Screening of GPCR drugs for repurposing in breast cancer

Drug repurposing can overcome both substantial costs and the lengthy process of new drug discovery and development in cancer treatment. Some Food and Drug Administration (FDA)-approved drugs have been found to have the potential to be repurposed as anti-cancer drugs. However, the progress is slow due to only a handful of strategies employed to identify drugs with repurposing potential. In this study, we evaluated GPCR-targeting drugs by high throughput screening (HTS) for their repurposing potential in triple-negative breast cancer (TNBC) and drug-resistant human epidermal growth factor receptor-2-positive (HER2+) breast cancer (BC), due to the dire need to discover novel targets and drugs in these subtypes. We assessed the efficacy and potency of drugs/compounds targeting different GPCRs for the growth rate inhibition in the following models: two TNBC cell lines (MDA-MB-231 and MDA-MB-468) and two HER2+ BC cell lines (BT474 and SKBR3), sensitive or resistant to lapatinib + trastuzumab, an effective combination of HER2-targeting therapies. We identified six drugs/compounds as potential hits, of which 4 were FDA-approved drugs. We focused on β-adrenergic receptor-targeting nebivolol as a candidate, primarily because of the potential role of these receptors in BC and its excellent long-term safety profile. The effects of nebivolol were validated in an independent assay in all the cell line models. The effects of nebivolol were independent of its activation of β3 receptors and nitric oxide production. Nebivolol reduced invasion and migration potentials which also suggests its inhibitory role in metastasis. Analysis of the Surveillance, Epidemiology and End Results (SEER)-Medicare dataset found numerically but not statistically significant reduced risk of all-cause mortality in the nebivolol group. In-depth future analyses, including detailed in vivo studies and real-world data analysis with more patients, are needed to further investigate the potential of nebivolol as a repurposed therapy for BC

Design, Optimization, and Validation of Multiplex Immunofluorescence Assay for Detecting Biomarker Expression on Circulating Tumor Cells in Breast Cancer

The purpose of this study was to design, optimize and validate a multiplex immunofluorescence (IF) assay to identify CTCsanddetect apanel of biomarkers onCTCs. This project was completed with contributions from Lacey E. Dobrolecki and Michael T. Lewis from Baylor College of Medicine.Pharmacy Practice and Translational Research, Department ofHonors Colleg

Developing an Immunofluorescence Assay for Detecting Rb and phospho-Rb on Circulating Tumor Cells in Breast Cancer

Endocrine therapies (ET) such as tamoxifen, fulvestrant, and aromatase inhibitors (AIs) are the standard-of-care first-line treatment in majority of estrogen receptor (ER)-positive breast cancers (BC). Recent clinical studies using cyclin-dependent kinase 4 and 6 inhibitors (CDK4/6i) plus AIs or fulvestrant have shown significant improvement in survival outcomes in patients with ER+ metastatic BC compared to standalone ETs. CDK4/6i exert their action by inhibiting the phosphorylation of retinoblastoma (Rb) protein and consequently inducing cell cycle arrest. However, not all patients respond to this combination therapy and those, who initially respond, eventually develop resistance. Emerging studies suggest that the intrinsic resistance to CDK4/6i could be due to the loss of Rb or its mutations. Therefore, CDK4/6i resistance can be evaluated by measuring expression of total and phospho-Rb and Rb mutations. However, repeated biopsies to evaluate biomarkers in tumors is not feasible in patients. In this research, we aimed to develop an immunofluorescence assay to evaluate the expression of Rb and phospho-Rb using circulating tumor cells (CTCs) which can help predict response and resistance to CDK4/6i. CTCs serve as representative of the tumor bulk in patients and animal models and allow for less-invasive, frequent blood collection and real-time monitoring of treatment response. MCF7 cells treated with vehicle or abemaciclib (500 nM) for 48 hours were spiked into blood from non-tumor bearing mice. The MCF7 cells-spiked blood was processed using ScreenCell® device and the cells were transferred to slides and stained with DAPI, pan-cytokeratin, CD45, estrogen receptor, Rb and phospho-Rb. Tumor cells were defined as pan-cytokeratin-positive, CD45-negative, and nuclear stain-positive cells. Various antibody combinations were examined to increase the sensitivity of the IF assay for individual markers as well as the multiplexed assay. We also developed quantitative assessment approach to detect per-cell intensity of various markers. Treatment with abemaciclib reduced the intensity of phospho/Total Rb from 2.6 +/- 0.6 to 0.8 +/- 0.2 units (p < 0.05, t-test, n=8-9) in the vehicle-treated samples. There was no significant difference in the Rb intensity between the treatment groups. Ongoing studies focus on validation of the assay using preclinical models and clinical samples.Pharmacy Practice and Translational Research, Department ofHonors Colleg

Spectroscopic and Molecular Methods to Differentiate Gender in Immature Date Palm (Phoenix dactylifera L.)

Phoenix dactylifera (date palm) is a well-known nutritious and economically important fruit tree found in arid regions of the Middle East and North Africa. Being diploid, it has extremely high divergence in gender, where sex differentiation in immature date palms (Phoenix dactylifera L.) has remained an enigma in recent years. Herein, new robust infrared (near-infrared reflectance spectroscopy (NIRS) and Fourier transform infrared attenuated total reflectance (FTIR/ATR)) and nuclear magnetic resonance (NMR) spectroscopy methods coupled with extensive chemometric analysis were used to identify the sex differentiation in immature date palm leaves. NIRS/FTIR reflectance and 1H-NMR profiling suggested that the signals of monosaccharides (glucose and fructose) and/or disaccharides (maltose and sucrose) play key roles in sex differentiation. The three kinds of spectroscopic data were clearly differentiated among known and unknown male and female leaves via principal component and partial least square discriminant analyses. Furthermore, sex-specific genes and molecular markers obtained from the lower halves of LG12 chromosomes showed enhanced transcript accumulation of mPdIRDP52, mPdIRDP50, and PDK101 in females compared with in males. The phylogeny showed that the mPdIRD033, mPdIRD031, and mPdCIR032 markers formed distinctive clades with more than 70% similarity in gender differentiation. The three robust analyses provide an alternative tool to differentiate sex in date palm trees, which offers a solution to the long-standing challenge of dioecism and could enhance in situ tree propagation programs

Complete chloroplast genomes of medicinally important Teucrium species and comparative analyses with related species from Lamiaceae

Teucrium is one of the most economically and ecologically important genera in the Lamiaceae family; however, it is currently the least well understood at the plastome level. In the current study, we sequenced the complete chloroplast (cp) genomes of T. stocksianum subsp. stenophyllum R.A.King (TSS), T. stocksianum subsp. stocksianum Boiss. (TS) and T. mascatense Boiss. (TM) through next-generation sequencing and compared them with the cp genomes of related species in Lamiaceae (Ajuga reptans L., Caryopteris mongholica Bunge, Lamium album L., Lamium galeobdolon (L.) Crantz, and Stachys byzantina K.Koch). The results revealed that the TSS, TS and TM cp genomes have sizes of 150,087, 150,076 and 150,499 bp, respectively. Similarly, the large single-copy (LSC) regions of TSS, TS and TM had sizes of 81,707, 81,682 and 82,075 bp, respectively. The gene contents and orders of these genomes were similar to those of other angiosperm species. However, various differences were observed at the inverted repeat (IR) junctions, and the extent of the IR expansion into ψrps19 was 58 bp, 23 bp and 61 bp in TSS, TS and TM, respectively. Similarly, in all genomes, the pbsA gene was present in the LSC at varying distances from the JLA (IRa-LSC) junction. Furthermore, 89, 72, and 92 repeats were identified in the TSS, TM and TS cp genomes, respectively. The highest number of simple sequence repeats was found in TSS (128), followed by TS (127) and TM (121). Pairwise alignments of the TSS cp genome with related cp genomes showed a high degree of synteny. However, relatively lower sequence identity was observed when various coding regions were compared to those of related cp genomes. The average pairwise divergence among the complete cp genomes showed that TSS was more divergent from TM (0.018) than from TS (0.006). The current study provides valuable genomic insight into the genus Teucrium and its subspecies that may be applied to a more comprehensive study

First complete chloroplast genomics and comparative phylogenetic analysis of Commiphora gileadensis and C. foliacea: Myrrh producing trees.

Commiphora gileadensis and C. foliacea (family Burseraceae) are pantropical in nature and known for producing fragrant resin (myrrh). Both the tree species are economically and medicinally important however, least genomic understanding is available for this genus. Herein, we report the complete chloroplast genome sequences of C. gileadensis and C. foliacea and comparative analysis with related species (C. wightii and Boswellia sacra). A modified chloroplast DNA extraction method was adopted, followed with next generation sequencing, detailed bioinformatics and PCR analyses. The results revealed that the cp genome sizes of C. gileadensis and C. foliacea, are 160,268 and 160,249 bp, respectively, with classic quadripartite structures that comprises of inverted repeat's pair. Overall, the organization of these cp genomes, GC contents, gene order, and codon usage were comparable to other cp genomes in angiosperm. Approximately, 198 and 175 perfect simple sequence repeats were detected in C. gileadensis and C. foliacea genomes, respectively. Similarly, 30 and 25 palindromic, 15 and 25 forward, and 20 and 25 tandem repeats were determined in both the cp genomes, respectively. Comparison of these complete cp genomes with C. wightii and B. sacra revealed significant sequence resemblance and comparatively highest deviation in intergenic spacers. The phylo-genomic comparison showed that C. gileadensis and C. foliacea form a single clade with previously reported C. wightii and B. sacra from family Burseraceae. Current study reports for the first time the cp genomics of species from Commiphora, which could be helpful in understanding genetic diversity and phylogeny of this myrrh producing species

A CTC-Cluster-Specific Signature Derived from OMICS Analysis of Patient-Derived Xenograft Tumors Predicts Outcomes in Basal-Like Breast Cancer

Circulating tumor cell clusters (CTCcl) have a higher metastatic potential compared to single CTCs and predict long-term outcomes in breast cancer (BC) patients. Because of the rarity of CTCcls, molecular characterization of primary tumors that give rise to CTCcl hold significant promise for better diagnosis and target discovery to combat metastatic BC. In our study, we utilized the reverse-phase protein array (RPPA) and transcriptomic (RNA-Seq) data of 10 triple-negative BC patient-derived xenograft (TNBC PDX) transplantable models with CTCs and evaluated expression of upregulated candidate protein Bcl2 (B-cell lymphoma 2) by immunohistochemistry (IHC). The sample-set consisted of six CTCcl-negative (CTCcl-) and four CTCcl-positive (CTCcl+) models. We analyzed the RPPA and transcriptomic profiles of CTCcl- and CTCcl+ TNBC PDX models. In addition, we derived a CTCcl-specific gene signature for testing if it predicted outcomes using a publicly available dataset from 360 patients with basal-like BC. The RPPA analysis of CTCcl+ vs. CTCcl- TNBC PDX tumors revealed elevated expression of Bcl2 (false discovery rate (FDR) 34); whereas, only one of six CTCcl- TNBC PDX primary tumors met this criterion. Evaluation of epithelial-mesenchymal transition (EMT)-specific signature did not show significant differences between CTCcl+ and CTCcl- tumors. However, a gene signature associated with the presence of CTCcls in TNBC PDX models was associated with worse relapse-free survival in the publicly available dataset from 360 patients with basal-like BC. In summary, we identified the multigene signature of primary PDX tumors associated with the presence of CTCcls. Evaluation of additional TNBC PDX models and patients can further illuminate cellular and molecular pathways facilitating CTCcl formation

DataSheet2_Screening of GPCR drugs for repurposing in breast cancer.PDF

Drug repurposing can overcome both substantial costs and the lengthy process of new drug discovery and development in cancer treatment. Some Food and Drug Administration (FDA)-approved drugs have been found to have the potential to be repurposed as anti-cancer drugs. However, the progress is slow due to only a handful of strategies employed to identify drugs with repurposing potential. In this study, we evaluated GPCR-targeting drugs by high throughput screening (HTS) for their repurposing potential in triple-negative breast cancer (TNBC) and drug-resistant human epidermal growth factor receptor-2-positive (HER2+) breast cancer (BC), due to the dire need to discover novel targets and drugs in these subtypes. We assessed the efficacy and potency of drugs/compounds targeting different GPCRs for the growth rate inhibition in the following models: two TNBC cell lines (MDA-MB-231 and MDA-MB-468) and two HER2+ BC cell lines (BT474 and SKBR3), sensitive or resistant to lapatinib + trastuzumab, an effective combination of HER2-targeting therapies. We identified six drugs/compounds as potential hits, of which 4 were FDA-approved drugs. We focused on β-adrenergic receptor-targeting nebivolol as a candidate, primarily because of the potential role of these receptors in BC and its excellent long-term safety profile. The effects of nebivolol were validated in an independent assay in all the cell line models. The effects of nebivolol were independent of its activation of β3 receptors and nitric oxide production. Nebivolol reduced invasion and migration potentials which also suggests its inhibitory role in metastasis. Analysis of the Surveillance, Epidemiology and End Results (SEER)-Medicare dataset found numerically but not statistically significant reduced risk of all-cause mortality in the nebivolol group. In-depth future analyses, including detailed in vivo studies and real-world data analysis with more patients, are needed to further investigate the potential of nebivolol as a repurposed therapy for BC.</p

Table1_Screening of GPCR drugs for repurposing in breast cancer.XLSX

Drug repurposing can overcome both substantial costs and the lengthy process of new drug discovery and development in cancer treatment. Some Food and Drug Administration (FDA)-approved drugs have been found to have the potential to be repurposed as anti-cancer drugs. However, the progress is slow due to only a handful of strategies employed to identify drugs with repurposing potential. In this study, we evaluated GPCR-targeting drugs by high throughput screening (HTS) for their repurposing potential in triple-negative breast cancer (TNBC) and drug-resistant human epidermal growth factor receptor-2-positive (HER2+) breast cancer (BC), due to the dire need to discover novel targets and drugs in these subtypes. We assessed the efficacy and potency of drugs/compounds targeting different GPCRs for the growth rate inhibition in the following models: two TNBC cell lines (MDA-MB-231 and MDA-MB-468) and two HER2+ BC cell lines (BT474 and SKBR3), sensitive or resistant to lapatinib + trastuzumab, an effective combination of HER2-targeting therapies. We identified six drugs/compounds as potential hits, of which 4 were FDA-approved drugs. We focused on β-adrenergic receptor-targeting nebivolol as a candidate, primarily because of the potential role of these receptors in BC and its excellent long-term safety profile. The effects of nebivolol were validated in an independent assay in all the cell line models. The effects of nebivolol were independent of its activation of β3 receptors and nitric oxide production. Nebivolol reduced invasion and migration potentials which also suggests its inhibitory role in metastasis. Analysis of the Surveillance, Epidemiology and End Results (SEER)-Medicare dataset found numerically but not statistically significant reduced risk of all-cause mortality in the nebivolol group. In-depth future analyses, including detailed in vivo studies and real-world data analysis with more patients, are needed to further investigate the potential of nebivolol as a repurposed therapy for BC.</p

DataSheet3_Screening of GPCR drugs for repurposing in breast cancer.PDF

Drug repurposing can overcome both substantial costs and the lengthy process of new drug discovery and development in cancer treatment. Some Food and Drug Administration (FDA)-approved drugs have been found to have the potential to be repurposed as anti-cancer drugs. However, the progress is slow due to only a handful of strategies employed to identify drugs with repurposing potential. In this study, we evaluated GPCR-targeting drugs by high throughput screening (HTS) for their repurposing potential in triple-negative breast cancer (TNBC) and drug-resistant human epidermal growth factor receptor-2-positive (HER2+) breast cancer (BC), due to the dire need to discover novel targets and drugs in these subtypes. We assessed the efficacy and potency of drugs/compounds targeting different GPCRs for the growth rate inhibition in the following models: two TNBC cell lines (MDA-MB-231 and MDA-MB-468) and two HER2+ BC cell lines (BT474 and SKBR3), sensitive or resistant to lapatinib + trastuzumab, an effective combination of HER2-targeting therapies. We identified six drugs/compounds as potential hits, of which 4 were FDA-approved drugs. We focused on β-adrenergic receptor-targeting nebivolol as a candidate, primarily because of the potential role of these receptors in BC and its excellent long-term safety profile. The effects of nebivolol were validated in an independent assay in all the cell line models. The effects of nebivolol were independent of its activation of β3 receptors and nitric oxide production. Nebivolol reduced invasion and migration potentials which also suggests its inhibitory role in metastasis. Analysis of the Surveillance, Epidemiology and End Results (SEER)-Medicare dataset found numerically but not statistically significant reduced risk of all-cause mortality in the nebivolol group. In-depth future analyses, including detailed in vivo studies and real-world data analysis with more patients, are needed to further investigate the potential of nebivolol as a repurposed therapy for BC.</p