Iqbal Mahmud

βArrestin2 Mediates Renal Cell Carcinoma Tumor Growth

Renal Cell Carcinoma (RCC) is one of the most lethal urological cancers worldwide. The disease does not present early clinical symptoms and is commonly diagnosed at an advanced stage. Limited molecular drivers have been identified for RCC, resulting in the lack of effective treatment for patients with progressive disease. Ubiquitous βArrestin2 (βArr2) is well established for its function in the desensitization and trafficking of G protein-coupled receptors. More recently, βArr2 has been implicated in the regulation of fundamental cellular functions, including proliferation and invasion. We used bioinformatic and genetic approaches to determine role of βArr2 in RCC tumor growth. Analysis of published human datasets shows that ARRB2 (gene encoding βArr2) expression is increased in RCC tumor compared to normal tissue and that high levels of ARRB2 correlate with worse patient survival. Experimentally, we show that knockout of ARRB2 decreases rate of RCC cell proliferation and migration in vitro and xenograft tumor growth in animals. Mechanistically, βArr2 regulates c-Src activity, Cyclin A expression and cell cycle progression that are involved in tumor growth. These results show that βArr2 is a critical regulator of RCC tumor growth and suggest its utility as a potential marker and drug target to treat advanced disease.

Abstract LB-312: GRK5 activity mediates in vivo and in vitro prostate cancer progression and chemoresistance

Despite decades of research targeting the activity of the androgen receptor (AR), patients diagnosed with locally advanced and metastatic prostate cancer face an incurable disease. The Daaka lab investigates constituents of G-protein-coupled receptor signaling cascades for their ability to provide mitogenic signaling, which promotes PCa progression. We previously identified G protein-coupled receptor 5 (GRK5) for its ability to regulate PCa progression, independent of AR activity. GRK5 partially partitions to the nucleus, wherein it has been shown to regulate the transcriptome in non-PCa models. To globally elucidate the mechanistic impact of GRK5 on PCa progression, we are investigating the impact of GRK5 on the PCa transcriptome. We hypothesize that GRK59s regulation of the PCa transcriptome promotes tumor progression. To assay the effect of GRK5 on the PCa transcriptome, RNA sequencing was performed in two cell lines: PC3 Control (PC3 shGFP) and PC3 GRK5 Knockdown (PC3 shGRK5).xa0 Ontologic analysis identified the epithelial–mesenchymal transition (EMT) as being affected when GRK5 is depleted. The epithelial–mesenchymal transition (EMT) is highly associated with promoting PCa progression and chemoresistance, thus we selected this pathway for further investigation. Confirmatory western blot analysis and quantitative PCR (qPCR) validated that depleting GRK5 suppresses the expression of the mesenchymal markers Vimentin and N-Cadherin. Similarly, overexpression of GRK5 increases the expression of these mesenchymal markers. Stable overexpression of GRK5 promotes cells to develop a spindle-like morphology, indicative of a mesenchymal state. GRK5 mediates this mesenchymal transition through increasing the expression of the EMT transcription factor, Twist1. Further analysis identifies that GRK5 activity promotes in vitro invasion. Cell lines overexpressing GRK5 demonstrate an increase in resistance to docetaxel, the mainstay chemotherapy for advanced PCa. Overexpression of GRK5 promotes lymphatic intravasation. Overexpression of mutated forms of GRK5 that are relegated to the nucleus are able recapitulated all aforesaid changes, arguing that the nuclear activity of GRK5 mediates this effect. Collectively, this data presents a novel mechanism promoting PCa progression, independent of AR activity. Citation Format: Joseph B. Black, Hamsa Thayele Purayil, Iqbal Mahmud, Daiqing Liao, Yehia Daaka. GRK5 activity mediates in vivo and in vitro prostate cancer progression and chemoresistance [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 LB-312.

Abstract B004: A combination of two chemically distinct inhibitors of class I HDACs is highly effective to suppress AR signaling and in vivo growth of prostate cancer xenograft

Class I HDACs 1-3 are critical for the AR-mediated transcriptional program. Thus, targeting class I HDACs is a promising strategy for treating prostate cancer. However, several FDA-approved HDAC inhibitors (HDACi) broadly inhibiting different HDACs have been shown to be ineffective and overtly toxic for treating castration-resistant prostate cancer in clinical trials. These observations suggest that HDACi selective for class I HDACs may be more effective but less toxic for treating prostate cancer than pan HDACi. Notably, we and others have shown that HDACi of different chemical classes individually exhibit nonoverlapping but limited effects on the acetylome in cancer cells. We hypothesize that a combination treatment strategy with class I HDAC-selective HDACi of different chemical classes is more effective to block AR signaling and prostate tumor growth than such HDACi individually. We have recently discovered the novel benzoylhydrazide chemo-type of class I HDAC-selective HDACi with a unique pharmacologic profile. Medicinal chemistry effort led to an optimized analog, SR-4370, with 6 nM potency to HDAC3. In support of our hypothesis, SR-4370 in combination with the clinical class I HDACi entinostat markedly suppressed AR signaling, prostate cancer cell proliferation in vitro, and tumor growth in vivo, and the tumor-suppressive effects by the HDACi combination were more pronounced than by SR-4370 and entinostat individually. Gene expression profiling experiments revealed that the SR-4370/entinostat combination profoundly downregulated AR, AR-V7, and AR target genes, while activating immune and cell death signaling pathways. In a C4-2 xenograft model using athymic nude mice, the SR-4370/entinostat combination was highly effective in suppressing tumor growth. Importantly, these HDACi alone or in combination did not cause observable adverse effects judged by body weight and blood chemistry tests of treated mice. Overall, our data suggest that the SR-4370/entinostat combination may be a translatable treatment for castration-resistant prostate cancer. Citation Format: Guimei Tian, Iqbal Mahmud, Ryan Stowe, Yushan Zhang, Hamsa Thayek Purayil, William Roush, Yehia Daaka, Daiqing Liao. A combination of two chemically distinct inhibitors of class I HDACs is highly effective to suppress AR signaling and in vivo growth of prostate cancer xenograft [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B004.

Abstract 1473: Role of acetyltransferases CBP and p300 in de novo fatty acid synthesis in colorectal cancer

CBP/p300 are two paralogous lysine acetyltransferases that acetylate protein substrates including histones, and serve as transcriptional co-activators for numerous signaling pathways involved in tumorigenesis and cancer progression. Recently, evidence has emerged that p300 regulates the transcription of genes involved in lipogenesis in cancers. Dysregulated lipogenesis and increased de novo production of fatty acids (FA) represent a major metabolic shift in cancer. De novo FA synthesis in cancer is suggested to be a source of lipids for membrane biogenesis, a reservoir of energy, and a source of pro-survival signaling molecules. Therefore, de novo FA synthesis has been linked to survival and increased proliferation of cancer cells, representing a potential area for therapeutic intervention. While emerging evidence indicates CBP/p300 may play a key role in regulating lipogenesis, little is known about the mechanism of how these proteins control lipid production in different cancers. Colorectal cancer (CRC) is a commonly diagnosed human cancer and is a major cause of cancer-related mortality. Key regulators for de novo FA synthesis, such as FASN, have been reported to be elevated in colorectal cancer. We analyzed publically available data sets and report that in colon adenocarcinoma, the mRNA levels of CBP/p300 positively correlate with the expression of genes involved in lipogenesis, such as FASN, SREBP1 and SREBP2. Furthermore, our analysis shows that EP300 expression is negatively correlated with patient survival. Strikingly, our preliminary data suggests that HDAC inhibitors may regulate p300 activity and could serve as a therapeutic approach for suppressing lipogenesis. We are currently utilizing small molecule inhibitors, genetic manipulation of the CREBBP/EP300 genes, and biochemical experiments to investigate the role of CBP/p300 in de novo FA synthesis in colorectal cancer. We hypothesize that inhibition of CBP/p300 could be an attractive therapeutic option for inhibiting tumor growth through downregulating de novo FA synthesis in colorectal cancer. (Supported by grants from James and Esther King Biomedical Research Program and Bankhead-Coly Cancer Research Program, Florida Department of Health.) Citation Format: Aaron Waddell, Iqbal Mahmud, Daiqing Liao. Role of acetyltransferases CBP and p300 in de novo fatty acid synthesis in colorectal 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 1473.

Abstract LB-266: DAXX promotes de novo lipogenesis in triple-negative breast cancer

Triple-negative breast cancers (TNBCs), defined by the absence of ER and PR expression and the lack of HER2 amplification, have no targeted treatment options, are highly aggressive, and exhibit poor prognosis. Identification of drivers and associated signaling pathways, particularly for TNBCs, is needed to assist the development of targeted therapies, to prolong disease-free survival, and to improve clinical outcome for cancer patients. Using high-throughput microarray-based global transcriptome profiling, we found that transcriptional regulator DAXX depletion strikingly impaired while DAXX overexpression enhanced the expression of genes in the de novo lipogenesis pathways in TNBCs cell line. Consistently, unbiased global metabolomics and lipidomics profiling by Ultra-High performance Liquid Chromatography coupled High-Resolution Mass Spectrometry (UHPLC-HRMS) also revealed that DAXX promotes de novo lipogenesis in both TNBC cell line as well as in corresponding xenograft tumors. In analyzing large number of genomic datasets including The Cancer Genome Atlas (TCGA), MolEcular Taxonomy of BReast cancer International Consortium (METABRIC), and The Cancer Cell Line Encyclopedia (CCLE) dataset, we found that DAXX expression is significantly higher in TNBC tumor samples and cell lines, and also DAXX gene expression positively correlates with that of key lipogenesis enzymes. The role of DAXX in cancer metabolism is previously unknown. Our integrated OMICs data analysis thus indicates that DAXX promotes de novo lipogenesis in TNBC. The mechanism through which DAXX regulates lipid metabolism is being investigated in our laboratory. Overall, our study identifies a potentially important pathway driving TNBC progression and suggests potential therapeutic targets for treating this aggressive form of breast cancer that still lacks curative treatment options. Citation Format: Iqbal Mahmud, Timothy J. Garrett, Daiqing Liao. DAXX promotes de novo lipogenesis in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-266. doi:10.1158/1538-7445.AM2017-LB-266

Abstract 1982: β-arrestin 2 mediates tumor growth and metastasis in renal cell carcinoma cells

Renal Cell Carcinoma (RCC) is one of the most lethal urological cancers worldwide, with incidence and mortality rates increasing in the past two decades. The disease does not present early clinical symptoms and is commonly diagnosed at the metastatic stage, leaving the 5-year survival rate at ≈ 10-20%. For a wide array of neoplasms, many key molecular determinants involved in mediating the process of tumor cell growth, invasion, and colonization at a secondary site (i.e. metastasis) have been reported. However, few molecular predictors have been identified for RCC, rendering the mechanism(s) underlying RCC metastases poorly understood. This results in the lack of effective treatment for patients with advanced RCC. G protein-coupled receptors (GPCRs) and their effectors, such as the arrestin proteins, have been implicated in tumor growth, metastasis, and angiogenesis. Arrestin proteins are well known for their function in the desensitization and trafficking of GPCRs, but have also been implicated in unique signaling pathways to regulate fundamental cellular functions, including cell cycle progression, cell migration, and survival. Furthermore, arrestin involvement has been identified in a number of breast, colorectal, lung, and hematological malignancies. However, the role of arrestin proteins in RCC is yet to be determined. Our preliminary data show that β-arrestin 2 (βarr2) protein levels correlate with growth and metastatic potential in several RCC cell lines, including ACHN and SN12C. We hypothesize that βarr2 regulates RCC tumor progression, specifically through involvement in proliferation, invasion, and metastatic processes. To test our hypothesis, we used genetic-based loss of function approaches such as interfering RNA and CRISPR/Cas9. βarr2 knockdown results indicated a role in RCC malignancy as it significantly reduces the migration and invasion of RCC cell lines in vitro. βarr2 knockout (KO) impaired 3D spheroid formation of these cells and induced morphological changes compared to control cells as observed under confocal microscopy imaging. Moreover, epithelial marker E-cadherin expression levels were elevated while mesenchymal markers twist1, twist2 and vimentin levels were decreased in the βarr2 KO cells compared to control, indicating a mesenchymal to epithelial transition. In vivo data support our hypothesis that βarr2 plays a critical role in tumor growth and metastasis. Our data suggests a role for βarr2 in RCC malignancy and present a possible target in development of therapies for patients with advanced RCC. Citation Format: Jude Masannat, Yushan Zhang, Hamsa Purayil, Iqbal Mahmud, Yehia Daaka. β-arrestin 2 mediates tumor growth and metastasis in renal cell carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1982. doi:10.1158/1538-7445.AM2017-1982

Comparison of global metabolite extraction strategies for soybeans using UHPLC-HRMS

AbstractMetabolism, downstream effectors of genomics, transcriptomics, and proteomics, can determine the potential of phenotype of an organism including plants. Profiling the global scenario of metabolism requires optimization of different solvent extraction methods. Here, we report an approach comparing three different metabolite extraction strategies, including ammonium acetate/methanol (AAM), water/methanol (WM), and sodium phosphate/methanol (PM) in soybean plant using ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS). Interestingly, both AAM and WM methods were found to cover a wider range of metabolites and provide better detection of molecular features than the PM method. Various clustering analyses based on multivariate statistical tools revealed that both AAM and WM methods showed tight and overlapping extraction strategy compared with the PM method. Using MatLab-based Mahalanobis distance (DM) calculation, statistically significant score plot separation was observed between AAM and PM, as well as WM and PM. However, no significant separation was observed between AAM and WM, which is expected from the overlap of principal component scores for these two methods. Using differential metabolite expression analysis, we identified that a large number of metabolites were extracted at a significantly higher level using AAM vs. PM. These comparative extraction methods suggest that AAM can effectively be applied for an LC/MS-based plant metabolomics profile study.n Graphical abstractStep-by-step outline of three different metabolite extraction methods and data analysis

Microarray gene expression profiling reveals potential mechanisms of tumor suppression by the class I HDAC-selective benzoylhydrazide inhibitors.

Histone deacetylase (HDAC) inhibitors (HDIs) have therapeutic potentials for treating cancer and other diseases. Modulation of gene expression by HDIs is a major mechanism underlying their therapeutic effects. A novel class of HDIs with a previously undescribed benzoylhydrazide scaffold has been discovered through a high throughput screening campaign. Using microarray profiling of gene expression, we have previously demonstrated that treatment of breast cancer cells with a lead benzoylhydrazide HDI UF010 results in cell cycle arrest and apoptosis, likely through activation of tumor suppression pathways with concurrent inhibition of oncogenic pathways. In this brief report, we show methodological and analytical details and discuss additional pathways such as immune signaling that are affected by UF010. Raw and processed data from the microarray were deposited in NCBIs Gene Expression Omnibus (GEO) database under the accession number: GSE56823.