Biology subjects

Lee, A. V.

Publications and source records attributed to Lee, A. V..

28 records · Page 2Linked to original sources

bioRxiv2020

Salicylate, Bile Acids and Extreme Acid Cause Fitness Tradeoffs for Multidrug Pumps in Escherichia coli K-12

The aspirin derivative salicylate selects against bacterial multidrug efflux pumps of Escherichia coli K-12 such as MdtEF-TolC and EmrAB-TolC, and acid stress regulators such as GadE. Salicylate uptake is driven by the transmembrane pH gradient ({Delta}pH) and the proton motive force (PMF) which drives many efflux pumps. We used flow cytometry to measure the fitness tradeoffs of salicylate, bile acids, and extreme low pH for E. coli cultured with pump deletants. The AcrAB-TolC efflux pump conferred a fitness advantage in the presence of bile acids, an efflux substrate. Without bile acids, AcrA incurred a small fitness cost. The fitness advantage with bile acids was eliminated by the PMF uncoupler CCCP. The Gad acid fitness island encodes components of MdtEF-TolC (an acid-adapted efflux pump) as well as acid regulator GadE. The fitness advantage of E. coli cocultured with a Gad deletant ({Delta}slp-gadX) was lost in the presence of salicylate. Salicylate caused an even larger fitness cost for GadE. MdtE incurred negative or neutral fitness under all media conditions, as did EmrA. But when the competition cycle included two hours at pH 2, MdtE conferred a fitness advantage. The MdtE advantage required the presence of bile acids. Thus, the MdtEF-TolC pump is useful to E. coli for transient extreme acid exposure comparable to passage through the acidic stomach. Salicylate selects against some multidrug efflux pumps, whereas bile acids selects for them; and these fitness tradeoffs are amplified by extreme acid. IMPORTANCEControl of drug resistance in gut microbial communities is a compelling problem for human health. Growth of gut bacteria is limited by host-produced acids such as bile acids, and may be modulated by plant-derived acids such as salicylic acid. Membrane-soluble organic acids can control bacterial growth by disrupting membranes, decreasing cell pH, and depleting PMF. Our flow cytometry assay measures the fitness effects of exposure to membrane-soluble organic acids, with growth cycles that may include a period of extreme acid. We find that extreme-acid exposure leads to a fitness advantage for a multidrug pump, MdtEF-TolC, which otherwise incurs a large fitness cost. Thus, organic acids and stomach acid may play important roles in controlling multidrug resistance in the gut microbiome. Therapeutic acids might be developed to limit the prevalence of multidrug resistance pumps in environmental and host-associated communities.

microbiology↗

bioRxiv2020

Computational response modeling reveals context dependent Akt activity in luminal breast cancer cells

Aberrant signaling through insulin (Ins) and insulin-like growth factor I (IGF1) receptors contributes to the risk and advancement of many cancer types by activating cell survival cascades. Mechanistic computational modeling of such pathways provides insights into each components role in the cell response. In previous computational models, the two receptors were treated as indistinguishable, missing the opportunity to delineate their distinct roles in cancer progression. Here, a dual receptor (IGF1R & InsR) computational model elucidated new experimental hypotheses on how differential early responses emerge. Complementary to our previous findings, the model suggested that the regulation of insulin receptor substrate (IRS) is critical in inducing differential MAPK and Akt activation. As predicted, perturbing ribosomal protein S6 kinase (RPS6K) kinase activity led to an increased Akt activation with insulin stimulation compared to IGF1 stimulation. Being able to discern differential downstream signaling, we can explore improved anti-IGF1R cancer therapies by eliminating emergence of compensation mechanisms, without disrupting InsR signaling. ImplicationsThe study shows, both experimentally and through computational models, that IGF1 and insulin receptor signaling pathways respond differently to RPS6K inhibition.

systems biology↗

bioRxiv2020

Acquired mutations and transcriptional remodeling in long-term estrogen-deprived locoregional breast cancer recurrences

Endocrine therapy resistance is a hallmark of advanced estrogen receptor (ER) positive breast cancer. In this study, we performed DNA/RNA hybrid-capture sequencing on 12 locoregional recurrences after long-term estrogen-deprivation along with each tumors matched primary. Despite being up to 7 years removed from the primary lesion, most recurrences harbored similar intrinsic transcriptional and copy number profiles. Only two genes, AKAP9 and KMT2C, were found to have single nucleotide variant (SNV) enrichments in more than one recurrence. Enriched mutations in single cases included SNVs within transcriptional regulators such as ARID1A, TP53, FOXO1, BRD1, NCOA1 and NCOR2 with one local recurrence gaining three PIK3CA mutations. In contrast to DNA-level changes, we discovered recurrent outlier mRNA expression alterations were common--including outlier gains in TP63 (n=5 cases [42%]), NTRK3 [n=5 [42%]), NTRK2 (n=4 [33%]), PAX3 (n=4 [33%]), FGFR4 (n=3 [25%]) and TERT (n=3 [25%]). Recurrent losses involved ESR1 (n=5 [42%]), RELN (n=5 [42%]), SFRP4 (n=4 [33%]) and FOSB (n=4 [33%]). ESR1-depleted recurrences harbored shared transcriptional remodeling events including upregulation of PROM1 and other basal cancer markers. Taken together, this study defines acquired genomic changes in long-term, estrogen-deprived disease, highlights longitudinal RNA-profiling and identifies a common endocrine-resistant breast cancer subtype with basal-like transcriptional reprogramming.

cancer biology↗

bioRxiv2020

Identifying Genomic Alterations in Stage IV Breast Cancer Patients using MammaSeq™: An International Collaborative Study

BackgroundIdentification of genomic alterations present in cancer patients may aid in cancer diagnosis and prognosis and may identify therapeutic targets. In this study, we aimed to identify clinically actionable variants present in stage IV breast cancer (BC) samples. Materials and MethodsDNA was extracted from formalin fixed paraffin embedded (FFPE) samples of BC (n=41). DNA was sequenced using MammaSeq, a BC specific next generation sequencing panel targeting 79 genes and 1369 mutations. Ion Torrent Suite 4.0 was used to make variant calls on the raw data and the resulting single nucleotide variants were annotated using CRAVAT toolkit. SNVs were filtered to remove common polymorphisms and somatic variants. CNVkit was employed to identify copy number variations. The Precision Medicine Knowledgebase (PMKB) and OncoKB Precision Oncology Database were used to associate clinical significance with the identified variants. ResultsA total of 41 Turkish BC patient samples were sequenced (read depth of 94 - 13340, median of 1529). These samples were from patients diagnosed with various BC subtypes including invasive ductal carcinoma (IDC), invasive lobular carcinoma (ILC), apocrine BC and micropapillary BC. In total, 59 different alterations (49 SNVs and 10 CNVs) were identified. From these, 8 alterations (3 CNVs - ERBB2, FGFR1 and AR copy number gains and 5 SNVs - IDH1.R132H, TP53.E204*, PI3KCA.E545K, PI3KCA.H1047R and PI3KCA.R88Q) were identified to have some clinical significance by PMKB and OncoKB. Moreover, the top five genes with most SNVs included PIK3CA, TP53, MAP3K1, ATM and NCOR1. Additionally, copy number gains and losses were found in ERBB2, GRB7, IGFR1, AR, FGFR1, MYC and IKBKB, and BRCA2, RUNX1 and RB1 respectively. ConclusionWe identified 59 unique alterations in 38 genes in 41 stage IV BC tissue samples using MammaSeq. Ten of these alterations were found to have some clinical significance by OncoKB and PKMB. This study highlights the potential use of cancer specific NGS panels in clinic to get better insight into the patient-specific genomic alterations. Highlights- 41 stage IV stage breast cancer patients of Turkish descent were sequenced using MammaSeq - 49 single nucleotide variations and 10 copy number variations identified - PIK3CA and TP53 mutations were present in 24% and 17% of the samples respectively - 37% of the samples had ERBB2/GRB7 gains and 7% had loss of BRCA2/RB1 locus - Eight clinically significant alterations were identified Micro AbstractWe performed targeted sequencing using DNA from FFPE samples of 41 stage IV breast cancer patients using MammaSeq, a breast cancer gene specific targeted sequencing panel. In total, 49 single nucleotide variations (SNVs) and 10 copy number variations (CNVs) were identified. Eight alterations (3 CNVs - ERBB2, FGFR1 and AR copy number gains and 5 SNVs - IDH1.R132H, TP53.E204*, PI3KCA.E545K, PI3KCA.H1047R and PI3KCA.R88Q) were identified to have clinical significance by PMKB and OncoKB databases.

genomics↗

bioRxiv2019

Proteomic and Transcriptomic Profiling Identifies Mediators of Anchorage-Independent Growth and Roles of Inhibitor of Differentiation Proteins in Invasive Lobular Breast Cancer

BACKGROUNDInvasive lobular carcinoma (ILC) is a histological subtype of breast cancer with distinct molecular and clinical features from the more common subtype invasive lobular carcinoma (IDC). We have previously shown that human ILC cells lines have a remarkably unique ability to grow in ultra-low attachment (ULA) suspension cultures as compared to IDC cells, the mediators of which remain unknown. METHODSUsing flow cytometry and immunoblotting in human ILC and IDC cell lines, we measured levels of apoptosis and cell proliferation in attached (2D) and suspension (ULA) cultures. siRNA-mediated knockdown and pharmacological inhibitors were utilized to assess the effects of known regulators of anchorage-independence. Reverse Phase Protein Arrays and RNA-Sequencing were performed to identify novel proteomic and transcriptomic mediators of ULA growth in ILC cells. RESULTSWe show that human ILC cell lines exhibit enhanced anoikis resistance and cell proliferation in ULA cultures as compared to IDC cells. Transient restoration of E-cadherin did not impact the 2D or ULA growth of human ILC cell lines, while transient E-cadherin knockdown in IDC cells partially rescued their growth defect in ULA culture. Inhibition of the Rho/ROCK, p120-catenin or YAP/Hippo pathways previously implicated in anoikis resistance did not have a major effect on the ULA growth of ILC cells. Proteomic comparison of ILC and IDC cell lines identified unique induction of PI3K/Akt and p90-RSK pathways in ULA culture in ILC cells. Transcriptional profiling uncovered unique upregulation of the Inhibitors of Differentiation family transcription factors ID1 and ID3 in ILC ULA culture, the knockdown of which diminished anchorage-independent growth. We find that ID1 and ID3 expression is higher in human ILC tumors as compared to IDC and correlated with a worse disease-specific survival uniquely in the ILC cohort. CONCLUSIONOur comprehensive study of 2D and ULA growth in human ILC cell lines revealed anoikis resistance, cell proliferation and novel mediators of anchorage-independence and provides possible mechanistic insights and clinical implications for metastatic dissemination of ILC. High expression in human ILC tumors and association with clinical outcome implicate ID1 and ID3 as novel drivers and therapeutic targets for lobular breast cancer.

cancer biology↗

bioRxiv2019

Molecular Biology Information Service: An innovative medical library-based bioinformatics support service for biomedical researchers

Biomedical researchers are increasingly reliant on obtaining bioinformatics training in order to conduct their research. Here we present a model that academic institutions may follow to provide such training for their researchers, based on the Molecular Biology Information Service (MBIS) of the Health Sciences Library System, University of Pittsburgh. The MBIS runs a four-facet service with the following goals: (1) identify, procure, and implement commercially-licensed bioinformatics software, (2) teach hands-on workshops using bioinformatics tools to solve research questions, (3) provide in-person and email consultations on software/databases, and (4) maintain a web portal providing overall guidance on the access and use of bioinformatics resources and MBIS-created webtools. This paper describes these facets of MBIS activities from 2006-2018, including outcomes from a survey measuring attitudes of University of Pittsburgh researchers about MBIS service and performance.

bioinformatics↗

bioRxiv2019

Metastatic breast cancers have reduced immune cell recruitment but harbor increased macrophages relative to their matched primary tumors

The interplay between the immune system and tumor progression is well recognized. However, current human breast cancer immunophenotyping studies are mostly focused on primary tumors with metastatic breast cancer lesions remaining largely understudied. To address this gap, we examined exome-capture RNA sequencing data from 50 primary breast tumors (PBTs) and their patient-matched metastatic tumors (METs) in brain, ovary, bone and gastrointestinal tract. We used gene expression signatures as surrogates for tumor infiltrating lymphocytes (TIL) and compared TIL patterns in PBTs and METs. Enrichment analysis and deconvolution methods both revealed that METs have a significantly lower abundance of total immune cells, including CD8+ T cells, regulatory T cells and dendritic cells. An exception was M2-like macrophages, which were significantly higher in METs across the organ sites examined. Multiplex immunohistochemistry results were consistent with data from the in-silico analysis and showed increased macrophages in METs. We confirmed the finding of a significant reduction in immune cells in brain (BRM) METs by pathologic assessment of TILs in a set of 49 patient-matched pairs of PBT/BRMs. These findings indicate that METs have an overall lower infiltration of immune cells relative to their matched PBTs, possibly due to immune escape. RNAseq analysis suggests that the relative levels of M2-like macrophages are increased in METs, and their potential role in promoting breast cancer metastasis warrants further study.

immunology↗

bioRxiv2018

Targeted mutation detection in breast cancer using MammaSeqTM

BackgroundBreast cancer is the most common invasive cancer among women worldwide. Next-generation sequencing (NGS) has revolutionized the study of cancer across research labs around the globe, however genomic testing in clinical settings remain limited. Advances in sequencing reliability, pipeline analysis, accumulation of relevant data, and the reduction of costs are rapidly increasing the feasibility of NGS-based clinical decision making.\n\nMethodsWe report the development of MammaSeq, a breast cancer specific NGS panel, targeting 79 genes and 1369 mutations, optimized for use in primary and metastatic breast cancer. To validate the panel, 46 solid tumor and 14 plasma circulating-free cfDNA samples were sequenced to a mean depth of 2311X and 1820 X respectively. Variants were called using Ion Torrent Suite 4.0 and annotated with cravat CHASM. CNVKit was used to call copy number variants in the solid tumor cohort. The oncoKB Precision Oncology Database was used to identify clinically actionable variants. ddPCR was used to validate select cfDNA mutations.\n\nResultsIn cohorts of 46 solid tumors and 14 cfDNA samples from patients with advanced breast cancer we identified 592 and 43 protein coding mutations. Mutations per sample in the solid tumor cohort ranged from 1 to 128 (median 3) and the cfDNA cohort ranged from 0 to 26 (median 2.5). Copy number analysis in the solid tumor cohort identified 46 amplifications and 35 deletions. We identified 26 clinically actionable variants (levels 1-3) annotated by OncoKB, distributed across 20 out of 46 cases (40%), in the solid tumor cohort. Allele frequencies of ESR1 and FOXA1 mutations correlated with CA.27.29 levels in patient matched blood draws.\n\nConclusionsIn solid tumors biopsies and cfDNA, MammaSeq detects clinicaly actionable mutations (oncoKB levels 1-3) in 22/46 (48%) solid tumors and in 4/14 (29%) of cfDNA samples. MammaSeq is a targeted panel suitable for clinically actionable mutation detection in breast cancer.

cancer biology↗

bioRxiv2018

Loss of E-cadherin enhances IGF1-IGF1R pathway activation and sensitizes breast cancers to anti-IGF1R inhibitors

PurposeInsulin-like growth factor I (IGF1) signaling regulates breast cancer initiation and progression and associated cancer phenotypes. We previously identified E-cadherin (CDH1) as a repressor of IGF1 signaling and in this study examined how loss of E-cadherin affects IGF1R signaling and response to anti-IGF1R therapies in breast cancer.\n\nExperimental DesignBreast cancer cell lines were used to assess how altered E-cadherin levels regulate IGF1R signaling and response to two anti-IGF1R therapies. In situ proximity ligation assay (PLA) was used to define interaction between IGF1R and E-cadherin. TCGA RNA-seq and RPPA data was used to compare IGF1R activation in estrogen receptor positive (ER+) invasive lobular carcinoma (ILC) and invasive ductal carcinoma (IDC) tumors. ER+ ILC cell lines and xenograft tumor explant cultures were used to evaluate efficacy to IGF1R pathway inhibition in combination with endocrine therapy.\n\nResultsDiminished functional E-cadherin increased both activation of IGF1R signaling and efficacy to anti-IGF1R therapies. PLA demonstrated a direct endogenous interaction between IGF1R and E-cadherin at points of cell-cell contact. Increased expression of IGF1 ligand and levels of IGF1R phosphorylation were observed in E-cadherin deficient ER+ ILC compared to IDC tumors. IGF1R pathway inhibitors were effective in inhibiting growth in ER+ ILC cell lines and synergized with endocrine therapy and similarly IGF1R inhibition reduced proliferation in ILC tumor explant culture.\n\nConclusionsWe provide evidence that loss of E-cadherin hyperactivates the IGF1R pathway and increases sensitivity to IGF1R targeted therapy, thus identifying the IGF1R pathway as a potential novel target in E-cadherin deficient breast cancers.\n\nSTATEMENT OF SIGNIFICANCEIGF1R signaling is an attractive therapeutic target in breast cancer due to its regulation of proliferation, migration, and invasion. However, clinical trials targeting IGF1R have largely been unsuccessful due to lack of biomarkers to stratify patients for therapeutic response. In this study, we demonstrate loss of E-cadherin as a potential biomarker for response to anti-IGF1R therapy, and show efficacy of IGF1R inhibition in ER+ ILC in combination with endocrine therapy. Patients with ER+ ILC have poorer long-term outcomes than patients with ER+ IDC and have a propensity for increased late recurrences, highlighting the need for improved therapeutic strategies for this subtype of breast cancer. Here, we credential IGF1R inhibition as a novel therapeutic strategy in combination with endocrine therapy for the treatment of ER+ ILC.

cancer biology↗

bioRxiv2017

Exome-Capture RNA-Sequencing Of Decade-Old Breast Cancers And Matched Decalcified Bone Metastases Identifies Clinically Actionable Targets

Bone metastases (BoM) are a significant cause of morbidity in patients with Estrogen-receptor (ER)-positive breast cancer, yet characterizations of human specimens are limited. In this study, exome-capture RNA-sequencing (ecRNA-seq) on aged (8-12 years), formalin-fixed paraffin-embedded (FFPE) and decalcified cancer specimens was first evaluated. Gene expression values and RNA-seq quality metrics from FFPE or decalcified tumor RNA showed minimal differences when compared to matched flash-frozen or non-decalcified tumors. ecRNA-seq was then applied on a longitudinal collection of 11 primary breast cancers and patient-matched de novo or recurrent BoM. BoMs harbored shifts to more Her2 and LumB PAM50 intrinsic subtypes, temporally influenced expression evolution, recurrently dysregulated prognostic gene sets and altered expression of clinically actionable genes, particularly in the CDK-Rb-E2F and FGFR-signaling pathways. Taken together, this study demonstrates the use of ecRNA-seq on decade-old and decalcified specimens and defines expression-based tumor evolution in long-term, estrogen-deprived metastases that may have immediate clinical implications.\n\nGrant SupportResearch funding for this project was provided in part by a Susan G. Komen Scholar award to AVL and to SO, the Breast Cancer Research Foundation (AVL and SO), the Fashion Footwear Association of New York, the Magee-Womens Research Institute and Foundation, and through a Postdoctoral Fellowship awarded to RJW from the Department of Defense (BC123242). NP was supported by a training grant from the NIH/NIGMS (2T32GM008424-21) and an individual fellowship from the NIH/NCI (5F30CA203095).\n\nConflicts of Interest DisclosureNo relevant conflicts of interest disclosed for this study.\n\nAuthor ContributionsStudy concept and design (NP, RJW, SO, AVL); acquisition, analysis, or interpretation of data (all authors); drafting of the manuscript (NP, RJW, SO, AVL); critical revision of the manuscript for important intellectual content (all authors); administrative, technical, or material support (PCL, AB, RB, KRW, WH, JK, MR, ZF, AMB).

cancer biology↗