Publications by authors named "Nadia A Lanman"

11 Publications

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Simplified MethylRAD Sequencing to Detect Changes in DNA Methylation at Enhancer Elements in Differentiating Embryonic Stem Cells.

Epigenomes 2020 Dec 1;4(4). Epub 2020 Oct 1.

Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA.

Differential DNA methylation is characteristic of gene regulatory regions, such as enhancers, which mostly constitute low or intermediate CpG content in their DNA sequence. Consequently, quantification of changes in DNA methylation at these sites is challenging. Given that DNA methylation across most of the mammalian genome is maintained, the use of genome-wide bisulfite sequencing to measure fractional changes in DNA methylation at specific sites is an overexertion which is both expensive and cumbersome. Here, we developed a MethylRAD technique with an improved experimental plan and bioinformatic analysis tool to examine regional DNA methylation changes in embryonic stem cells (ESCs) during differentiation. The transcriptional silencing of pluripotency genes (PpGs) during ESC differentiation is accompanied by PpG enhancer (PpGe) silencing mediated by the demethylation of H3K4me1 by LSD1. Our MethylRAD data show that in the presence of LSD1 inhibitor, a significant fraction of LSD1-bound PpGe fails to gain DNA methylation. We further show that this effect is mostly observed in PpGes with low/intermediate CpG content. Underscoring the sensitivity and accuracy of MethylRAD sequencing, our study demonstrates that this method can detect small changes in DNA methylation in regulatory regions, including those with low/intermediate CpG content, thus asserting its use as a method of choice for diagnostic purposes.
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http://dx.doi.org/10.3390/epigenomes4040024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8023688PMC
December 2020

Aprepitant, an antiemetic agent, interferes with metal ion homeostasis of and displays potent synergistic interactions with azole drugs.

Virulence 2020 12;11(1):1466-1481

Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University , West Lafayette, IN, USA.

With the rapid increase in the frequency of azole-resistant species, combination therapy appears to be a promising tool to augment the antifungal activity of azole drugs against resistant species. Here, we report the effect of aprepitant, an antiemetic agent, on the antifungal activities of azole drugs against the multidrug-resistant . Aprepitant reduced the minimum inhibitory concentration (MIC) of itraconazole , by up to eight-folds. Additionally, the aprepitant/itraconazole combination interfered significantly with the biofilm-forming ability of by 95 ± 0.13%, and significantly disrupted mature biofilms by 52 ± 0.83%, relative to the untreated control. In a infection model, the aprepitant/itraconazole combination significantly prolonged the survival of infected nematodes by ~90% (five days post-infection) and reduced the fungal burden by ~92% relative to the untreated control. Further, this novel drug combination displayed broad-spectrum synergistic interactions against other medically important species such as , and (ƩFICI ranged from 0.08 to 0.31). Comparative transcriptomic profiling and mechanistic studies indicated aprepitant/itraconazole interferes significantly with metal ion homeostasis and compromises the ROS detoxification ability of . This study presents aprepitant as a novel, potent, and broad-spectrum azole chemosensitizing agent that warrants further investigation.
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http://dx.doi.org/10.1080/21505594.2020.1838741DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7588212PMC
December 2020

Potent Synergistic Interactions between Lopinavir and Azole Antifungal Drugs against Emerging Multidrug-Resistant Candida auris.

Antimicrob Agents Chemother 2020 12 16;65(1). Epub 2020 Dec 16.

Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, Indiana, USA

The limited therapeutic options and the recent emergence of multidrug-resistant species present a significant challenge to human medicine and underscore the need for novel therapeutic approaches. Drug repurposing appears as a promising tool to augment the activity of current azole antifungals, especially against multidrug-resistant In this study, we evaluated the fluconazole chemosensitization activities of 1,547 FDA-approved drugs and clinical molecules against azole-resistant This led to the discovery that lopinavir, an HIV protease inhibitor, is a potent agent capable of sensitizing to the effect of azole antifungals. At a therapeutically achievable concentration, lopinavir exhibited potent synergistic interactions with azole drugs, particularly with itraconazole against (fractional inhibitory concentration index [ΣFICI] ranged from 0.04 to 0.09). Additionally, the lopinavir/itraconazole combination enhanced the survival rate of -infected by 90% and reduced the fungal burden in infected nematodes by 88.5% (0.05) relative to that of the untreated control. Furthermore, lopinavir enhanced the antifungal activity of itraconazole against other medically important species, including , , , and Comparative transcriptomic profiling and mechanistic studies revealed that lopinavir was able to significantly interfere with the glucose permeation and ATP synthesis. This compromised the efflux ability of and consequently enhanced the susceptibility to azole drugs, as demonstrated by Nile red efflux assays. Altogether, these findings present lopinavir as a novel, potent, and broad-spectrum azole-chemosensitizing agent that warrants further investigation against recalcitrant infections.
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http://dx.doi.org/10.1128/AAC.00684-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7927799PMC
December 2020

Inhibition of the erythropoietin-producing receptor EPHB4 antagonizes androgen receptor overexpression and reduces enzalutamide resistance.

J Biol Chem 2020 04 17;295(16):5470-5483. Epub 2020 Mar 17.

Department of Toxicology and Cancer Biology, University of Kentucky, Lexington, Kentucky 40536; Markey Cancer Center, University of Kentucky, Lexington, Kentucky 40536. Electronic address:

Prostate cancer (PCa) cells heavily rely on an active androgen receptor (AR) pathway for their survival. Enzalutamide (MDV3100) is a second-generation antiandrogenic drug that was approved by the Food and Drug Administration in 2012 to treat patients with castration-resistant prostate cancer (CRPC). However, emergence of resistance against this drug is inevitable, and it has been a major challenge to develop interventions that help manage enzalutamide-resistant CRPC. Erythropoietin-producing human hepatocellular (Eph) receptors are targeted by ephrin protein ligands and have a broad range of functions. Increasing evidence indicates that this signaling pathway plays an important role in tumorigenesis. Overexpression of EPH receptor B4 (EPHB4) has been observed in multiple types of cancer, being closely associated with proliferation, invasion, and metastasis of tumors. Here, using RNA-Seq analyses of clinical and preclinical samples, along with several biochemical and molecular methods, we report that enzalutamide-resistant PCa requires an active EPHB4 pathway that supports drug resistance of this tumor type. Using a small kinase inhibitor and RNAi-based gene silencing to disrupt EPHB4 activity, we found that these disruptions re-sensitize enzalutamide-resistant PCa to the drug both and Mechanistically, we found that EPHB4 stimulates the AR by inducing proto-oncogene c-Myc (c-Myc) expression. Taken together, these results provide critical insight into the mechanism of enzalutamide resistance in PCa, potentially offering a therapeutic avenue for enhancing the efficacy of enzalutamide to better manage this common malignancy.
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http://dx.doi.org/10.1074/jbc.RA119.011385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7170517PMC
April 2020

Oct4-Mediated Inhibition of Lsd1 Activity Promotes the Active and Primed State of Pluripotency Enhancers.

Cell Rep 2020 02;30(5):1478-1490.e6

Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA; Purdue University Center for Cancer Research, Purdue University, West Lafayette, IN 47907, USA. Electronic address:

An aberrant increase in pluripotency gene (PpG) expression due to enhancer reactivation could induce stemness and enhance the tumorigenicity of cancer stem cells. Silencing of PpG enhancers (PpGe) during embryonic stem cell differentiation involves Lsd1-mediated H3K4me1 demethylation and DNA methylation. Here, we observed retention of H3K4me1 and DNA hypomethylation at PpGe associated with a partial repression of PpGs in F9 embryonal carcinoma cells (ECCs) post-differentiation. H3K4me1 demethylation in F9 ECCs could not be rescued by Lsd1 overexpression. Given our observation that H3K4me1 demethylation is accompanied by strong Oct4 repression in P19 ECCs, we tested if Oct4 interaction with Lsd1 affects its catalytic activity. Our data show a dose-dependent inhibition of Lsd1 activity by Oct4 and retention of H3K4me1 at PpGe in Oct4-overexpressing P19 ECCs. These data suggest that Lsd1-Oct4 interaction in cancer stem cells could establish a "primed" enhancer state that is susceptible to reactivation, leading to aberrant PpG expression.
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http://dx.doi.org/10.1016/j.celrep.2019.11.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7316367PMC
February 2020

Heterogeneity of human prostate carcinoma-associated fibroblasts implicates a role for subpopulations in myeloid cell recruitment.

Prostate 2020 02 25;80(2):173-185. Epub 2019 Nov 25.

Department of Surgery, NorthShore University HealthSystem, Evanston, Illinois.

Background: Carcinoma-associated fibroblasts (CAF) are a heterogeneous group of cells within the tumor microenvironment (TME) that can promote tumorigenesis in the prostate. By understanding the mechanism(s) by which CAF contributes to tumor growth, new therapeutic targets for the management of this disease may be identified. These studies determined whether unique sub-populations of human prostate CAF can be identified and functionally characterized.

Methods: Single-cell RNA-seq of primary human prostate CAF followed by unsupervised clustering was utilized to generate cell clusters based on differentially expressed (DE) gene profiles. Potential communication between CAF and immune cells was analyzed using in vivo tissue recombination by combining CAF or normal prostate fibroblasts (NPF) with non-tumorigenic, initiated prostate epithelial BPH-1 cells. Resultant grafts were assessed for inflammatory cell recruitment.

Results: Clustering of 3321 CAF allows for visualization of six subpopulations, demonstrating heterogeneity within CAF. Sub-renal capsule recombination assays show that the presence of CAF significantly increases myeloid cell recruitment to resultant tumors. This is supported by significantly increased expression of chemotactic chemokines CCL2 and CXCL12 in large clusters compared to other subpopulations. Bayesian analysis topologies also support differential communication signals between chemokine-related genes of individual clusters. Migration of THP-1 monocyte cells in vitro is stimulated in the presence of CAF conditioned medium (CM) compared with NPF CM. Further in vitro analyses suggest that CAF-derived chemokine CCL2 may be responsible for CAF-stimulated migration of THP-1 cells, since neutralization of this chemokine abrogates migration capacity.

Conclusions: CAF clustering based on DE gene expression supports the concept that clusters have unique functions within the TME, including a role in immune/inflammatory cell recruitment. These data suggest that CCL2 produced by CAF may be involved in the recruitment of inflammatory cells, but may also directly regulate the growth of the tumor. Further studies aimed at characterizing the subpopulation(s) of CAF which promote immune cell recruitment to the TME and/or stimulate prostate cancer growth and progression will be pursued.
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http://dx.doi.org/10.1002/pros.23929DOI Listing
February 2020

Distinct expression patterns of SULT2B1b in human prostate epithelium.

Prostate 2019 08 18;79(11):1256-1266. Epub 2019 Jun 18.

Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, Indiana.

Background: SULT2B1b (sulfotransferase family cytosolic 2B member 1b) catalyzes the sulfate conjugation of substrates such as cholesterol and oxysterols. Our laboratory has previously shown that SULT2B1b inhibition modulates androgen receptor signaling and induces apoptosis in prostate cancer cells. However, the functions of SULT2B1b in the prostate remain poorly understood.

Methods: We characterized the expression pattern of SULT2B1b in human benign prostate hyperplasia (BPH) as well as prostate cancer to determine the relationship between SULT2B1b and prostate diseases, using immunohistochemistry, immunofluorescence staining, immunoblot, and real-time polymerase chain reaction.

Results: SULT2B1b was strongly detected in the prostate epithelium but was absent in the stroma. Significantly lower SULT2B1b was found in primary cancer cells compared with adjacent normal epithelial cells. SULT2B1b further decreased in metastatic cancer cells. Most interestingly, we found, for the first time, that SULT2B1b was much more concentrated in the luminal layer than in the basal layer in both normal prostate and BPH samples. The stronger presence of SULT2B1b in luminal epithelial cells was confirmed by costaining with luminal and basal markers and in sorted paired luminal and basal cells. SULT2B1b expression was induced with prostate organoid differentiation.

Conclusions: SULT2B1b inversely correlates with prostate cancer status, with the highest level in the normal epithelium and lowest in the advanced metastatic prostate cancer. Furthermore, SULT2B1b is mostly located within the luminal layer of the prostate epithelium, suggesting that it may be implicated in luminal differentiation.
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http://dx.doi.org/10.1002/pros.23829DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7064034PMC
August 2019

Three Genes Define a Bacterial-Like Arsenic Tolerance Mechanism in the Arsenic Hyperaccumulating Fern Pteris vittata.

Curr Biol 2019 05 9;29(10):1625-1633.e3. Epub 2019 May 9.

Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907, USA. Electronic address:

Arsenic is a carcinogenic contaminant of water and food and a growing threat to human health in many regions of the world. This study focuses on the fern Pteris vittata (Pteridaceae), which is extraordinary in its ability to tolerate and hyperaccumulate very high levels of arsenic that would kill any other plant or animal outside the Pteridaceae. Here, we use RNA-seq to identify three genes (GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE (PvGAPC1), ORGANIC CATION TRANSPORTER 4 (PvOCT4), and GLUTATHIONE S-TRANSFERASE (PvGSTF1) that are highly upregulated by arsenic and are necessary for arsenic tolerance, as demonstrated by RNAi. The proteins encoded by these genes have unexpected properties: PvGAPC1 has an unusual active site and a much greater affinity for arsenate than phosphate; PvGSTF1 has arsenate reductase activity; and PvOCT4 localizes as puncta in the cytoplasm. Surprisingly, PvGAPC1, PvGSTF1, and arsenate localize in a similar pattern. These results are consistent with a model that describes the fate of arsenate once it enters the cell. It involves the conversion of arsenate into 1-arseno-3-phosphoglycerate by PvGAPC1. This "chemically trapped" arsenate is pumped into specific arsenic metabolizing vesicles by the PvOCT4 protein. Once inside these vesicles, 1-arseno-3-phosphoglycerate hydrolyses to release arsenate, which is then reduced by PvGSTF1 to arsenite, the form of arsenic stored in the vacuoles of this fern. This mechanism is strikingly similar to one recently described Pseudomonas aeruginosa, whose tolerance to arsenic also involves the biosynthesis and transport of 1-arseno-3-phosphoglycerate, indicating that P. vittata has evolved a simple, bacterial-like mechanism for arsenic tolerance.
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http://dx.doi.org/10.1016/j.cub.2019.04.029DOI Listing
May 2019

Thrombin Signaling Promotes Pancreatic Adenocarcinoma through PAR-1-Dependent Immune Evasion.

Cancer Res 2019 07 2;79(13):3417-3430. Epub 2019 May 2.

Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital, Cincinnati, Ohio.

Pancreatic ductal adenocarcinoma (PDAC) is associated with robust activity of the coagulation system. To determine mechanisms by which clotting factors influence PDAC tumor progression, we generated and characterized C57Bl/6-derived KPC ( ) cell lines. Tissue factor (TF) and protease-activated receptor-1 (PAR-1) were highly expressed in primary KPC pancreatic lesions and KPC cell lines similar to expression profiles observed in biopsies of patients with PDAC. In allograft studies, tumor growth and metastatic potential were significantly diminished by depletion of or in cancer cells or by genetic or pharmacologic reduction of the coagulation zymogen prothrombin in mice. Notably, PAR-1-deleted KPC cells (KPC-Par-1) failed to generate sizable tumors, a phenotype completely rescued by restoration of expression. Expression profiling of KPC and KPC-Par-1 cells indicated that thrombin-PAR-1 signaling significantly altered immune regulation pathways. Accordingly, KPC-Par-1 cells failed to form tumors in immune-competent mice but displayed robust tumor growth comparable to that observed with control KPC cells in immune-compromised mice. Immune cell depletion studies indicated that CD8 T cells, but not CD4 cells or natural killer cells, mediated elimination of KPC-Par-1 tumor cells in C57Bl/6 mice. These results demonstrate that PDAC is driven by activation of the coagulation system through tumor cell-derived TF, circulating prothrombin, and tumor cell-derived PAR-1 and further indicate that one key mechanism of thrombin/PAR-1-mediated tumor growth is suppression of antitumor immunity in the tumor microenvironment. SIGNIFICANCE: The tissue factor-thrombin-PAR-1 signaling axis in tumor cells promotes PDAC growth and disease progression with one key mechanism being suppression of antitumor immunity in the microenvironment.
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http://dx.doi.org/10.1158/0008-5472.CAN-18-3206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6699516PMC
July 2019

NOTCH signaling is activated in and contributes to resistance in enzalutamide-resistant prostate cancer cells.

J Biol Chem 2019 05 2;294(21):8543-8554. Epub 2019 Apr 2.

Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907; Department of Comparative Pathobiology, Purdue University, West Lafayette, Indiana 47907; Department of Toxicology and Cancer Biology, University of Kentucky, Lexington, Kentucky 40536. Electronic address:

Prostate cancer is the second leading cause of cancer death among men in the United States. The androgen receptor (AR) antagonist enzalutamide is a Food and Drug Administration-approved drug for treatment of patients with late-stage prostate cancer and is currently under clinical study for early-stage prostate cancer treatment. After a short positive response period, tumors will develop drug resistance. In this study using RNA-Seq and bioinformatics analyses, we observed that NOTCH signaling is a deregulated pathway in enzalutamide-resistant cells. and c- gene expression positively correlated with expression in samples from patient with hormone refractory disease in which AR expression levels correspond to those typically observed in enzalutamide resistance. Cleaved NOTCH1, HES1 (Hes family BHLH transcription factor 1), and c-MYC protein expression levels are elevated in two enzalutamide-resistant cell lines, MR49F and C4-2R, indicating NOTCH signaling activation. Moreover, inhibition of the overexpressed ADAM metallopeptidase domain 10 (ADAM10) in the resistant cells induces an exclusive reduction in cleaved NOTCH1 expression. Furthermore, exposure of enzalutamide-resistant cells to both PF-03084014 and enzalutamide increased cell death, decreased colony formation ability, and resensitized cells to enzalutamide. Knockdown of in C4-2R increased enzalutamide sensitivity by decreasing cell proliferation and increasing cleaved PARP expression. In a 22RV1 xenograft model, PF-03084014 and enzalutamide decreased tumor growth through reducing cell proliferation and increasing apoptosis. These results indicate that NOTCH1 signaling may contribute to enzalutamide resistance in prostate cancer, and inhibition of NOTCH signaling can resensitize resistant cells to enzalutamide.
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http://dx.doi.org/10.1074/jbc.RA118.006983DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6544854PMC
May 2019

Cholesterol Sulfotransferase SULT2B1b Modulates Sensitivity to Death Receptor Ligand TNFα in Castration-Resistant Prostate Cancer.

Mol Cancer Res 2019 06 1;17(6):1253-1263. Epub 2019 Mar 1.

Department of Comparative Pathobiology, Purdue University, West Lafayette, Indiana.

Cholesterol sulfotransferase, SULT2B1b, has been demonstrated to modulate both androgen receptor activity and cell growth properties. However, the mechanism(s) by which SULT2B1b alters these properties within prostate cancer cells has not been described. Furthermore, specific advantages of SULT2B1b expression in prostate cancer cells are not understood. In these studies, single-cell mRNA sequencing was conducted to compare the transcriptomes of SULT2B1b knockdown (KD) versus Control KD LNCaP cells. Over 2,000 differentially expressed genes were identified along with alterations in numerous canonical pathways, including the death receptor signaling pathway. The studies herein demonstrate that SULT2B1b KD increases TNFα expression in prostate cancer cells and results in NF-κB activation in a TNF-dependent manner. More importantly, SULT2B1b KD significantly enhances TNF-mediated apoptosis in both TNF-sensitive LNCaP cells and TNF-resistant C4-2 cells. Overexpression of SULT2B1b in LNCaP cells also decreases sensitivity to TNF-mediated cell death, suggesting that SULT2B1b modulates pathways dictating the TNF sensitivity capacity of prostate cancer cells. Probing human prostate cancer patient datasets further supports this work by providing evidence that SULT2B1b expression is inversely correlated with TNF-related genes, including , and . Together, these data provide evidence that SULT2B1b expression in prostate cancer cells enhances resistance to TNF and may provide a growth advantage. In addition, targeting SULT2B1b may induce an enhanced therapeutic response to TNF treatment in advanced prostate cancer. IMPLICATIONS: These data suggest that SULT2B1b expression enhances resistance to TNF and may promote prostate cancer.
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http://dx.doi.org/10.1158/1541-7786.MCR-18-1054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6548593PMC
June 2019