NR4A1 Antagonists Inhibit β1-Integrin-Dependent Breast Cancer Cell Migration

Overexpression of the nuclear receptor 4A1 (NR4A1) in breast cancer patients is a prognostic factor for decreased survival and increased metastasis, and this has been linked to NR4A1-dependent regulation of transforming growth factor β (TGF-β) signaling. Results of RNA interference studies demonstrate that basal migration of aggressive SKBR3 and MDA-MB-231 breast cancer cells is TGF-β independent and dependent on regulation of β1-integrin gene expression by NR4A1 which can be inhibited by the NR4A1 antagonists 1,1-bis(3′-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH) and a related p-carboxymethylphenyl [1,1-bis(3′-indolyl)-1-(p-carboxymethylphenyl)methane (DIM-C-pPhCO(2)Me)] analog. The NR4A1 antagonists also inhibited TGF-β-induced migration of MDA-MB-231 cells by blocking nuclear export of NR4A1, which is an essential step in TGF-β-induced cell migration. We also observed that NR4A1 regulates expression of both β1- and β3-integrins, and unlike other β1-integrin inhibitors which induce prometastatic β3-integrin, NR4A1 antagonists inhibit expression of both β1- and β3-integrin, demonstrating a novel mechanism-based approach for targeting integrins and integrin-dependent breast cancer metastasis

Abstract 3832: Synergistic anticancer effects of epigenetic drugs JQ1 and SAHA in adenoid cystic carcinoma of the lacrimal gland

Abstract Background: Adenoid cystic carcinoma is the most common epithelial malignancy of the lacrimal gland and has poor prognosis despite current treatment strategies. The aims of this study were to evaluate the anticancer efficacy of the dual inhibition of bromodomain proteins (by JQ1) and HDAC proteins (by SAHA or Vorinostat) in lacrimal gland adenoid cystic carcinoma (LGACC) and to elucidate the underlying mechanism of action. Methods: LGACC tissue samples were collected from patients and tissue digestion was done by a collagenase digestion method. Molecular characterization of LGACC primary cell cultures was done by immunocytochemistry. LGACC cells were treated with JQ1 and SAHA individually or in combination. The synergistic effect of JQ1 and SAHA combination was determined by the CellTiter-Glo luminescent cell viability assay using Combenefit software. Migration capability of LGACC cells after drug treatment was examined by a wound healing assays. The expression levels of genes associated with LGACC malignancy were investigated after treatment with JQ1 and SAHA for 48hrs by real time PCR. The expression of c-MYB protein was detected by western blot analysis. Results: LGACC primary cultures were successfully established in-vitro. Immunofluorescence of these cells revealed positive antigenicity for epithelial, proliferative and tumor markers such as FGFR1, MYB and NICD; slightly positive antigenicity for CK-5 and CK-7; and negative antigenicity for E-cadherin.. We found that synergism between JQ1 and SAHA was highly significant as shown by synergy matrix plot and 3D surface plot. Interestingly, migration capacity of LGACC cells was significantly (p<0.002) inhibited by the combination treatment compared to either drug alone.. Our gene expression analysis revealed that the expression levels of Notch1 (p<0.04), Hes1 (p<0.05), Hey1 (p<0.008), Maml1 (p<0.013) and CDKNB (p<0.04) were downregulated significantly in LGACC cells treated with the combination compared to single drug treatment. Furthermore, western blot analysis revealed that combination treatment significantly (p<0.05) downregulated MYB expression (0.9 fold). Currently, investigations are being conducted to evaluate the synergistic anticancer effect of JQ1 and SAHA in LGACC tumor xenografts in nude mice. Conclusion: In conclusion, we successfully generated LGACC cell lines in-vitro. Our results demonstrated that the combination of bromodomain inhibitor JQ1 and histone deacetylase inhibitor SAHA behaves in a synergistic manner and has superior anticancer activity against LGACC cancer cells via targeting the Notch pathway. Taken together, these findings indicate that LGACC is strikingly sensitive to epigenetic inhibitors, which may be quickly implemented for patient use. Citation Format: Ravi Doddapaneni, Wensi Tao, David Tse, Daniel Pelaez. Synergistic anticancer effects of epigenetic drugs JQ1 and SAHA in adenoid cystic carcinoma of the lacrimal gland [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3832

Ribitol alters multiple metabolic pathways of central carbon metabolism with enhanced glycolysis: A metabolomics and transcriptomics profiling of breast cancer

Breast cancer is heterogenous in development and cell population with prognoses being highly dependent on numerous factors from driving mutations, biomarker expression and variation in extracellular environment, all affecting response to therapies. Recently, much attention has been given to the role of metabolic alteration in cancers, expanding from the Warburg effect to highlight unique patterns in different cancer cell populations for improving diagnostic and therapeutic approaches. We recently reported on modulation of mannosylation of α-dystroglycan with the metabolite ribitol in breast cancer lines. Here we investigate the effects of pentose sugars ribitol, ribose, and xylitol media supplementation in breast cancer cells by metabolomics and differential gene expression profiling. This combined approach revealed distinctive patterns of alterations in metabolic pathways by ribitol, contrasted with the closely related pentose ribose and pentitol xylitol. Significantly, ribitol supplementation enhances utilization of glucose by glycolysis, whereas ribose improves oxidative phosphorylation and fatty acid synthesis. Ribitol supplementation also increased levels of reduced glutathione (associated with a decrease in oxidative phosphorylation, gluconeogenesis), where ribose supplementation elevated levels of oxidized glutathione (GSSG) indicating an increase in oxidative stress. Treatment with ribitol also enhanced nucleotide biosynthesis. The apparent TCA cycle dysregulation, with distinctive pattern in response to the individual pentitol and pentose, such as ribitol increasing succinate and fumarate while decreasing citrate, demonstrate the adaptive capability of cancer cells to nutritional environment. This metabolic reprogramming presents new avenues for developing targeted therapies to cancers with metabolites, especially in combination with other drug treatments

Mass spectrometry database of lacrimal gland adenoid cystic carcinoma and normal lacrimal gland tissue identifies extracellular matrix remodeling in these tumors

Adenoid cystic carcinoma of the lacrimal gland (LGACC) is a slow-growing but aggressive orbital malignancy. Due to the rarity of LGACC, it is poorly understood, which makes diagnosing, treating, and monitoring disease progression difficult. The aim is to understand the molecular drivers of LGACC further to identify potential targets for treating this cancer. Mass spectrometry was performed on LGACC and normal lacrimal gland samples to examine the differentially expressed proteins to understand this cancer's proteomic characteristics. Downstream gene ontology and pathway analysis revealed the extracellular matrix is the most upregulated process in LGACC. This data serves as a resource for further understanding LGACC and identifying potential treatment targets. This dataset is publicly available

Constitutive Optimized Production of Streptokinase in Saccharomyces cerevisiae Utilizing Glyceraldehyde 3-Phosphate Dehydrogenase Promoter of Pichia pastoris

A novel expression vector constructed from genes of Pichia pastoris was applied for heterologous gene expression in Saccharomyces cerevisiae. Recombinant streptokinase (SK) was synthesized by cloning the region encoding mature SK under the control of glyceraldehyde 3-phosphate dehydrogenase (GAP) promoter of Pichia pastoris in Saccharomyces cerevisiae. SK was intracellularly expressed constitutively, as evidenced by lyticase-nitroanilide and caseinolytic assays. The functional activity was confirmed by plasminogen activation assay and in vitro clot lysis assay. Stability and absence of toxicity to the host with the recombinant expression vector as evidenced by southern analysis and growth profile indicate the application of this expression system for large-scale production of SK. Two-stage statistical approach, Plackett-Burman (PB) design and response surface methodology (RSM) was used for SK production medium optimization. In the first stage, carbon and organic nitrogen sources were qualitatively screened by PB design and in the second stage there was quantitative optimization of four process variables, yeast extract, dextrose, pH, and temperature, by RSM. PB design resulted in dextrose and peptone as best carbon and nitrogen sources for SK production. RSM method, proved as an efficient technique for optimizing process conditions which resulted in 110% increase in SK production, 2352 IU/mL, than for unoptimized conditions

Fibroblast growth factor receptor 1 (FGFR1) as a therapeutic target in adenoid cystic carcinoma of the lacrimal gland

Identification of molecular targets is the first step in developing efficacious therapeutic strategies for tumors. A tumors' biological response to perturbagens yields important information on the molecular determinants for tumor growth. The aim of this study was to characterize the response of adenoid cystic carcinoma of the lacrimal gland (LGACC) to intra-arterial cytoreductive chemotherapy (IACC) in order to identify novel targets to enhance therapy. We performed high-throughput proteomic analysis on paired samples from pre-IACC diagnostic biopsies and post-IACC excised tumor samples from 6 LGACC patients. This proteomic analysis provides a comprehensive landscape of the cellular compartments contained within the excised tumors. Interestingly, we found a strong upregulation across the fibroblast growth factor (FGF) signaling pathway, with FGF receptor 1 (FGFR1) exhibiting a consistent and significant upregulation in all post-IACC samples. We thus evaluated the therapeutic efficacy of a novel FGFR1 selective inhibitor, AZD4547, in combination with cisplatin on LGACC cells in-vitro. The combination index (CI) value (<0.895) demonstrated synergistic effect of AZD4547 and cisplatin in inhibiting cell growth and viability (p<0.02), with a differential response seen in post-IACC cultures when compared to pre-IACC cultures. The combination approach showed synergy of the drugs in the migration assay. Western blot analysis indicated a significant upregulation of cleaved caspase-3 and downregulation the expression of FGFR1 (p<0.05) with the combination treatment as compared to either agent independently. Our findings demonstrate that FGFR1 inhibition potentiates the cytoreductive effects of cisplatin and suggest a potential therapeutic benefit of using AZD4547 in the management of LGACC

Therapeutic targeting of oncogenic transcription factors by natural products in eye cancer

[Display omitted] Carcinogenesis has a multifactorial etiology, and the underlying molecular pathogenesis is still not entirely understood, especially for eye cancers. Primary malignant intraocular neoplasms are relatively rare, but delayed detection and inappropriate management contribute to poor outcomes. Conventional treatment, such as orbital exenteration, chemotherapy, or radiotherapy, alone results in high mortality for many of these malignancies. Recent sequential multimodal therapy with a combination of high-dose chemotherapy, followed by appropriate surgery, radiotherapy, and additional adjuvant chemotherapy has helped dramatically improve management. Transcription factors are proteins that regulate gene expression by modulating the synthesis of mRNA. Since transcription is a dominant control point in the production of many proteins, transcription factors represent key regulators for numerous cellular functions, including proliferation, differentiation, and apoptosis, making them compelling targets for drug development. Natural compounds have been studied for their potential to be potent yet safe chemotherapeutic drugs. Since the ancient times, plant-derived bioactive molecules have been used to treat dreadful diseases like cancer, and several refined pharmaceutics have been developed from these compounds. Understanding targeting mechanisms of oncogenic transcription factors by natural products can add to our oncologic management toolbox. This review summarizes the current findings of natural products in targeting specific oncogenic transcription factors in various types of eye cancer