bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1800: Progesterone receptor attenuates STAT1-mediated interferon signaling in breast cancer
Authors: Goodman Merit , Trinca Gloria , Walter Katherine , Papachristou Evangelia , D'Santos Clive , Li Tianbao , Liu Qi , Lai Zhao , Chalise Prabhakar , Madan Rashna , Jin Victor , Carroll Jason , Hagan Christy
Published: August 2018

Why some tumors remain indolent and others progress to clinical relevance remains a major unanswered question in cancer biology. Interferon signaling in nascent tumors, mediated by STAT1, is a critical step through which the surveilling immune system can recognize and destroy developing tumors, thereby blocking their progression. We used an unbiased approach aimed at identifying nuclear protein interactions, called RIME, to identify novel progesterone receptor (PR) protein-protein interactions in T47D breast cancer cells. Using this technique, we identified a novel interaction between PR and STAT1. This interaction led to decreased interferon-induced STAT1 phosphorylation, a phenotype that was further potentiated in the presence of PR ligand. In human breast cancer samples, PR-positive tumors exhibited lower levels of phospho-STAT1 as compared to their PR-negative counterparts, indicating that this phenotype translates to human tumors. Breast cancer cells lacking PR expression exhibited higher levels of interferon-stimulated gene (ISG) RNA, the transcriptional endpoint of interferon activation, indicating that unliganded PR alone could decrease transcription of ISGs. Moreover, the absence of PR led to increased recruitment of STAT1, STAT2 and IRF9 (key transcription factors necessary for ISG transcription) to ISG promoters. These data indicate that PR, both in the presence and absence of ligand, attenuates interferon-induced STAT1 signaling, culminating in significantly abrogated activation of genes transcribed in response to interferons. PR-positive tumors may use downregulation of STAT1-mediated interferon signaling to escape immune surveillance, leading to the development of clinically relevant PR-positive tumors. Selective immune evasion of PR-positive tumors may be one explanation as to why over 70% of breast cancers are PR-positive at the time of diagnosis.

Citation Format: Merit Goodman, Gloria Trinca, Katherine Walter, Evangelia Papachristou, Clive D'Santos, Tianbao Li, Qi Liu, Zhao Lai, Prabhakar Chalise, Rashna Madan, Victor Jin, Jason Carroll, Christy Hagan. Progesterone receptor attenuates STAT1-mediated interferon signaling in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1800.
Abstract 1811: O-GlcNAc-dependent regulation of progesterone receptor function in breast cancer
Authors: Trinca Gloria M. , Goodman Merit L. , Papachristou Evangelia K. , D'Santos Clive S. , Chalise Prabhakar , Madan Rashna , Slawson Chad , Hagan Christy R.
Published: August 2018

Emerging clinical trial data implicate progestins in the development of breast cancer. While the role for the progesterone receptor (PR) in this process remains controversial, it is clear that PR, a steroid-activated nuclear receptor, alters the transcriptional landscape of breast cancer. PR interacts with many different types of proteins, including transcriptional co-activators and co-repressors, transcription factors, nuclear receptors and proteins that post-translationally modify PR (i.e. kinases and phosphatases). Herein, we identify a novel interaction between PR and OGT, the enzyme that catalyzes the addition of a single N-acetylglucosamine sugar, referred to as O-GlcNAc, to acceptor serines and threonines in target proteins. This interaction between PR and OGT leads to the post-translational modification of PR by O-GlcNAc. Moreover, we show that O-GlcNAcylated PR is more transcriptionally active on PR-target genes, despite the observation that PR mRNA and protein levels are decreased when O-GlcNAc levels are high. O-GlcNAcylation in breast cancer is clinically relevant, as we show that O-GlcNAc levels are higher in breast cancer as compared to matched normal tissues, and PR-positive breast cancers have higher levels of OGT. These data predict that under conditions where O-GlcNAc levels are high (breast cancer), PR, through an interaction with the modifying enzyme OGT, will exhibit increased O-GlcNAcylation and potentiated transcriptional activity. Therapeutic strategies aimed at altering cellular O-GlcNAc levels may have profound effects on PR transcriptional activity in breast cancer.

Citation Format: Gloria M. Trinca, Merit L. Goodman, Evangelia K. Papachristou, Clive S. D'Santos, Prabhakar Chalise, Rashna Madan, Chad Slawson, Christy R. Hagan. O-GlcNAc-dependent regulation of progesterone receptor function in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1811.
Abstract 1824: Mechanisms of resistance to anti-EGFR/HER2 therapy in breast cancer
Authors: Borrero-García Luis Daniel , Vidal Brian , Dharmawardhane Suranganie
Published: August 2018

Therapy resistance to epidermal growth factor receptor family (EGFR and HER2) targeted therapies has been an obstacle for treatment success in breast cancer. Therefore, our goal is to investigate the mechanisms of resistance to these therapeutics and look for alternative ways to overcome such resistance. We previously created and characterized syngeneic gefitinib and lapatinib (EGFR and HER2 therapeutics) therapy resistant variants of the therapy sensitive SKBR3 human breast cancer cell line. We found that key mechanisms of resistance involved downregulation of miR-200a and upregulation of miR-221/222 families, which are known to regulate epithelial to mesenchymal transition (EMT). To validate the role of miR-221/222 in therapy resistance, western blots were performed for the miR-221/222 target Trichorhinophalangeal syndrome type I (TRPS1) protein. Downregulation of TRPS1 is known to promote EMT and metastasis. As expected, our results showed a downregulation of TRPS1 in the therapy resistant cells compare to the therapy sensitive cells. Since miR-200a was significantly downregulated in the therapy resistant cells, we ectopically expressed miR-200a in the EGFR therapy resistant SKBR3 cells using a lentiviral vector. MiR-200a expressing cells were selected by puromycin resistance. Preliminary data showed a decrease in the fold resistance of cells resistant to lapatinib transfected with lentiviral vector overexpressing miR-200a. These results suggest that overexpression of miR-200a in lapatinib resistant cells can be used to overcome therapy resistance in breast cancer.

Citation Format: Luis Daniel Borrero-García, Brian Vidal, Suranganie Dharmawardhane. Mechanisms of resistance to anti-EGFR/HER2 therapy in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1824.
Abstract 1834: Src inhibition overcomes neratinib resistance in HER2-positive breast cancer
Authors: Conlon Neil , Lowry Michelle , Breslin Susan , O'Driscoll Lorraine , Eustace Alexander J. , Crown John , O'Donovan Norma , Collins Denis M.
Published: August 2018

Introduction: Despite the recent successes of HER2-targeted therapies, drug resistance still represents a significant clinical problem. Neratinib, an irreversible pan-HER tyrosine kinase inhibitor, has been recently FDA approved for the extended adjuvant treatment of early-stage HER2-positive breast cancer and little is known of its mechanism of resistance in breast cancer. Using a neratinib-resistant cell line model of HER2-positive breast cancer, HCC1954-N, the aim of this study was to characterise cross-resistance to other HER-targeting tyrosine kinase inhibitors (TKIs) and investigate the role of Src in acquired neratinib resistance.

Methods: The effect of HER-family targeting TKIs neratinib, lapatinib, afatinib and, the Src inhibitor, dasatinib on proliferation of HCC1954-N and HCC1954-Par cells was assessed by acid phosphatase assays and three-dimensional Matrigel assays. Cell cycle progression was examined by flow cytometry using a propidium iodide assay. Caspase 3/7-Glo assay was used to assess apoptosis induction. To assess the prevention of the development of neratinib resistance, HCC1954 cells were treated twice weekly with neratinib, dasatinib, or the combination and stained with crystal violet when confluent.

Results: The HCC1954-N cell line was resistant to neratinib and to afatinib and lapatinib compared to parental HCC1954, but neratinib was still the most potent of the three anti-proliferative agents. The combination of neratinib and dasatinib was synergistic in the neratinib-resistant HCC1954-N cell line in both adherent (CI value = 0.1 ± 0.03) and three-dimensional (CI value = 0.36 ± 0.02) conditions. Neratinib plus dasatinib did not result in cell cycle arrest; however, the combination caused a significant increase in the sub-G1 cell cycle population (p value = 0.018), indicating induction of apoptosis. This was further validated by increased activation of caspase 3 and 7 (p value = 0.015) in HCC1954-N cells treated with neratinib plus dasatinib. The addition of dasatinib to neratinib treatment prevented the development of neratinib resistance in parental HCC1954 cells.

Conclusions: Further pre-clinical investigation of the combination of neratinib plus dasatinib is warranted as a potential therapeutic intervention for patients with neratinib-resistant HER2-positive breast cancer.

Citation Format: Neil Conlon, Michelle Lowry, Susan Breslin, Lorraine O'Driscoll, Alexander J. Eustace, John Crown, Norma O'Donovan, Denis M. Collins. Src inhibition overcomes neratinib resistance in HER2-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1834.
Abstract 1851: Combined inhibition of PARP and the unfolded protein response synergistically reduces viability in BRCA1-mutant breast cancer cells
Authors: Endress Jennifer E. , Harris Isaac S. , Stewart-Ornstein Jacob , Lahav Galit , Brugge Joan S.
Published: August 2018

As single agents, small molecule inhibitors of poly-ADP-ribose polymerase (PARP) have shown remarkable efficacy in BRCA1/2-mutant tumors due to their synthetic lethality in DNA damage response-deficient cells. While PARP inhibitors, such as olaparib, have gained FDA-approval for gynecological malignancies, recurrent disease remains a challenge. To identify drug combinations with the potential to improve the initial response to PARP inhibition, we carried out a high-throughput drug screen in which we examined the ability of olaparib to synergize with more than 500 small molecules in BRCA1-mutant breast cancer cells. The compounds investigated included not only conventional “anti-cancer” agents but also lesser-studied metabolic and stress-inducing drugs. All compounds were tested across 10-point dose curves using an image-based readout to avoid false positives/negatives associated with more traditional metabolic readouts of cell viability (i.e. Cell Titer Glo). This screening approach revealed that inhibition of the unfolded protein response (UPR) sensitizes breast cancer cells to PARP inhibition. We have further characterized interaction of one class of inhibitors that target IRE1 (inositol-requiring enzyme 1), which initiates adaptive responses to ER stress through unconventional mRNA splicing of the transcription factor X-box binding protein 1 (XBP1). Combining olaparib with an inhibitor of IRE1-mediated XBP1 splicing (STF-0831010) synergistically reduced cancer cell viability in BRCA1-mutant SUM149 breast cancer cells. This combination led to increased DNA damage, indicated by increased γ-H2AX staining. This study provides evidence that UPR inhibition sensitizes cells to PARP inhibition, suggesting a previously un-recognized connection between PARP inhibition and protein homeostasis. Furthermore, it identifies a potential new drug combination to increase the efficacy of PARP inhibitors in the clinic.

Citation Format: Jennifer E. Endress, Isaac S. Harris, Jacob Stewart-Ornstein, Galit Lahav, Joan S. Brugge. Combined inhibition of PARP and the unfolded protein response synergistically reduces viability in BRCA1-mutant breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1851.
Abstract 1858: EDNRB isoform 3 confers temozolomide resistance in human melanoma cells by modulating membrane potential, reactive oxygen species and mitochondrial Ca2+
Authors: Cui Long , Liang Bo , Chen Xiaoting , Wang Chi Chiu
Published: August 2018

Background: The role of Endothelin receptor type B (EDNRB) isoform 3 involved in Temozolomide (TMZ) induced melanoma cell death has not yet been elucidated.

Methods: The subcellular localization of EDNRB isoform 3 was determined by confocal and immunoblotting assays. Silencing EDNRB isoform 3 was performed by CRISPR/Cas9. Apoptosis was assessed by Annexin V/PI staining and caspases 3/7/9 activity. Mitochondrial membrane potential, reactive oxygen species and mitochondrial Ca2+ were measured by flow cytometry. Apoptosis protein array was applied.

Results: Confocal and immunoblot analyses indicate mitochondrial localization of EDNRB isoform 3 and the first N-terminal (1-22) amino acids are sufficient for its mitochondrial targeting. EDNRB isoform 3 depleted A375 cells significantly inhibit apoptosis. EDNRB isoform 3 mediates chemoresistance appears to be acting via a mitochondrial apoptosis pathway as manifested by the prevention in mitochondrial depolarization, reduced reactive oxygen species, enhanced mitochondrial Ca2+ uptake and decreased caspase 9 activation. Additionally, apoptosis array shows that lack of EDNRB isoform 3 has relatively lower expression of phosphorylation of p53 at S392 and a slightly higher expression of Paraoxonase 2.

Conclusion: Our findings raise the possibility of targeting EDNRB isoform 3 as new therapeutic strategy in combination with TMZ for melanoma treatment.

Citation Format: Long Cui, Bo Liang, Xiaoting Chen, Chi Chiu Wang. EDNRB isoform 3 confers temozolomide resistance in human melanoma cells by modulating membrane potential, reactive oxygen species and mitochondrial Ca2+ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1858.
Abstract 1866: Small molecule NSC59984 is a radio-sensitizer dependent on ERK2 and DDR but independent of wild-type p53
Authors: Zhang Shengliang , Zhou Lanlan , Finnberg Niklas , El-Deiry Wafik
Published: August 2018

Radiotherapy is a common and effective therapeutic strategy applied to various types of tumors. Mutations that commonly occur in p53 as a tumor suppressor gene, are a hallmark of tumor, and confer cellular radio-resistance. Targeted therapy is considered as a promising approach to increase efficacy and specificity of radiotherapy. We explore utilization of a small molecule targeting mutant p53 to enhance efficacy of radiotherapy in colorectal cancer. Colorectal cancer cells carrying with mutant p53 were treated with NSC59984 followed by ionizing radiation treatment. Combined NSC59984 with radiation significantly reduced colony formation in mutant p53-expressing colorectal cancer cells in a dose-dependent manner, suggesting that NSC59984 enhances the efficacy of radiotherapy. NSC59984 induces mutant p53 degradation via the ERK2 pathway. We investigated the role of ERK2 in the efficacy of the combination of NSC59984 and radiation treatment. ERK2 blockade partially abrogated the effect of NSC59984 on the radio-sensitivity, and this correlated with the rescue of mutant p53. Our results suggest that NSC59984 increases the efficacy of radiotherapy via activation of ERK2 or ERK2-dependant mutant p53 degradation. Mutant p53 and ERK2 are important regulators involved in repair of DNA double-strand breaks. We examined the DNA damage response (DDR) system and found that reduced Rad51 protein in cells treated with NSC59984, and the Rad51 protein was increased by the blockade of the ERK2 pathway. On the basis of the above results, we conclude that small-molecule NSC59984 enhances cancer cell sensitivity to radiotherapy via the ERK2 pathway and effects on DDR.

Citation Format: Shengliang Zhang, Lanlan Zhou, Niklas Finnberg, Wafik El-Deiry. Small molecule NSC59984 is a radio-sensitizer dependent on ERK2 and DDR but independent of wild-type p53 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1866.
Abstract 1894: Identification of novel OTX015-containing combinations for lymphoma treatment
Authors: Gaudio Eugenio , Tarantelli Chiara , Spriano Filippo , Arribas Alberto J. , Cascione Luciano , Zucca Emanuele , Stathis Anastasios , Bertoni Francesco
Published: August 2018

Introduction. Epigenetics represents and intriguing target of therapy for lymphomas. The epigenetic mechanisms are regulated by several players (e.g. HDAC, HAT, BET) and among them the BET proteins became of interest a few years ago with demonstrated preclinical anti-cancer activity with the first compounds and early clinical responses first in class clinical-grade BET inhibitor OTX015 (MK-8628/birabresib). Despite a widespread anti-proliferative activity, the cytotoxicity effect of BET inhibitors is limited, and, here, we aimed to identify drugs that improve OTX015 activity performing a screening with the BET inhibitor with a library 348 compounds in two lymphoma cell lines.

Methods. Two cell lines derived from germinal center B cell (GCB) diffuse large B cell lymphoma (DLBCL) (OCI-LY-19 and WSU-DLCL2) were exposed to OTX015 (single dose, 100 nM) in combination with two different doses (20 and 1,000 nM) of 348 compounds. Compounds giving a 1.5-fold decreased proliferation with the combination than with the individual compounds were further evaluated in two additional lymphoma cell lines, exposed (72h) to increasing doses of OTX015 alone and in combination with increasing doses of other selected compounds. Synergy was assessed with Chou-Talalay combination index (CI): synergism (<0.9), additive (0.9-1.1), antagonism/no benefit (> 1.1).

Results. OCI-LY-19 and WSU-DLCL2 were treated with OTX015 (100 nM) and 348 compounds as single agents and in combination. The combinations of OTX015 with a series of compounds achieved improved anti-tumor activity than single agents. Besides HDAC and mTOR inhibitors, in accordance with what previously reported (Boi, Gaudio, Bonetti et al, Clinical Cancer Res 2015), the ABL/SRC inhibitor dasatinib, the AKT1/2/3 inhibitor MK-2206, the JAK2 inhibitor TG101209 and the LRRK2 inhibitor LRRK2-IN appeared interesting combination partners. These screening results were validated in the DLBCL cell lines with targeted experiments, as well as in chronic lymphocytic leukemia (CLL) (OTX015 plus MK-2206) and mantle cell lymphoma (MCL) (REC1) (Dasatinib, LRRK2-IN). Experiments in additional cell lines and assessing the effects on the apoptosis are on-going and will be presented.

Conclusion. A chemical screening has identified novel OTX015-containing combinations active in lymphoma cell lines that are worth of further investigations.

Work supported by a San Salvatore Foundation grant.

Citation Format: Eugenio Gaudio, Chiara Tarantelli, Filippo Spriano, Alberto J. Arribas, Luciano Cascione, Emanuele Zucca, Anastasios Stathis, Francesco Bertoni. Identification of novel OTX015-containing combinations for lymphoma treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1894.
Abstract 1902: Potential role of DYRK family kinases in harmine-induced apoptosis in neuroblastoma
Authors: Uhl Katie L. , Schultz Chad R. , Geerts Dirk , Bachmann Andre S.
Published: August 2018

The purpose of this study was to determine the effect of the natural product Harmine in human neuroblastoma (NB). NB is an early childhood malignancy arising from the developing peripheral nervous system. In up to 25% of cases MYCN gene amplification is correlated to high tumor stage and poor patient prognosis. High stage NB relapses frequently in spite of multimodal therapy and is then virtually untreatable. There is a clear need for specific, novel therapeutics. Harmine is a tricyclic β-carboline alkaloid from the harmal plant with cytostatic and cytotoxic effects on tumor cells. It is capable of blocking the activity of mitogen activate protein kinase (MAPK) and the dual specificity tyrosine-phosphorylation-regulated kinase (DYRK) family proteins. These kinases inhibit apoptosis and encourage proliferation. Four human NB cell lines were used to study the effects of Harmine treatment: SK-N-BE(2)-C and KELLY (MYCN amplified) as well as SK-N-AS and SK-N-FI (MYCN single copy). Molecular interaction models of Harmine bound to DYRK family kinases were generated by computational docking using x-ray structures. The anti-cancer properties of Harmine were analyzed by real-time cell viability assays, in a dose-dependent manner, over a 72 hour period. The IC50 values were 169.9, 170.8, and 791.7 μM for SK-N-BE(2)-C, KELLY, and SK-N-FI, respectively, after 72 hours. Apoptosis assays for caspase activation, PARP cleavage, and annexin V induction, were performed using Western blot and flow cytometry. NB cell line exposure to 100 μM Harmine resulted in caspase-3/7 and caspase 9 activation, PARP cleavage, and annexin V-positive stained cells as early as 24 hours after treatment, indicating apoptosis induction. These results led us to investigate the clinical correlations of DYRK family gene expression in NB tumors. The patient results support our hypothesis that Harmine induces NB cell death through a cellular mechanism that involves DYRK family kinases and triggers caspase-mediated apoptosis.

Citation Format: Katie L. Uhl, Chad R. Schultz, Dirk Geerts, Andre S. Bachmann. Potential role of DYRK family kinases in harmine-induced apoptosis in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1902.
Abstract 1903: Antiproliferative effect of cannabinoid receptor agonists on rectal cancer lines
Authors: Gómez Alette Ortega , Luna-Merlos Pedro , Hernández Victor Manuel García , García Abelardo Meneses , Vega Horacio Astudillo- de la , López Eric Alejandro García , Angel Abel Santamaría Del , García Erika Betzabé Ruiz
Published: August 2018

Rectal cancer is considered a major health issue in several countries that present advanced and metastatic stages and a limited response to standard treatments. Though the antineoplasic activity of cannabinoids has been documented in several studies, scarce information is available about the effect of these agents in cell lines of rectal cancer. In this work, we explored the antiproliferative effects of cannabinoid receptor agonists in rectal tumor cells. The effects of the three agonists anandamide, methanandamide and CP55940 were evaluated in the SW837 rectal tumor cell line. The three agents tested showed time and concentration dependent effects on cell viability, induction of cell death and morphological changes. The three agonists were effective to induce late apoptosis and necrosis and internucleosomal DNA fragmentation. In addition, we evaluated for the first time the patterns of CB1 and CB2 cannabinoid receptor expression in SW837. Though some differences in their effects were found, our data support the concept that these agents mostly share common antiproliferative/cell death mechanisms. Our results also confirm the antineoplasic potential of cannabinoids receptor agonists against rectal cancer, and suggest the design of cannabinoid based coadjutant therapies at the clinical level.

Citation Format: Alette Ortega Gómez, Pedro Luna-Merlos, Victor Manuel García Hernández, Abelardo Meneses García, Horacio Astudillo- de la Vega, Eric Alejandro García López, Abel Santamaría Del Angel, Erika Betzabé Ruiz García. Antiproliferative effect of cannabinoid receptor agonists on rectal cancer lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1903.
Abstract 1912: A microbial glucuronidase target implicated in intestinal reactivation of tyrosine kinase inhibitors
Authors: Wallace Bret David , Hymes Jeffrey , Redinbo Matthew , Peterson Ward
Published: August 2018

Many drugs are inactivated by liver UGT enzymes and reactivated by enteric bacterial β-glucuronidases (GUS), an axis of drug metabolism that contributes to gut toxicities of anti-neoplastic agents, NSAIDs, and potentially other drug classes. For example, toxic reactivation of irinotecan by E. coli GUS is the causative event leading to intense, delayed diarrhea. Receptor tyrosine kinase (RTK)-targeted cancer drugs that undergo this UGT-GUS axis of metabolism are also associated with a high incidence of diarrhea. However, glucuronides of RTK inhibitors (RTKi) are not substrates of E. coli GUS. We recently reported that the human gut microbiome expresses nearly 300 distinct GUS enzymes (GUSome). The objective of this work is to identify the GUS enzyme(s) capable of metabolizing the glucuronide of the RTKi, sorafenib, that may contribute to sorafenib-induced diarrhea.

We used UGT1A9 supersomes to make sorafenib-glucuronide (S-G) from commercially purchased sorafenib. S-G was screened against a library of 15 purified GUSome enzymes to identify the specific GUS orthologs that can cleave S-G, which was determined by monitoring sorafenib absorbance at 280 nm. Similar approaches were used to identify GUS enzymes that can cleave other RTKi's.

Of the GUS enzymes evaluated for cleavage of S-G, only the GUS enzyme from an uncultured Eubacterium species was active. The structurally similar regorafenib-glucuronide and two glucuronides of other RTKi's (axitinib and imatinib) are also substrates of Eubacterium GUS. All four RTKi's are associated with diarrhea in at least 40-60% of patients. In contrast to E. coli GUS, very little is known about Eubacterium GUS.

We have identified a set of RTKi-glucuronides as the first substrates of Eubacterium GUS, which may contribute to diarrhea associated with targeted anti-cancer drugs.

Citation Format: Bret David Wallace, Jeffrey Hymes, Matthew Redinbo, Ward Peterson. A microbial glucuronidase target implicated in intestinal reactivation of tyrosine kinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1912.
Abstract 1921: The sensitivity of ovarian cancer cells to the HDAC inhibitor belinostat is improved by inhibiting Bcl-xL or Mcl-1 anti-apoptotic proteins
Authors: Duboc Charlène , Abeilard Edwige , Voisin-Chiret Anne Sophie , Gauduchon Pascal , Poulain Laurent , Villedieu Marie
Published: August 2018

The identification of novel therapeutic strategies is an important urgent requirement for the clinical management of ovarian cancers, which remain the leading cause of death from gynecologic cancer. Our previous studies have shown that the anti-apoptotic proteins Bcl-xL and Mcl-1 cooperate to protect resistant ovarian cancer cells from apoptosis as their concomitant inhibition results in massive apoptotic cell death. Moreover, their BH3-only pro-apoptotic partners Bim and Puma are crucial actors in the induced cell death. This suggests that ovarian cancer cell apoptosis can be triggered by inhibiting Bcl-xL and Mcl-1 and/or by promoting their BH3-only partners, especially Bim and Puma. The expression of these Bcl-2 family proteins has been reported to be modulated by HDAC (histone deacetylases) inhibitors in various cancer models. The objective of the present study was to evaluate the efficacy of the FDA-approved pan-HDAC inhibitor belinostat (i) to reduce the [Bcl-xL and Mcl-1] / BH3-only proteins ratio and (ii) to trigger apoptosis in chemoresistant ovarian cancer cell lines, alone or in combination with other pharmacological molecules that modulate these Bcl-2 family proteins. HDAC inhibition by belinostat led to a G2/M blockade and consequently reduced cell proliferation. Moreover, belinostat used at high concentrations (from 1000 to 2000 nM) efficiently triggered apoptosis in chemoresistant ovarian cancer cells. This cytotoxic effect was associated with both an up-regulation of Bim and Puma protein expression and an inhibition of Bcl-xL protein expression. Used at sub-toxic concentrations, belinostat also up-regulated Bim and Puma proteins but failed to down-regulate Bcl-xL and Mcl-1 protein expression. We therefore investigated whether Bcl-xL or Mcl-1 inhibition could sensitize ovarian cancer cells to sub-toxic concentrations of belinostat. Interestingly, the combination of belinostat with the BH3-mimetic ABT-737, that inhibits Bcl-xL, induced massive apoptosis. Moreover, Mcl-1 direct or indirect inhibition was also very efficient to sensitize ovarian cancer cells to belinostat. These results suggest that belinostat, alone or in association with pharmacological molecules that inhibit Mcl-1 or Bcl-xL, represents a promising approach for the treatment of chemoresistant ovarian cancers.

Citation Format: Charlène Duboc, Edwige Abeilard, Anne Sophie Voisin-Chiret, Pascal Gauduchon, Laurent Poulain, Marie Villedieu. The sensitivity of ovarian cancer cells to the HDAC inhibitor belinostat is improved by inhibiting Bcl-xL or Mcl-1 anti-apoptotic proteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1921.
Abstract 1974: Inhibition of the P-glycoprotein with a novel agent in overexpressing P-glycoprotein chemotherapy resistant colorectal cancer cells
Authors: Farrell Christopher L. , Mahan Rebecca , Blauvelt Catherine , Servais Taylor Kaye , Lane Jessica
Published: August 2018

Multidrug resistance (MDR) plays a key role in chemotherapy failure and poor patient outcome. One of the ways multidrug resistance occurs is through the efflux of chemotherapeutic agents using the P-glycoprotein transporter protein on the surface of the tumor cell. There are several medications that work through P-glycoprotein (Pgp), which can either induce or inhibit the activity of the transporter. The purpose of this study was to evaluate known P-glycoprotein inhibitors and their effects on chemotherapy resistance in Caco2 cells, a colorectal cancer cell line. The experiments consisted of feeding the colorectal cancer cells with commonly known drugs that are Pgp substrates such as vinblastine for 4-8 months to establish an overexpressing Pgp transporter. An untreated negative control was used to show that Pgp was not overly expressed. Once the treated cells were overexpressing Pgp, the cells were seeded into a 96 well plate with a matrix of increasing chemotherapy and bergamottin, a known P-glycoprotein inhibitor. A cell proliferation assay was performed according to manufacturer protocol, with a Presto-Blue Cell Proliferation Assay kit to assess the effects of the chemotherapy agent, vinblastine and the bergamottin. Absorbance was recorded at 595 nanometers. This assay was tested on several multidrug resistant colorectal cell with different substrates and normal P-glycoprotein expressing cancer cells. The overexpressing Pgp cell lines required higher doses of vinblastine to exhibit cell death compared to the negative cells. The positive cells treated with bergamottin only showed change in cell death with increasing bergamottin concentration. From these results, it was determined that the use of the novel P-glycoprotein inhibitor was able to reverse the chemotherapy induced multidrug resistance in the cells with high Pgp expression. Patients whose tumor cells develop MDR have a poor prognosis compared to patients whose cells are responsive to chemotherapy. Because the chemotherapy agents are ineffective in killing the all of the tumor cells including cells which have become metastatic, the overall survival rate for patients with MDR-positive tumors is markedly decreased. Identify these patients as potential candidates for the Pgp inhibitor could significant increase their chances of survival with this aggressive form of cancer.

Citation Format: Christopher L. Farrell, Rebecca Mahan, Catherine Blauvelt, Taylor Kaye Servais, Jessica Lane. Inhibition of the P-glycoprotein with a novel agent in overexpressing P-glycoprotein chemotherapy resistant colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1974.
Abstract 1979: HQP1351, a novel multikinase inhibitor in clinical development, overcomes drug resistance for the treatment of gastrointestinal stromal tumors in preclinical models
Authors: Wang Guangfeng , Qiu Haibo , Min Ping , Wu Miaoyi , Dang Shuo , Yang Chunyang , Zhang Fei , Zhuang Wei , Zhou Zhiwei , Fang Douglas D. , Yang Dajun , Zhai Yifan
Published: August 2018

Gastrointestinal stromal tumors (GIST) harbor driver mutations of signal transduction kinases such as KIT. Besides surgical resection for primary localized tumors, imatinib remains the first-line treatment for advanced and metastatic GISTs. Imatinib targets a few kinases including KIT that often carries the primary driver mutations commonly located on exon 11 and exon 9. Unfortunately, a large proportion of the patients ultimately develop progressive disease owing to the secondary resistance mutations in KIT gene. We have developed an orally bioavailable multikinase inhibitor HQP1351, which is currently in clinical trials for the treatment of T315I mutant CML. Here, we demonstrated that HQP1351 inhibited both wild type and mutant (i.e., KITL576P, KITV559D, and KITV559D/T670I) KIT in biochemical assays. Using a panel of imatinib-resistant and sensitive GIST cancer cell lines derived from patient samples, we showed that HQP1351 exhibited more potent anti-proliferative activities than ponatinib (range of IC50 values: 0.027-0.133 µM vs. 0.021-0.730 µM), the latter of which is currently in clinical development in GIST to overcome the resistance to imatinib (NCT03171389). In addition, the treatment of GIST cancer cells with HQP1351 in vitro led to more profound inhibition of pharmacodynamic markers, including p-c-KIT, p-AKT, p-ERK1/2, and p-AKT, in comparison with ponatinib. Correspondingly, in multiple xenograft tumor models derived from these GIST cancer cell lines carrying the secondary mutations of KIT, HQP1351 exhibited superior antitumor activities to ponatinib. Collectively, considering that HQP1351 has already been in clinical trials, the above results suggest that therapeutic application of HQP1351 in imatinib-resistant GIST patients deserves further investigation in human.

Citation Format: Guangfeng Wang, Haibo Qiu, Ping Min, Miaoyi Wu, Shuo Dang, Chunyang Yang, Fei Zhang, Wei Zhuang, Zhiwei Zhou, Douglas D. Fang, Dajun Yang, Yifan Zhai. HQP1351, a novel multikinase inhibitor in clinical development, overcomes drug resistance for the treatment of gastrointestinal stromal tumors in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1979.
Abstract 198: MUC13 promotes pancreatic tumor-stromal interactions by influencing tumor microenvironment
Authors: Khan Sheema S. , Doxtater Kyle , Kumari Sonam , Setua Saini , Sikander Mohammed , Malik Shabnam , Yallapu Murali , Behrman Stephen , Chauhan Subhash , Jaggi Meena
Published: August 2018

Objective: Pancreatic cancer (PanCa) is the third leading cause of cancer-related deaths in the U.S. MUC13, mucin, is aberrantly expressed in PanCa and its overexpression leads to tumorigenic and metastatic behavior of cancer cells. Herein, we investigate the role of MUC13 in tumor microenvironment (TME) that favors PanCa progression/metastasis and drug resistance.

Methods: MUC13 expressing (Panc-1) and knockdown PanCa cells (HPAF-II) and pancreatic stromal cells (PSCs), derived from freshly collected human pancreatic tissues, were utilized for the study. Using immunoblotting, ELISA, qPCR and RT-PCR techniques, we assessed the MUC13-induced alterations in the expression of important molecules that are involved in promotion of pancreatic tumor stromal interactions and drug resistance. Cytation 3 live cell imaging was performed for 3D tumor-stromal cell co-culture and invasion assays. Xenograft mice tumor tissues generated using ectopically expressing MUC13 and null cells were used for the study.

Results: Our results demonstrate that MUC13 activates SHH signaling pathway in PanCa cells via upregulation of SHH and its related important downstream targets such as CXCR-4, Gli-1, SMO, PTCH1/2, NFκB and p-AKT in PanCa cells using immunoblotting and PCR. We also observed increased expression of SHH, Gli-1, CXCR-4 and α-SMA in MUC13-expressing xenograft tumors using Immunohistochemistry. Ectopic MUC13-expressing PanCa cells grown in co-culture with PSCs showed enhanced secretion of IL-6 and CXCL-12 (ELISA), increase in invasion (Matrigel invasion assay) and migratory (Boyden chamber assay) potential of both the cultured PanCa as well as stromal cells. Additionally, ectopically MUC13-expressing cells were resistant to treatment with gemcitabine as depicted by proliferation assay and qPCR results showed enhanced expression of ribonucleoside-diphosphate reductase (RRM1/2) and miR-21, which are known to be involved in gemcitabine resistance. Overall, our results suggest that MUC13 modulates tumor-stromal crosstalk, promoting tumor growth, invasion and drug resistance in TME.

Conclusion: Our results suggest that MUC13 expression might influence signaling supportive of interactive TME, leading to increased tumor growth and drug resistance. These findings illustrate mechanisms by which MUC13 promotes tumorigenic TME and contributes to PanCa cell survival, metastasis and drug resistance.

Citation Format: Sheema S. Khan, Kyle Doxtater, Sonam Kumari, Saini Setua, Mohammed Sikander, Shabnam Malik, Murali Yallapu, Stephen Behrman, Subhash Chauhan, Meena Jaggi. MUC13 promotes pancreatic tumor-stromal interactions by influencing tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 198.
Abstract 1984: Cell fate reprogramming of liver tumor-initiating stem-like cells via phosphorylated NUMB and TBC1D15
Authors: Machida Keigo , Siddique Hifzur R. , Zheng Mengmei , Winer Peleg , Kumar Dinesh Babu Uthaya , Rokan Ahmed , Sher Linda , Tahara Stanley M. , Elowitz Michael , Liang Chengyu , Tsukamoto Hidekazu
Published: August 2018

Tumor-initiating stem-like cells (TICs) are defective in their control of asymmetric division. Stem cells are maintained through asymmetric self-renewing divisions in which one daughter cell commits to a specific fate while the other retains the multipotent characteristics of its parent. The p53 interacting protein NUMB preserves this intrinsic cellular asymmetry and functions as a vital barrier against unchecked expansion as seen in TICs. How TICs overcome this control of asymmetric division to cause cancer is unknown. The hypothesis is that accentuated TLR4-NANOG-mediated NUMB phosphorylation and TBC1D15 upregulation promote self-renewal of TICs in liver oncogenesis caused by HCV and alcohol. Here, we determined the causal roles of NUMB phosphorylation and TBC1D15 oncoprotein in alcohol-induced liver tumorigenesis in HCV-Tg mice. Liver-specific TBC1D15 deficiency or non-phosphorylatable mutations of NUMB, reduce liver tumor incidence and tumor-associated NANOG+ TICs in HCV NS5A Tg mice fed alcohol Western diet. TICs lost asymmetric division capacity and overexpressed TBC1D15, which associated with NUMB to promote aPKCζ activity and NUMB phosphorylation. Thus, TIC self-renewal is dependent on TBC1D15 and NUMB phosphorylation. Similarly, aPKCζ-mediated NUMB phosphorylation and induced TBC1D15 are mutually required for self-renewal. Furthermore, TBC1D15 activated NOTCH via binding to NOTCH intracellular domain to trigger its proteolytic. TBC1D15 interacts with all NOTCH isoforms, activates NOTCH pathway, and induces Nanog and TIC self-renewal in a NOTCH-dependent manner. TBC1D15 cooperates with the NOTCH pathway to support TIC tumorigenic activity and an inhibitor of this interaction is potentially therapeutic. Candidate small molecule antagonists have been tested for their ability to block the interaction of TBC1D15-NOTCH/NICD. The originality of this findings lies within the discovery of the novel oncoprotein TBC1D15 and its unique oncogenic activities involving p53 degradation and cooperation with the NOTCH pathway in TICs and CD133+ Huh7 cells. These mechanistic findings have laid down the foundation for a new translational path for discovery of new therapeutic targets.

Citation Format: Keigo Machida, Hifzur R. Siddique, Mengmei Zheng, Peleg Winer, Dinesh Babu Uthaya Kumar, Ahmed Rokan, Linda Sher, Stanley M. Tahara, Michael Elowitz, Chengyu Liang, Hidekazu Tsukamoto. Cell fate reprogramming of liver tumor-initiating stem-like cells via phosphorylated NUMB and TBC1D15 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1984.
Abstract 1996: Nuclear factor (erythroid-derived 2)-like 2 promotes tumor-initiating cell lineage and drug resistance in hepatocellular carcinoma
Authors: Leung Hoi Wing , Lee Terence Kin Wah
Published: August 2018

Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide. The long-term prognosis of HCC remains unsatisfactory due to high recurrence rates and chemoresistance. The efficacy of sorafenib, the only FDA-approved molecularly targeted drug in advanced hepatocellular carcinoma (HCC) is limited by acquired resistance. Increasing evidences showed that liver tumor-initiating cells (T-ICs) is the source of acquired resistance and metastasis. We have previously established sorafenib-resistant HCC cells both in vitro and PDTX models and demonstrated that these cells are endowed with enhanced T-IC properties. Interestingly, nuclear factor (erythroid-derived 2)-like 2 (NRF2) expression was elevated in these resistant cells. The transcription factor NRF2 has long been considered as a tumour suppressor, as it can regulate cellular antioxidant response to protect cells from oxidative stress, chemotherapeutic agents and radiotherapy. In HCC, NRF2 level was up-regulated, and its expression was correlated with tumor differentiation, metastasis, and tumor size, revealing its oncogenic role. By lentiviral based knockdown approach, we found that NRF2 repression suppressed liver T-IC properties including self-renewal, in vivo tumorigenicity, HCC invasiveness and expression of liver T-IC markers. In addition, we found that NRF2 repression not only sensitized HCC cells to sorafenib but also to other chemotherapeutic drugs including doxorubicin and fluorouracil. Mechanistically, the signaling cascade related to sonic hedgehog pathway was greatly suppressed in NRF2 knockdown HCC cells, when compared with control counterparts. Collectively, NRF2 was involved in drug resistance, invasion and migration, tumorigenicity and self-renewal via regulating T-IC properties at least in part through direct regulation of sonic hedgehog pathway in HCC. Targeting NRF2 mediated signaling cascade alone or in combination with other treatment modalities may be a new a potential therapeutic approach for treatment of HCC.

Citation Format: Hoi Wing Leung, Terence Kin Wah Lee. Nuclear factor (erythroid-derived 2)-like 2 promotes tumor-initiating cell lineage and drug resistance in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1996.
Abstract 2001: Transcriptional integrator p300 in human prostate tumor-initiating cells
Authors: Gruber Martina , Handle Florian , Culig Zoran
Published: August 2018

Introduction

p300 is a well-known coactivator of the androgen receptor (AR) and has been shown to play an important role in prostate cancer (PCa). The expression of p300 has been correlated to tumor volume and PCa progression. Therefore, we hypothesize that p300 may play an important role in tumor-initiating cells and therapy resistance. Due to its multifunctional role p300 may serve as a promising new therapeutic target for treatment of PCa. Ultimately, it is our aim to find new innovative therapeutic targets for castration resistant PCa.

Material & Methods

The tumor-initiating capacity of the AR-negative PCa cell lines PC3 and Du145, as well as primary prostate epithelial cells was characterized by seeding a limiting number of cells per flask. Colony formation efficiency, colony type and detailed morphological information about each colony were determined by an automated pipeline that we have developed. Stable cell lines with doxycycline inducible p300 sh RNAs were generated by lentiviral transduction. Knockdown of p300 was verified by Western Blot and reduced p300 activity was confirmed by measuring histone h3 acetylation.

Results

In concordance with the literature we identified three colony types (holo-, mero- and paraclones) in PC3 and Du145 cell lines as well as primary PCa cells. Serial clonogenic assays confirmed in both cell lines that holoclones show the highest colony formation potential and maintain their tumor-initiating capacity over numerous rounds. In PC3 we detected 10% and in Du145 30% holoclones. Under optimal growth conditions p300 was detected in all colony types, however no differences in protein expression of p300 as well as on mRNA level were observed. Knockdown of p300 did not alter colony formation efficiency or the relative distribution of colony types.

Conclusion

Taken together, we confirmed the presence of tumor-initiating holoclones in PCa cell lines and primary prostate epithelial cells and established stable cell lines with inducible knockdown of p300. In contrast to several other types of malignancies, p300 has no specific role in regulation of stemness in our PCa models at the tested conditions. As a next step we will determine the effects of p300 knockdown during chemotherapy and radiation therapy to evaluate if p300 depletion may enhance the treatment efficacy and which role p300 plays in therapy resistance.

Citation Format: Martina Gruber, Florian Handle, Zoran Culig. Transcriptional integrator p300 in human prostate tumor-initiating cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2001.