O’Connor, Miriam Tosetto, Blathnaid Nolan, David Fennelly, Kieran Sheahan, Glen Doherty, Elizabeth J. Ryan. The tumor-permissive role of KHSRP in colorectal cancer: Mechanistic insights from a gene expression study. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2878.
Abstract 2881: Deubiquitinase Usp9x controls tumorigenicity through regulation of the Ets-1 transcription factor in melanoma
Authors: Potu Harish , Peterson Luke F. Peterson , Kandarpa Malathi Kandarpa , Pal Anupama Pal , Sun Hanshi Sun , Harms Paul W. Harms , Hollenhorst Peter C. Hollenhorst , Eskiocak Ugur Eskiocak , Talpaz Moshe Talpaz , Donato Nicholas J. Donato
Published: July 2016

Transcription factors are frequently deregulated in cancer cells and may be good therapeutic targets, but few successful targeting strategies have been reported. Deubiquitinases (DUBs) are specialized enzymes that regulate the ubiquitin (Ub) content on many proteins and DUB expression and activity are elevated in a number of cancers where they can act to alter tumor suppressor and/or oncoprotein levels. We previously described Usp9x activity and expression in melanoma; here we sought to investigate its role in primary melanoma and metastatic disease. Usp9x was upregulated in tumor cells compared to normal melanocytes and Usp9x expression and activity were found to be essential for 3D growth and melanoma tumor expansion in vivo. We defined the Usp9x ubiquitinated protein landscape and demonstrate that Usp9x regulates Ets-1, a cancer-promoting transcription factor. Usp9x binds, deubiquitinates and thereby stabilizes Ets-1 protein, and primary tissue and tumor analysis demonstrated elevated and coincident Usp9x/Ets-1 protein expression in melanoma compared to normal skin or benign nevi. Usp9x knockdown or Usp9x inhibition with small molecule G9 reduced Ets-1 protein levels and blocked tumor growth in vitro and in vivo. Conversely, Usp9x overexpression in melanoma cells increased Ets-1 protein levels and enhanced 3D tumor growth in vitro and in vivo, which were all reversible by treatment with G9. We conclude that Usp9x is essential for Ets-1 protein stability and may be therapeutically exploited with small molecule Usp9x inhibitors to reduce Ets-1-dependent gene expression and tumorigenicity.

Citation Format: Harish Potu, Luke F. Peterson Peterson, Malathi Kandarpa Kandarpa, Anupama Pal Pal, Hanshi Sun Sun, Paul W. Harms Harms, Peter C. Hollenhorst Hollenhorst, Ugur Eskiocak Eskiocak, Moshe Talpaz Talpaz, Nicholas J. Donato Donato. Deubiquitinase Usp9x controls tumorigenicity through regulation of the Ets-1 transcription factor in melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2881.
Abstract 2882: HnRNP Q1, a novel translational regulator for Aurora-A protein synthesis in colorectal cancer
Authors: Lai Chien-Hsien , Hung Liang-Yi
Published: July 2016

HnRNP Q1 is a multifunctional RNA-binding protein and has an abundant expression level compared with hnRNP Q member. In colorectal cancer, hnRNP Q has been reported to associate with cell proliferation, but the underlying mechanism is still unclear. By RNA-immunoprecipitation assay following next-generation sequencing (NGS), a group of cell cycle-related genes was identified to be targeted by hnRNP Q1, including Aurora-A kinase. Aurora-A is a cell cycle dependent kinase involved in regulating centrosome duplication, maturation and spindle assembly during mitosis. Abnormally elevated Aurora-A will cause mitotic defects and aneuploidy, which might lead to genomic instability and tumorigenesis. Our previous work has demonstrated that Aurora-A can be translational up-regulated in colorectal cancer. In order to investigate the role of hnRNP Q1 in Aurora-A expression, GFP-hnNRP Q1 was overexpressed in colorectal cancer cells. We found that Aurora-A protein, but not the mRNA level, was up-regulated in those GFP-hnRNP Q1-overexpressing cells. By biotin pull down and Biacore analysis, we revealed that hnRNP Q1 binds to the 5’-UTRs of Aurora-A mRNA directly, and their interacting domain was mapped. Importantly, we found that hnRNP Q1 can enhance Aurora-A protein synthesis through IRES-mediated translational regulation in a cell cycle-regulated manner by ribosomal protein S6 immunoprecipitation, polysome fractionation and bicistronic reporter assay. CCK-8 assay, colony formation and in vivo xenograft assay all suggested that overexpression of hnRNP Q1 can promote cell proliferation and tumorigenesis of colorectal cancer cells. In addition, the expression level of hnRNP Q1 is positively correlated with Aurora-A in clinical colorectal cancer tissues. Taken together, our data indicate that hnRNP Q1 is a novel trans-acting factor that binds to Aurora-A 5’-UTRs and regulates its translation, which may increase cell proliferation and contribute to the tumorigenesis of colorectal cancer.

Citation Format: Chien-Hsien Lai, Liang-Yi Hung. HnRNP Q1, a novel translational regulator for Aurora-A protein synthesis in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2882.
Abstract 2886: KLF8 phosphorylation at Serine 48 by ERK2 is critical for its stability and function
Authors: Lahiri Satadru , Lu Heng , Mukherjee Debarati , Yu Lin , Zhao Jihe
Published: July 2016

Krüppel-like factor 8 (KLF8) is a member of Krüppel like family transcription factor which was found to be upregulated in different types of cancers, primarily breast and ovarian cancer. It transcriptionally activates a host of downstream effectors like CyclinD1, EGFR, MMP9, and EPSTI1 to promote cell proliferation, cancer cell invasion and migration. Additionally, KLF8 transcriptionally represses E-cadherin to induce epithelial to mesenchymal transition and oncogenic transformation of cell. As such, KLF8 plays a central role in cancer progression. Several types of post-translational modifications including sumoylation, acetylation, ubiquitylation and PARylation have been discovered that regulate KLF8's function KLF8 has been shown to consistently migrate as doublet on SDS-PAGE. However, the reason for this motility shift of KLF8 has remained mysterious. We performed a series of experiments to determine the mechanism(s) behind this motility shift of KLF8. Truncation mutation showed that deletion of the first 50 amino acid residues, but not other regions, caused disappearance of the top band of the doublet, suggesting that the N50 region is responsible for the motility shift. Phosphatase treatment resulted in the same change in wild-type KLF8 protein, indicating that phosphorylation in the N50 region is causal to the motility shift. Further deletion and point mutation in this region identified Serine 48 as a novel phosphorylation site for KLF8. Interestingly, these experiments also identified KLF8 N terminal 31-40 region to play an important role in regulating the S48 phosphorylation and the motility shift. Our study identified that phosphorylation at Serine 48 is critical in maintaining KLF8 stability. Finally, we demonstrated that abolishing KLF8 S48 phosphorylation inhibits KLF8 mediated cancer cell migration. Kinase inhibitors treatment showed that ERK2 is the kinase responsible for KLF8 Serine 48 phosphorylation. Furthermore we identified that phosphorylated KLF8 acts as a mask to protect unphosphorylated KLF8. Overall this study identifies a novel phosphorylation site of KLF8 and a hitherto unknown link between ERK2 and KLF8 that is critical for maintaining its overall stability and function.

Citation Format: Satadru Lahiri, Heng Lu, Debarati Mukherjee, Lin Yu, Jihe Zhao. KLF8 phosphorylation at Serine 48 by ERK2 is critical for its stability and function. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2886.
Abstract 2888: Identification of an NKX3.1-G9a-UTY regulatory network that controls proper prostate differentiation
Authors: Le Magnen Clementine , Dutta Aditya , Mitrofanova Antonina , Califano Andrea , Abate-Shen Cory
Published: July 2016

Maintenance of cell differentiation is tightly coordinated by specific gene expression programs, which are often deregulated during tumorigenesis. While the homeodomain-containing transcription factor, NKX3.1, plays essential roles in both prostate differentiation and prostate cancer, the mechanisms governing these events are still unclear. Here, we aim to elucidate detailed mechanisms governing Nkx3.1-driven prostate epithelial differentiation and explain how deregulation of such can lead to prostate tumorigenesis.

Using an Nkx3.1 mutant mouse model, we find that loss-of-function of Nkx3.1 in the prostate results in the down-regulation of genes associated with prostate differentiation as well as the up-regulation of genes that are not normally expressed in prostate. Interestingly, these mice also develop pre-malignant lesions resembling prostatic intraepithelial neoplasia (PIN). Conversely, gain of function of Nkx3.1 in a fully differentiated non-prostate epithelium is sufficient to induce prostate differentiation in renal grafts in vivo. Using a DNA binding mutant, we show that these activities are mediated via NKX3.1 homeodomain in vitro and in vivo. Exploiting mass spectrometry to identify NKX3.1-interacting proteins, we find that NKX3.1 interacts with the histone methyltransferase G9a and that this interaction is required for induction of prostate differentiation in vitro and in vivo. Finally, we show that these NKX3.1-driven activities are mediated, at least in part, by regulation of target genes such as the male-specific factor UTY (KDM6c).

We propose that the NKX3.1-G9a-UTY network is essential for proper prostate differentiation whereas disruption of these activities predisposes to cancer.

Citation Format: Clementine Le Magnen, Aditya Dutta, Antonina Mitrofanova, Andrea Califano, Cory Abate-Shen. Identification of an NKX3.1-G9a-UTY regulatory network that controls proper prostate differentiation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2888.
Abstract 2893: PARP-1 regulates cell cycle by downregulating E2F-1 transcriptional activity in vitro and in vivo
Authors: Iglesias Pablo , Seoane Marcos , Castro-Piedras Isabel , Arce Victor M. , Costoya Jose A.
Published: July 2016

Although PARP-1 has been traditionally related to DNA repair, in the last years an increasing number of studies have linked this polymerase to other cellular processes such as metabolism and cell mitosis.

In this study we wanted to investigate the biological importance of the interaction between PARP-1 and the transcription factor E2F-1, and more specifically in scenarios where the activity, or hyperactivity, of E2F-1 is of critical importance such as embryonic development or oncogenesis. In this regard, we have found that the treatment either with enzymatic inhibitors of PARP-1 has an effect on the transcriptional activity of E2F-1 as well as the proliferative rate of treated cells. This effect is significantly increased with the treatment with gossypol, a specific inhibitor of PARP-1 protein-protein interactions occurring through the BRCT domain, as in the case of E2F-1. This effect is also observed in vivo since the severity of histological malformations in Rb-/- embryos is significantly reduced in Parp-1-/- Rb-/- embryos, which phenotype closely resembles that of E2f-1-/- Rb-/- mice. Finally, we also found that the deletion or inhibition of PARP-1 in an animal model of gliomagenesis helps the cell to block oncogenic stimuli derived from E2F-1 hyperactivity, by reactivating critical signalling pathways involved in oncogene-induced senescence.

In summary, the importance of the relationship between PARP-1 and E2F-1 in different biological contexts leads us to believe that the search for ways of altering or disrupting this interaction could be a novel strategy for the development of molecules of therapeutic potential.

Citation Format: Pablo Iglesias, Marcos Seoane, Isabel Castro-Piedras, Victor M. Arce, Jose A. Costoya. PARP-1 regulates cell cycle by downregulating E2F-1 transcriptional activity in vitro and in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2893.
Abstract 29: MEK and PI3K signaling cooperate through mTORC1/2 to promote PIK3CA mutant melanoma cell proliferation
Authors: Silva Jillian M. , Deuker Marian M. , Baguley Bruce C. , McMahon Martin
Published: July 2016

Oncogenic transformation of mutationally activated BRAF or NRAS melanocytes often requires the cooperation of genetic aberrations in the phosphoinositide 3-kinase (PI3K) pathway to promote melanomagenesis. Activating point mutations in the p110α catalytic subunit of PI3K are detected at a low frequency in both BRAF and NRAS mutant melanomas, yet the principle mechanism of this cooperation remains elusive. Thus, using BRAFV600E/PIK3CAH1047R, NRASQ61H/PIK3CAH1047R, and PIK3CAE545K mutant melanoma cells derived from metastatic melanoma patients treated with pathway-targeted inhibitors, we examined the contribution of mutational activation of PIK3CA to melanoma maintenance and signaling and the consequential response of these PIK3CA mutant cells to α-specific PI3K inhibition. Combined MEK and isoform-selective PI3K inhibition elicited more potent anti-proliferative effects and greater suppression of S-phase progression of the cell cycle compared to single-agent inhibition of either pathway. Analysis of signaling downstream of MEK or PI3K revealed that these pathways cooperated to regulate PIK3CA cell proliferation through mTORC1-mediated effects on ribosomal protein S6 and 4E-BP1 phosphorylation in an AKT-dependent manner. Despite the profound anti-proliferative and biochemical effects of α-specific or PI3Kβ-sparing class I PI3K inhibition in vitro, these agents elicited largely cytostatic effects on PIK3CA xenograft tumors. However, combined inhibition of PI3Kβ-sparing class I PI3K and MEK did significantly cooperate to reduce tumor growth compared to the corresponding monotherapies. Furthermore, this study provides a biochemical mechanism to explain how mTORC1/2 moderates the cooperation of MEK and PI3K signaling for the maintenance of PIK3CA mutant melanoma cell proliferation.

Citation Format: Jillian M. Silva, Marian M. Deuker, Bruce C. Baguley, Martin McMahon. MEK and PI3K signaling cooperate through mTORC1/2 to promote PIK3CA mutant melanoma cell proliferation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 29.
Abstract 2906: Targeting FoxA1 in estrogen receptor-positive breast cancer: biological characterization of kinase regulators
Authors: Johnston Simon J. , Holmes Kelly A. , Carroll Jason S.
Published: July 2016

Estrogen receptor (ER) is the driving and defining transcription factor in 75% of breast cancers and is directly inhibited by targeted therapies such as tamoxifen. DNA interactions and transcriptional potential of ER depend on the pioneer factor, FoxA1, which thereby plays an essential role in determining tumour growth and progression - even when resistance to existing drugs has developed. The aim of this study is to characterize kinase regulators of FoxA1 function in order to identify druggable targets for the indirect inhibition of ER.

To identify kinase regulators in ER+ breast cancer, primary screening of multiple cell lines was performed using an siRNA library of the human kinome. After secondary screening of ER+ specific growth-regulatory kinases using a stable luciferase reporter of FoxA1 activation, 50 putative kinase regulators of FoxA1 were identified. These were cross-referenced with kinase inhibitors of FoxA1 activation discovered via a separate cell-based compound screen and competitive-binding kinase displacement assay.

This two-pronged screening approach revealed 6 common putative FoxA1-regulatory kinase enzymes. These were characterized by chromatin immunoprecipitation (ChIP) of FoxA1 and ER with quantitative PCR of archetypal ER enhancers, 48 hours after siRNA knockdown of the kinase. This revealed 3 kinases whose inhibition prevented FoxA1 and ER from accessing the chromatin, and 1 kinase that had the opposite effect.

Mass spectrometry-based proteomic analysis of endogenous FoxA1-protein interactors using ChIP is underway to assess the change in FoxA1 co-factors following inhibition of these 4 candidate kinases. We used a proteomic purification method called RIME which permits analysis of phosphorylation events on FoxA1. We are currently assessing whether silencing of specific kinases influences phosphorylation of the putative substrate, FoxA1.

In summary, the identified kinases demonstrate promising FoxA1 specificity, and may provide druggable targets for the inhibition of ER via FoxA1. This study has potential clinical value for patients with tumours that have become resistant to existing ER-targeted therapies.

Citation Format: Simon J. Johnston, Kelly A. Holmes, Jason S. Carroll. Targeting FoxA1 in estrogen receptor-positive breast cancer: biological characterization of kinase regulators. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2906.
Abstract 2907: Eugenol potentiates the effect of cisplatin on cancer stem-like cells through targeting the NF-κB pathway
Authors: Islam Syed S. , Ibtehaj Al-Sharif , Ahlam Sultan , Aboussekhra Abdelilah
Published: July 2016

Triple-negative subtype of breast cancer (TNBC) contains exclusively tumorigenic and often endowed with self-renewing and resistance to cytotoxic chemotherapy, such as, paclitaxel resulting in relative increases in CSCs phenotype. Signaling through NF-kB may be essential for the CSCs self-renewal and could present potential target for novel treatment in breast cancer patients. We explored the possible involvement of NF-kB pathway that contribute to the functions and maintenance of breast CSCs and targeting this pathway by cisplatin combining with a natural product eugenol both in vitro and in vivo, using estrogen dependent (MCF-7) and independent (MDA-MB-231, MDA-MB-468 and BT-20) cell lines and xenografted both subcutaneous and orthotopic (fat pad) sites in nude mice. Chromatin immunoprecipitation (ChIP) assay was utilized to assess the NF-kB associated DNA binding capacity in promoter region. Surprisingly, neither cisplatin nor eugenol was capable of complete diminishing the CSCs populations. Cisplatin alone administration partially diminished apoptosis, mammospheres formation, aldehyde dehydrogenase 1 (ALDH1) activity, invasion and growth on nude mice. We observed cisplatin-induced activation of NF-kB associated with survival and regrowth of mamospheres. For effective elimination of CSCs phenotype by cisplatin, we cotreated cells with eugenol. Combination of eugenol and cisplatin significantly eradicated cisplatin induced NF-kB DNA binding capability assed by EMSA and ChIP assays, which was associated with abrogated apoptosis, mammospheres formation, ALDH1 activity and invasion. In vivo, combination therapy reduced the tumor size in synergistic manner. This was possibly due to induction of apoptosis, inhibition of proliferation, angiogenesis and downregulation of cisplatin-induced expression of proteins of NF-kB target genes. Although treatment of mammospheres by cisplatin partially induced the CSCs population through interleukin (IL)-6 pathway, however, only coadministration of eugenol and cisplatin reduced the numbers of CSCs to virtually undeletable levels in vitro and in vivo. Our data suggest that eugenol may be well suited to increase targeting of breast CSCs by cisplatin.

Citation Format: Syed S. Islam, Al-Sharif Ibtehaj, Sultan Ahlam, Abdelilah Aboussekhra. Eugenol potentiates the effect of cisplatin on cancer stem-like cells through targeting the NF-κB pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2907.
Abstract 2924: Metastasis-associated protein 1 induces tamoxifen resistance in MCF7 breast cancer cells
Authors: Lee Min-Ho , Koh Dahae , Lee Mi-Ock
Published: July 2016

Tamoxifen is most commonly used to treat estrogen receptor-alpha positive breast cancer, but its therapeutic benefit is limited by the development of drug resistance. Metastasis associated protein 1 (MTA1) is a cancer progression-related gene and has been reported to be overexpressed in a variety of human cancers including breast cancer. Here, we showed that transient overexpression of MTA1 in MCF7 breast cancer cells results in tamoxifen resistance, whereas si-RNA mediated knockdown of MTA1 was more susceptible to tamoxifen-induced growth inhibition. Moreover, stable overexpression of MTA1 in MCF7 cells promoted cell proliferation and reduced tamoxifen sensitivity. To explore the mechanisms of MTA1-mediate tamoxifen resistance, we used microarray analysis to profile the gene expression patterns of MTA1 overexpress MCF7 stable cells after tamoxifen treatment. From microarray data analysis, 538 genes were selected as associated with MTA1-mediated tamoxifen resistance (P < 0.05, LPE test; cutoff ≥ 1.5-fold). The Database for Annotation, Visualization and Integrated Discovery (DAVID) analysis revealed that the predominant biological processes associated with these genes are M phase, negative regulation of cell proliferation, mitosis and M phase of mitotic cell cycle. Further, we found that cell-cycle regulator Cyclin D1 and cyclin B1 protein levels were increased in MTA1 overexpression MCF7 stable cells. Analyses of publicly available patient data sets indicated that MTA1 levels are higher in patients after endocrine therapy failure (recurrence), compared to those of responders (non-recurrence). These results suggest that MTA1 may contributed tamoxifen resistance through the regulation of cell cycle components.

Citation Format: Min-Ho Lee, Dahae Koh, Mi-Ock Lee. Metastasis-associated protein 1 induces tamoxifen resistance in MCF7 breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2924.
Abstract 2926: The regulatory mechanism of human LY6K related to carcinogenesis and metastasis in breast cancer
Authors: Kong Hyun Kyung , Son Da Som , Park Jong Hoon
Published: July 2016

The human lymphocyte antigen 6 complex locus K (LY6K) belongs to the lympocyte antigen 6/urokinase-type plasminogen activator receptor (Ly-6/uPAR) superfamily which are associated with development of the immune system and carcinogenesis. LY6K is a cancer biomarker and a therapeutic target that induces invasion and metastasis by activating the Raf-1/MEK/ERK signaling pathway and that stimulates cytotoxic T lymphocytes. However, the molecular mechanisms including genetic and epigenetic that determine human LY6K transcription are complete unknown. To elucidate the genetic mechanisms involved in human LY6K gene regulation and expression, multiple cis elements were predicted using TRANSFAC software, and the LY6K regulatory region was identified using the lucifearse assay in the human LY6K gene promoter. We performed chromatin immunoprecipitation, electrophoretic mobility shift analysis, and supershift assays to investigate the transcription factor activity on the LY6K promoter, and the effect of a single nucleotide polymorphism (SNP) and CpG site methylation on AP-1 transcription factor binding affinity. AP-1 and the CREB transcription factor bound to LY6K promoter within -550/-1, which was essential for LY6K expression but only the AP-1 heterodimer, JunD and Fra-1, modulates LY6K gene transcriptional level. A decrease in LY6K was associated with the SNP242 C allele, a polymorphic G/C-SNP at the 242 nucleotide in the LY6K promoter region (rs2585175), or methylation of the CpG site, which was closely located with the AP-1 site by interfering with binding of the AP-1 transcription factor to the LY6K promoter. In summary, AP-1 activation is important role in promoting LY6K gene expression that regulates cell mobility of breast cancer cells, whereas the SNP242 C allele or methylation of the CpG site may reduce the risk of invasion or metastasis by interfering AP-1 activation. And DNA methylation and histone modification in the 5’-flanking region of the LY6K gene plays an important role in LY6K expression in both breast cancer cells and breast cancer. An understanding of genetic and epigenetic changes in LY6K may contribute to the diagnosis of carcinogenic risk and to an enhanced prediction of outcome in patients with breast cancer.

Citation Format: Hyun Kyung Kong, Da Som Son, Jong Hoon Park. The regulatory mechanism of human LY6K related to carcinogenesis and metastasis in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2926.
Abstract 2940: The p53 mutation R273H contributed to drug resistance of EGFR tyrosine kinase inhibitors
Authors: Fang Yueh-Fu , Lin Chun-Yu , Lee Peichih
Published: July 2016

Tyrosine kinase inhibitors (TKIs) of epidermal growth factor receptor (EGFR) were the standard first-line treatments for lung cancers with activating EGFR mutations. Emergent drug resistance is the major problem in the patients who received treatment of EGFR TKIs. Second point mutation T790M mutations could be found in 50-60% patients who had drug resistance to EGFR TKIs. Alternative pathway of Met signaling, transformation to small cell lung cancer, over-expression of other HER family receptors or epithelial mesenchymal transit were reported in these patients who had drug resistance to EGFR TKIs. NFKB pathway played a role of early resistance to EGFR TKIs in the patients.

The p53 mutation could be found in 70-80% lung adenocarcinoma patients. These mutations could be found in lung cancer patients who had wild type or mutated EGFR. Lung adenocarcinomas with mutated p53 lose tumor suppressor function of wild type p53 and have new function from mutated p53. We thought p53 mutation may have gain of function and result in drug resistance to EGFR TKIs in lung cancer patients.

We knocked down p53 in three cancer cell line, MDA-MB-468 (p53 R273H), BT-549 (p53 R249S) and H322 (p53 R248L). We found knockdown of p53 R273H in MDA-MB-468 increased sensitivity to gefitinib. Knockdown of p53 R249S and p53 R248L showed the same response to gefitinib in cancer cell lines BT-549 and H322.

Lung cancer cell line HCC827 had EGFR exon 19 deletion and was sensitive to gefitinib. We overexpressed p53 R273H, p53 R157H and p53 R249S in HCC827 lung cancer cell line. Overexpression of p53 R273H increased resistance to gefitinib in HCC827 lung cancer cell line. Overexpression of p53 R157H and p53 R249S did not increased the resistance to gefitinib.

Conclusion: Gain of function of p53 R273H increased resistance to EGFR TKIs in cancer cell lines. Further immunohistochemistry staining and gene analysis of mutated p53 will be done in lung adenocarcinoma from lung cancer patients who received EGFR TKIs.

Citation Format: Yueh-Fu Fang, Chun-Yu Lin, Peichih Lee. The p53 mutation R273H contributed to drug resistance of EGFR tyrosine kinase inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2940.
Abstract 2946: Increased ALDH7A1 expression enhances the resistance to the anticancer drugs and colony formation in lung cancer cell lines
Authors: Sakamori Yuichi , Ozasa Hiroaki , Kunii Eiji , Yagi Yoshitaka , Tsuji Takahiro , Nomizo Takeshi , Nagai Hiroki , Kim Young Hak , Maeno Ken , Oguri Tetsuya , Mishima Michiaki
Published: July 2016

Background: Aldehyde dehydrogenase (ALDH) forms a superfamily of enzymes that catalyze conversion of aldehydes into carboxylic acids, and they are categorized into 19 families in human. Increased ALDH activity was reported to be a potential marker of cancer stem cell (CSC) in various solid tumors including lung cancer. Furthermore, High ALDH expression has been shown to be involved in drug resistance to conventional cytotoxic drugs. Recent studies revealed that ALDH7A1 is highly expressed in prostate cancer and associated with recurrence in patients with surgically resected non-small-cell lung cancer. However, there is not a fundamental link between ALDH7A1 expression and malignant phenotype, including drug resistance and colony formation.

Material and methods: Anticancer drug resistant cell lines were established from lung cancer cell lines (PC-6, DMS53, PC-14, PC-9, NCI-H23 and NCI-H2228 cells) by exposure to various anticancer drug, including 7-ethyl-10-hydroxycamptothesin (SN-38), gemcitabine (GEM), cisplatin (CDDP), etoposide, paclitaxel (TXL), pemetrexed, amrubicin, erlotinib and crizotinib. Protein expression was determined by western blotting and gene expression was examined by RT-PCR. ALDH enzyme activity was measured by ALDEFLOUR TM assay. Proliferation was measured using MTS assay. ALDH7A1encoding plasmid was stably transfected into PC-14 cell. These transfected cells displayed sphere formation on ultralow binding plates and survived for more than 3 weeks.

Results: We found that the levels of ALDH7A1 expression were higher in various anticancer drug-resistant lung cancer cell lines compared with the parental cells. ALDH enzyme activity was about 4-9 fold higher in SN-38, TXL, CDDP and GEM resistant cell lines (PC6/SN38, PC6/TXL, PC14/CDDP and PC14/GEM) than the parents cells. Up-regulation of ALDH7A1 expression by using a forced expression vector in PC14 cell altered cytotoxicity to SN-38 and TXL. Furthermore, overexpression of ALDH7A1in PC-14 cells enhanced sphere formation relative to the cells transfected with control vector.

Conclusion: These results suggested that increased ALDH7A1 expression in lung cancer cell lines might be one of the mechanisms of acquired resistance to anticancer agents and enhance colony formation.

Citation Format: Yuichi Sakamori, Hiroaki Ozasa, Eiji Kunii, Yoshitaka Yagi, Takahiro Tsuji, Takeshi Nomizo, Hiroki Nagai, Young Hak Kim, Ken Maeno, Tetsuya Oguri, Michiaki Mishima. Increased ALDH7A1 expression enhances the resistance to the anticancer drugs and colony formation in lung cancer cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2946.
Abstract 2954: MAP-kinase pathway activation facilitates survival of persistent FGFR1-amplified lung cancer cells upon FGFR inhibition
Authors: Malchers Florian , Ercanoglu Meryem Seda , Thomas Roman Kurt
Published: July 2016

One recurrent alteration of squamous cell lung cancer is the amplified region of the 8p12-p11 locus. The tyrosine kinase fibroblast growth factor receptor 1 (FGFR1) seems to be one of the most prominent targets of this amplification. Thus, small molecules inhibiting FGFRs have been employed to treat FGFR1-amplified squamous cell lung cancer. However, only about 16% of such FGFR1-amplified tumors respond to single agent inhibitor therapy. Several tumors exhibited insufficient tumor shrinkage, compatible with the existence of persistence of inhibitor-resistant tumor cells in the tumor. To investigate the mechanism of FGFR-inhibitor persistence we studied the lung cancer cell lines DMS114 and H1581. Both cell lines demonstrate GI50 values in the nanomolar range upon three different FGFR-inhibitors. However, both cell lines exhibit sustained cell viability within 0.5 to 5 μM inhibitor treatment, indicating a subpopulation of persistent cells. We therefore isolated these persistent cells by treating with a high dose of FGFR inhibitors. After 8 to 12 weeks the cells were completely resistant against all FGFR inhibitors tested. Genetic identity with the original cell line was confirmed by fingerprinting. We found that while parental cell lines showed depleted pERK signals, persistent cells were marked by a constant pERK level upon treatment. In H1581 cells, reactivation of the mitogen-activated protein kinase (MAPK) pathway was demonstrated by an active RAS-pulldown assay. Whole exome sequencing (WES) revealed high and focal NRAS amplification and DUSP6 depletion, leading to MAPK-pathway reactivation. Furthermore, retroviral overexpression of wild type RAS-isoforms induced FGFR-inhibitor resistance in parental H1581 cells. In DMS114 we observed MET and IGF-1R activation as possible mechanisms of persistence. Furthermore, co-inhibition of FGFR and MEK was a highly effective combination therapy to treat FGFR-inhibitor persistent cells. This study associates the MAPK-pathway as a key pathway for FGFR-dependent cell lines. Furthermore, these results suggest a beneficial FGFR / MEK combination treatment to avoid the outgrowth of FGFR-inhibitor persistent cells.

Citation Format: Florian Malchers, Meryem Seda Ercanoglu, Roman Kurt Thomas. MAP-kinase pathway activation facilitates survival of persistent FGFR1-amplified lung cancer cells upon FGFR inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2954.
Abstract 2955: Herceptarg, a novel heterodimeric biparatopic common light chain IgG1 antibody based on trastuzumab and pertuzumab, exerts potent anti-tumoral activity
Authors: Moessner Ekkehard , Hofer Thomas , Waldhauer Inja , Scheuer Werner , Hosse Ralf , Duerner Lydia , He Mi , Zick Karlheinz , Fischer Jens , Sustmann Claudio , Weinzierl Tina , Bacac Marina , Gerdes Christian , Umana Pablo , Klein Christian
Published: July 2016

Introduction: Trastuzumab and pertuzumab are humanized antibodies recognizing different functional HER2 epitopes. Preclinical data demonstrated strong anti-tumoral efficacy when combining them (Scheuer et al., Canc Res, 2009). The CLEOPATRA trial showed that the addition of pertuzumab to trastuzumab + docetaxel strongly improves overall survival of HER2+ metastatic breast cancer patients (Swain et al., NEJM, 2015). Herceptarg is a novel heterodimeric 1+1 biparatopic common light chain IgG1 antibody based on trastuzumab and pertuzumab.

Methods: Using consensus light chains, pertuzumab heavy chain affinity maturation via phage display and knob-into-holes technology, a heterodimeric biparatopic common light chain antibody based on trastuzumab and pertuzumab was generated (Figure 1A). This bispecific antibody was characterized in direct comparison to the respective parental antibodies and their combination in vitro by surface plasmon resonance, proliferation, ADCC and CDC assays and in vivo using the orthotopic KPL-4 breast cancer xenograft model. KPL-4 cells were provided by Prof. Kurebayashi (Kawasaki Medical School, Kurashiki, Japan).

Results: In vitro, Herceptarg has the highest binding affinity for HER2 on cells, mediates potent ADCC activity, comparable or superior growth inhibition activity of breast and gastric cancer cells and CDC superior to the combination of pertuzumab and trastuzumab. In vivo, Herceptarg (10 mg/kg, q1w) mediates anti-tumoral efficacy in the orthotopic KPL-4 breast cancer xenograft model resulting in tumor regression comparable to the combination of trastuzumab and pertuzumab (10 mg/kg, q1w, each), and superior to the respective single agent therapies.

Conclusions: Taken together, these data demonstrate that Herceptarg, as a single IgG1 bispecific antibody is superior (or at least comparable) to the combination of trastuzumab and pertuzumab in vitro and in vivo and may ultimately improve outcome of breast and gastric cancer patients.

Citation Format: Ekkehard Moessner, Thomas Hofer, Inja Waldhauer, Werner Scheuer, Ralf Hosse, Lydia Duerner, Mi He, Karlheinz Zick, Jens Fischer, Claudio Sustmann, Tina Weinzierl, Marina Bacac, Christian Gerdes, Pablo Umana, Christian Klein. Herceptarg, a novel heterodimeric biparatopic common light chain IgG1 antibody based on trastuzumab and pertuzumab, exerts potent anti-tumoral activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2955.