Publications by authors named "Melanie H Cobb"

126 Publications

Beth Levine M.D. Prize in Autophagy Research.

Autophagy 2021 Sep 30;17(9):2053. Epub 2021 Aug 30.

Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA.

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http://dx.doi.org/10.1080/15548627.2021.1962170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8496531PMC
September 2021

The Pancreatic ß-cell Response to Secretory Demands and Adaption to Stress.

Endocrinology 2021 Nov;162(11)

Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.

Pancreatic β cells dedicate much of their protein translation capacity to producing insulin to maintain glucose homeostasis. In response to increased secretory demand, β cells can compensate by increasing insulin production capability even in the face of protracted peripheral insulin resistance. The ability to amplify insulin secretion in response to hyperglycemia is a critical facet of β-cell function, and the exact mechanisms by which this occurs have been studied for decades. To adapt to the constant and fast-changing demands for insulin production, β cells use the unfolded protein response of the endoplasmic reticulum. Failure of these compensatory mechanisms contributes to both type 1 and 2 diabetes. Additionally, studies in which β cells are "rested" by reducing endogenous insulin demand have shown promise as a therapeutic strategy that could be applied more broadly. Here, we review recent findings in β cells pertaining to the metabolic amplifying pathway, the unfolded protein response, and potential advances in therapeutics based on β-cell rest.
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http://dx.doi.org/10.1210/endocr/bqab173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8459449PMC
November 2021

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Mol Pharmacol 2021 Jul 26. Epub 2021 Jul 26.

Pharmacology, University of Texas Southwestern Medical Center, United States

The WNK (with-no lysine (K)) kinases and their downstream effector kinases, OSR1 (oxidative stress responsive 1) and SPAK (SPS/STE20-related proline-alanine rich kinase), have well-established functions in the maintenance of cell volume and ion homeostasis. Mutations in these kinases have been linked to an inherited form of hypertension, neurological defects, and other pathologies. A rapidly expanding body of evidence points to the involvement of WNKs in regulating multiple diverse cellular processes as well as the progression of some forms of cancer. How OSR1/SPAK contribute to these processes is well understood in some cases, but completely unknown in others. OSR1 and SPAK are targeted to both WNKs and substrates via their conserved C-terminal (CCT) protein interaction domains. Considerable effort has been put forth to understand the structure, function, and interaction specificity of the CCT domains in relation to WNK signaling, and multiple inhibitors of WNK signaling target these domains. The domains bind RFxV and RxFxV protein sequence motifs with the consensus sequence R-F-x-V/I or R-x-F-x-V/I, but residues outside the core motif also contribute to specificity. CCT interactions are required for OSR1 and SPAK activation and deactivation as well as cation-chloride cotransporter substrate phosphorylation. All four WNKs also contain CCT-like domains that have similar structures and conserved binding residues when compared to CCT domains, but their functions and interaction specificities are mostly unknown. A better understanding of the varied actions of these domains and their interactions will better define the known signaling mechanisms of the WNK pathway as well as uncover new ones. WNK kinases and downstream effector kinases, OSR1 and SPAK, have been shown to be involved in an array of diverse cellular processes. Here we review the function of modular protein interaction domains found in OSR1 and SPAK as well as related domains found in WNKs.
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http://dx.doi.org/10.1124/molpharm.121.000307DOI Listing
July 2021

WNK1 Enhances Migration and Invasion in Breast Cancer Models.

Mol Cancer Ther 2021 Oct 12;20(10):1800-1808. Epub 2021 Jul 12.

Department of Pharmacology, UT Southwestern Medical Center, Dallas, Texas.

Metastasis is the major cause of mortality in patients with breast cancer. Many signaling pathways have been linked to cancer invasiveness, but blockade of few protein components has succeeded in reducing metastasis. Thus, identification of proteins contributing to invasion that are manipulable by small molecules may be valuable in inhibiting spread of the disease. The protein kinase with no lysine (K) 1 (WNK1) has been suggested to induce migration of cells representing a range of cancer types. Analyses of mouse models and patient data have implicated WNK1 as one of a handful of genes uniquely linked to invasive breast cancer. Here, we present evidence that inhibition of WNK1 slows breast cancer metastasis. We show that depletion or inhibition of WNK1 reduces migration of several breast cancer cell lines in wound healing assays and decreases invasion in collagen matrices. Furthermore, WNK1 depletion suppresses expression of AXL, a tyrosine kinase implicated in metastasis. Finally, we demonstrate that WNK inhibition in mice attenuates tumor progression and metastatic burden. These data showing reduced migration, invasion, and metastasis upon WNK1 depletion in multiple breast cancer models suggest that WNK1 contributes to the metastatic phenotype, and that WNK1 inhibition may offer a therapeutic avenue for attenuating progression of invasive breast cancers.
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http://dx.doi.org/10.1158/1535-7163.MCT-21-0174DOI Listing
October 2021

Control of Podocyte and Glomerular Capillary Wall Structure and Elasticity by WNK1 Kinase.

Front Cell Dev Biol 2020 2;8:618898. Epub 2021 Feb 2.

Department of Internal Medicine, Division of Nephrology, University of Texas Southwestern Medical Center, Dallas, TX, United States.

Cytoskeletal structure and its regulation are essential for maintenance of the differentiated state of specific types of cells and their adaptation to physiologic and pathophysiologic conditions. Renal glomerular capillaries, composed of podocytes, endothelial cells, and the glomerular basement membrane, have distinct structural and biophysical properties and are the site of injury in many glomerular diseases. Calcineurin inhibitors, immunosuppressant drugs used for organ transplantation and auto-immune diseases, can protect podocytes and glomerular capillaries from injury by preserving podocyte cytoskeletal structure. These drugs cause complications including hypertension and hyperkalemia which are mediated by WNK (With No Lysine) kinases as well as vasculopathy with glomerulopathy. WNK kinases and their target kinases oxidative stress-responsive kinase 1 (OSR1) and SPS1-related proline/alanine-rich kinase (SPAK) have fundamental roles in angiogenesis and are activated by calcineurin inhibitors, but the actions of these agents on kidney vasculature, and glomerular capillaries are not fully understood. We investigated WNK1 expression in cultured podocytes and isolated mouse glomerular capillaries to determine if WNK1 contributes to calcineurin inhibitor-induced preservation of podocyte and glomerular structure. WNK1 and OSR1/SPAK are expressed in podocytes, and in a pattern similar to podocyte synaptopodin in glomerular capillaries. Calcineurin inhibitors increased active OSR1/SPAK in glomerular capillaries, the Young's modulus (E) of glomeruli, and the F/G actin ratio, effects all blocked by WNK inhibition. In glomeruli, WNK inhibition caused reduced and irregular synaptopodin-staining, abnormal capillary and foot process structures, and increased deformability. In cultured podocytes, FK506 activated OSR1/SPAK, increased lamellipodia, accelerated cell migration, and promoted traction force. These actions of FK506 were reduced by depletion of WNK1. Collectively, these results demonstrate the importance of WNK1 in regulation of the podocyte actin cytoskeleton, biophysical properties of glomerular capillaries, and slit diaphragm structure, all of which are essential to normal kidney function.
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http://dx.doi.org/10.3389/fcell.2020.618898DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7884762PMC
February 2021

α-Adrenergic Disruption of β Cell BDNF-TrkB Receptor Tyrosine Kinase Signaling.

Front Cell Dev Biol 2020 15;8:576396. Epub 2020 Oct 15.

Department of Pharmacology, UT Southwestern Medical Center, Dallas, TX, United States.

Adrenergic signaling is a well-known input into pancreatic islet function. Specifically, the insulin-secreting islet β cell expresses the G-linked α-adrenergic receptor, which upon activation suppresses insulin secretion. The use of the adrenergic agonist epinephrine at micromolar doses may have supraphysiological effects. We found that pretreating β cells with micromolar concentrations of epinephrine differentially inhibited activation of receptor tyrosine kinases. We chose TrkB as an example because of its relative sensitivity to the effects of epinephrine and due to its potential regulatory role in the β cell. Our characterization of brain-derived neurotrophic factor (BDNF)-TrkB signaling in MIN6 β cells showed that TrkB is activated by BDNF as expected, leading to canonical TrkB autophosphorylation and subsequent downstream signaling, as well as chronic effects on β cell growth. Micromolar, but not nanomolar, concentrations of epinephrine blocked BDNF-induced TrkB autophosphorylation and downstream mitogen-activated protein kinase pathway activation, suggesting an inhibitory phenomenon at the receptor level. We determined epinephrine-mediated inhibition of TrkB activation to be G-dependent using pertussis toxin, arguing against an off-target effect of high-dose epinephrine. Published data suggested that inhibition of potassium channels or phosphoinositide-3-kinase signaling may abrogate the negative effects of epinephrine; however, these did not rescue TrkB signaling in our experiments. Taken together, these results show that (1) TrkB kinase signaling occurs in β cells and (2) use of epinephrine in studies of insulin secretion requires careful consideration of concentration-dependent effects. BDNF-TrkB signaling in β cells may underlie pro-survival or growth signaling and warrants further study.
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http://dx.doi.org/10.3389/fcell.2020.576396DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7593622PMC
October 2020

The CK1α Activator Pyrvinium Enhances the Catalytic Efficiency (/) of CK1α.

Biochemistry 2019 12 10;58(51):5102-5106. Epub 2019 Dec 10.

Molecular Oncology Program, The DeWitt Daughtry Family Department of Surgery, Miller School of Medicine , University of Miami , Miami , Florida 33136 , United States.

The serine/threonine protein kinase casein kinase 1α (CK1α) functions as a negative regulator of Wnt signaling, phosphorylating β-catenin at serine 45 (P-S45) to initiate its eventual ubiquitin-mediated degradation. We previously showed that the repurposed, FDA-approved anthelminthic drug pyrvinium potently inhibits Wnt signaling and Moreover, we proposed that pyrvinium's Wnt inhibitory activity was the result of its function as an activator of CK1α. An understanding of the mechanism by which pyrvinium activates CK1α is important because pyrvinium was given an orphan drug designation by the FDA to treat familial adenomatous polyposis, a precancerous condition driven by constitutive Wnt signaling. In the current study, we show that pyrvinium stimulates the phosphorylation of S45 β-catenin, a known CK1α substrate, in a cell-based assay, and does so in a dose- and time-dependent manner. Alternative splicing of CK1α results in four forms of the protein with distinct biological properties. We evaluated these splice products and identified the CK1α splice variant, CK1αS, as the form that exhibits the most robust response to pyrvinium in cells. Kinetic studies indicate that pyrvinium also stimulates the kinase activity of purified, recombinant CK1αS , increasing its catalytic efficiency (/) toward substrates. These studies provide strong and clear mechanistic evidence that pyrvinium enhances CK1α kinase activity.
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http://dx.doi.org/10.1021/acs.biochem.9b00891DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6942998PMC
December 2019

Subtype-specific secretomic characterization of pulmonary neuroendocrine tumor cells.

Nat Commun 2019 07 19;10(1):3201. Epub 2019 Jul 19.

Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, 75390, TX, USA.

Pulmonary neuroendocrine (NE) cancer, including small cell lung cancer (SCLC), is a particularly aggressive malignancy. The lineage-specific transcription factors Achaete-scute homolog 1 (ASCL1), NEUROD1 and POU2F3 have been reported to identify the different subtypes of pulmonary NE cancers. Using a large-scale mass spectrometric approach, here we perform quantitative secretome analysis in 13 cell lines that signify the different NE lung cancer subtypes. We quantify 1,626 proteins and identify IGFBP5 as a secreted marker for ASCL1 SCLC. ASCL1 binds to the E-box elements in IGFBP5 and directly regulates its transcription. Knockdown of ASCL1 decreases IGFBP5 expression, which, in turn, leads to hyperactivation of IGF-1R signaling. Pharmacological co-targeting of ASCL1 and IGF-1R results in markedly synergistic effects in ASCL1 SCLC in vitro and in mouse models. We expect that this secretome resource will provide the foundation for future mechanistic and biomarker discovery studies, helping to delineate the molecular underpinnings of pulmonary NE tumors.
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http://dx.doi.org/10.1038/s41467-019-11153-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6642156PMC
July 2019

Measuring Relative Insulin Secretion using a Co-Secreted Luciferase Surrogate.

J Vis Exp 2019 06 25(148). Epub 2019 Jun 25.

Department of Pharmacology, University of Texas Southwestern Medical Center.

Performing antibody-based assays for secreted insulin post-sample collection usually requires a few hours to a day of assay time and can be expensive, depending on the specific assay. Secreted luciferase assays expedite results and lower the assay cost per sample substantially. Here we present a relatively underused approach to gauge insulin secretory activity from pancreatic β cells by using Gaussia luciferase genetically inserted within the C-peptide. During proteolytic processing of proinsulin, the C-peptide is excised releasing the luciferase within the insulin secretory vesicle where it is co-secreted with insulin. Results can be obtained within minutes after sample collection because of the speed of luciferase assays. A limitation of the assay is that it is a relative measurement of insulin secretion and not an absolute quantitation. However, this protocol is economical, scalable, and can be performed using most standard luminescence plate readers. Analog and digital multichannel pipettes facilitate multiple steps of the assay. Many different experimental variations can be tested simultaneously. Once a focused set of conditions are decided upon, insulin concentrations should be measured directly using antibody-based assays with standard curves to confirm the luciferase assay results.
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http://dx.doi.org/10.3791/59926DOI Listing
June 2019

Functional divergence caused by mutations in an energetic hotspot in ERK2.

Proc Natl Acad Sci U S A 2019 07 11;116(31):15514-15523. Epub 2019 Jul 11.

Department of Pharmacology, UT Southwestern Medical Center, Dallas, TX 75390;

The most frequent extracellular signal-regulated kinase 2 (ERK2) mutation occurring in cancers is E322K (E-K). ERK2 E-K reverses a buried charge in the ERK2 common docking (CD) site, a region that binds activators, inhibitors, and substrates. Little is known about the cellular consequences associated with this mutation, other than apparent increases in tumor resistance to pathway inhibitors. ERK2 E-K, like the mutation of the preceding aspartate (ERK2 D321N [D-N]) known as the sevenmaker mutation, causes increased activity in cells and evades inactivation by dual-specificity phosphatases. As opposed to findings in cancer cells, in developmental assays in , only ERK2 D-N displays a significant gain of function, revealing mutation-specific phenotypes. The crystal structure of ERK2 D-N is indistinguishable from that of wild-type protein, yet this mutant displays increased thermal stability. In contrast, the crystal structure of ERK2 E-K reveals profound structural changes, including disorder in the CD site and exposure of the activation loop phosphorylation sites, which likely account for the decreased thermal stability of the protein. These contiguous mutations in the CD site of ERK2 are both required for docking interactions but lead to unpredictably different functional outcomes. Our results suggest that the CD site is in an energetically strained configuration, and this helps drive conformational changes at distal sites on ERK2 during docking interactions.
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http://dx.doi.org/10.1073/pnas.1905015116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6681740PMC
July 2019

WNK pathways in cancer signaling networks.

Cell Commun Signal 2018 11 3;16(1):72. Epub 2018 Nov 3.

Department of Pharmacology, The University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, TX, 75390-9041, USA.

Background: The with no lysine [K] (WNK) pathway consists of the structurally unique WNK kinases, their downstream target kinases, oxidative stress responsive (OSR)1 and SPS/Ste20-related proline-alanine-rich kinase (SPAK), and a multitude of OSR1/SPAK substrates including cation chloride cotransporters.

Main Body: While the best known functions of the WNK pathway is regulation of ion transport across cell membranes, WNK pathway components have been implicated in numerous human diseases. The goal of our review is to draw attention to how this pathway and its components exert influence on the progression of cancer, specifically by detailing WNK signaling intersections with major cell communication networks and processes.

Conclusion: Here we describe how WNKs and associated proteins interact with and influence PI3K-AKT, TGF-β, and NF-κB signaling, as well as its unanticipated role in the regulation of angiogenesis.
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http://dx.doi.org/10.1186/s12964-018-0287-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6215617PMC
November 2018

Chromomycin A potently inhibits glucose-stimulated insulin secretion from pancreatic β cells.

J Gen Physiol 2018 12 23;150(12):1747-1757. Epub 2018 Oct 23.

Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX.

Modulators of insulin secretion could be used to treat diabetes and as tools to investigate β cell regulatory pathways in order to increase our understanding of pancreatic islet function. Toward this goal, we previously used an insulin-linked luciferase that is cosecreted with insulin in MIN6 β cells to perform a high-throughput screen of natural products for chronic effects on glucose-stimulated insulin secretion. In this study, using multiple phenotypic analyses, we found that one of the top natural product hits, chromomycin A2 (CMA2), potently inhibited insulin secretion by at least three potential mechanisms: disruption of Wnt signaling, interference of β cell gene expression, and partial suppression of Ca influx. Chronic treatment with CMA2 largely ablated glucose-stimulated insulin secretion even after washout, but it did not inhibit glucose-stimulated generation of ATP or Ca influx. However, by using the K channel opener diazoxide, we uncovered defects in depolarization-induced Ca influx that may contribute to the suppressed secretory response. Glucose-responsive ERK1/2 and S6 phosphorylation were also disrupted by chronic CMA2 treatment. By querying the FUSION bioinformatic database, we revealed that the phenotypic effects of CMA2 cluster with a number of Wnt-GSK3 pathway-related genes. Furthermore, CMA2 consistently decreased GSK3β phosphorylation and suppressed activation of a β-catenin activity reporter. CMA2 and a related compound, mithramycin, are known to have DNA interaction properties, possibly abrogating transcription factor binding to critical β cell gene promoters. We observed that CMA2 but not mithramycin suppressed expression of PDX1 and UCN3. However, neither expression of INSI/II nor insulin content was affected by chronic CMA2. The mechanisms of CMA2-induced insulin secretion defects may involve components both proximal and distal to Ca influx. Therefore, CMA2 is an example of a chemical that can simultaneously disrupt β cell function through both noncytotoxic and cytotoxic mechanisms. Future therapeutic applications of CMA2 and similar aureolic acid analogues should consider their potential effects on pancreatic islet function.
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http://dx.doi.org/10.1085/jgp.201812177DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6279362PMC
December 2018

Pulling a MAST1 on Cisplatin Resistance.

Cancer Cell 2018 08;34(2):183-185

Department of Pharmacology, UT Southwestern Medical Center, Dallas, TX 75390, USA. Electronic address:

In this issue of Cancer Cell, Jin and colleagues use a kinome-wide screen to identify MAST1 as a cause of cisplatin resistance. They demonstrate that this kinase is a novel activator of MEK1 and the MAPK cascade and that it can harness pathway output to block cisplatin-induced cell death.
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http://dx.doi.org/10.1016/j.ccell.2018.07.010DOI Listing
August 2018

OSR1 regulates a subset of inward rectifier potassium channels via a binding motif variant.

Proc Natl Acad Sci U S A 2018 04 26;115(15):3840-3845. Epub 2018 Mar 26.

Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390;

The with-no-lysine (K) (WNK) signaling pathway to STE20/SPS1-related proline- and alanine-rich kinase (SPAK) and oxidative stress-responsive 1 (OSR1) kinase is an important mediator of cell volume and ion transport. SPAK and OSR1 associate with upstream kinases WNK 1-4, substrates, and other proteins through their C-terminal domains which interact with linear R-F-x-V/I sequence motifs. In this study we find that SPAK and OSR1 also interact with similar affinity with a motif variant, R-x-F-x-V/I. Eight of 16 human inward rectifier K channels have an R-x-F-x-V motif. We demonstrate that two of these channels, Kir2.1 and Kir2.3, are activated by OSR1, while Kir4.1, which does not contain the motif, is not sensitive to changes in OSR1 or WNK activity. Mutation of the motif prevents activation of Kir2.3 by OSR1. Both siRNA knockdown of OSR1 and chemical inhibition of WNK activity disrupt NaCl-induced plasma membrane localization of Kir2.3. Our results suggest a mechanism by which WNK-OSR1 enhance Kir2.1 and Kir2.3 channel activity by increasing their plasma membrane localization. Regulation of members of the inward rectifier K channel family adds functional and mechanistic insight into the physiological impact of the WNK pathway.
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http://dx.doi.org/10.1073/pnas.1802339115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5899495PMC
April 2018

Enteroid Monolayers Reveal an Autonomous WNT and BMP Circuit Controlling Intestinal Epithelial Growth and Organization.

Dev Cell 2018 03 1;44(5):624-633.e4. Epub 2018 Mar 1.

Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address:

The intestinal epithelium maintains a remarkable balance between proliferation and differentiation despite rapid cellular turnover. A central challenge is to elucidate mechanisms required for robust control of tissue renewal. Opposing WNT and BMP signaling is essential in establishing epithelial homeostasis. However, it has been difficult to disentangle contributions from multiple sources of morphogen signals in the tissue. Here, to dissect epithelial-autonomous morphogenic signaling circuits, we developed an enteroid monolayer culture system that recapitulates four key properties of the intestinal epithelium, namely the ability to maintain proliferative and differentiated zones, self-renew, polarize, and generate major intestinal cell types. We systematically perturb intrinsic and extrinsic sources of WNT and BMP signals to reveal a core morphogenic circuit that controls proliferation, tissue organization, and cell fate. Our work demonstrates the ability of intestinal epithelium, even in the absence of 3D tissue architecture, to control its own growth and organization through morphogen-mediated feedback.
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http://dx.doi.org/10.1016/j.devcel.2018.01.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5849535PMC
March 2018

The Epithelial Sodium Channel (αENaC) Is a Downstream Therapeutic Target of ASCL1 in Pulmonary Neuroendocrine Tumors.

Transl Oncol 2018 Apr 2;11(2):292-299. Epub 2018 Feb 2.

Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas. Electronic address:

Small cell lung cancer (SCLC) is an aggressive neuroendocrine carcinoma, designated as a recalcitrant cancer by the National Cancer Institute, in urgent need of new rational therapeutic targets. Previous studies have determined that the basic helix-loop-helix transcription factor achaete-scute homolog 1 (ASCL1) is essential for the survival and progression of a fraction of pulmonary neuroendocrine cancer cells, which include both SCLC and a subset of non-SCLC. Previously, to understand how ASCL1 initiates tumorigenesis in pulmonary neuroendocrine cancer and identify the transcriptional targets of ASCL1, whole-genome RNA-sequencing analysis combined with chromatin immunoprecipitation-sequencing was performed with a series of lung cancer cell lines. From this analysis, we discovered that the gene SCNN1A, which encodes the alpha subunit of the epithelial sodium channel (αENaC), is highly correlated with ASCL1 expression in SCLC. The product of the SCNN1A gene ENaC can be pharmacologically inhibited with amiloride, a drug that has been used clinically for close to 50 years. Amiloride inhibited growth of ASCL1-dependent SCLC more strongly than ASCL1-independent SCLC in vitro and slowed growth of ASCL1-driven SCLC in xenografts. We conclude that SCNN1A/αENaC is a direct transcriptional target of the neuroendocrine lung cancer lineage oncogene ASCL1 that can be pharmacologically targeted with antitumor effects.
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http://dx.doi.org/10.1016/j.tranon.2018.01.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5884185PMC
April 2018

Differential abundance of CK1α provides selectivity for pharmacological CK1α activators to target WNT-dependent tumors.

Sci Signal 2017 Jun 27;10(485). Epub 2017 Jun 27.

Molecular Oncology Program, Department of Surgery, Miller School of Medicine, University of Miami, Miami, FL 33136, USA.

Constitutive WNT activity drives the growth of various human tumors, including nearly all colorectal cancers (CRCs). Despite this prominence in cancer, no WNT inhibitor is currently approved for use in the clinic largely due to the small number of druggable signaling components in the WNT pathway and the substantial toxicity to normal gastrointestinal tissue. We have shown that pyrvinium, which activates casein kinase 1α (CK1α), is a potent inhibitor of WNT signaling. However, its poor bioavailability limited the ability to test this first-in-class WNT inhibitor in vivo. We characterized a novel small-molecule CK1α activator called SSTC3, which has better pharmacokinetic properties than pyrvinium, and found that it inhibited the growth of CRC xenografts in mice. SSTC3 also attenuated the growth of a patient-derived metastatic CRC xenograft, for which few therapies exist. SSTC3 exhibited minimal gastrointestinal toxicity compared to other classes of WNT inhibitors. Consistent with this observation, we showed that the abundance of the SSTC3 target, CK1α, was decreased in WNT-driven tumors relative to normal gastrointestinal tissue, and knocking down CK1α increased cellular sensitivity to SSTC3. Thus, we propose that distinct CK1α abundance provides an enhanced therapeutic index for pharmacological CK1α activators to target WNT-driven tumors.
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http://dx.doi.org/10.1126/scisignal.aak9916DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5555225PMC
June 2017

Assaying Protein Kinase Activity with Radiolabeled ATP.

J Vis Exp 2017 05 26(123). Epub 2017 May 26.

Department of Pharmacology, University of Texas Southwestern Medical Center;

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering allosteric details of their regulation. Kinases comprise signaling networks whose defects are often hallmarks of multiple forms of cancer and related diseases, making an assay platform amenable to manipulation of upstream regulatory factors and validation of reaction requirements critical in the search for improved therapeutics. Here, we describe a basic kinase assay that can be easily adapted to suit specific experimental questions including but not limited to testing the effects of biochemical and pharmacological agents, genetic manipulations such as mutation and deletion, as well as cell culture conditions and treatments to probe cell signaling mechanisms. This assay utilizes radiolabeled [γ-P] ATP, which allows for quantitative comparisons and clear visualization of results, and can be modified for use with immunoprecipitated or recombinant kinase, specific or typified substrates, all over a wide range of reaction conditions.
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http://dx.doi.org/10.3791/55504DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608137PMC
May 2017

Mechanisms of the amplifying pathway of insulin secretion in the β cell.

Pharmacol Ther 2017 Nov 18;179:17-30. Epub 2017 May 18.

Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX, United States.

Pancreatic islet β cells secrete insulin in response to nutrient secretagogues, like glucose, dependent on calcium influx and nutrient metabolism. One of the most intriguing qualities of β cells is their ability to use metabolism to amplify the amount of secreted insulin independent of further alterations in intracellular calcium. Many years studying this amplifying process have shaped our current understanding of β cell stimulus-secretion coupling; yet, the exact mechanisms of amplification have been elusive. Recent studies utilizing metabolomics, computational modeling, and animal models have progressed our understanding of the metabolic amplifying pathway of insulin secretion from the β cell. New approaches will be discussed which offer in-roads to a more complete model of β cell function. The development of β cell therapeutics may be aided by such a model, facilitating the targeting of aspects of the metabolic amplifying pathway which are unique to the β cell.
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http://dx.doi.org/10.1016/j.pharmthera.2017.05.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7269041PMC
November 2017

WNK1 is an unexpected autophagy inhibitor.

Autophagy 2017 May 15;13(5):969-970. Epub 2017 Feb 15.

a Department of Pharmacology , University of Texas Southwestern Medical Center , Dallas , TX , USA.

Autophagy is a cellular degradation pathway that is essential to maintain cellular physiology, and deregulation of autophagy leads to multiple diseases in humans. In a recent study, we discovered that the protein kinase WNK1 (WNK lysine deficient protein kinase 1) is an inhibitor of autophagy. The loss of WNK1 increases both basal and starvation-induced autophagy. In addition, the depletion of WNK1 increases the activation of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex, which is required to induce autophagy. Moreover, the loss of WNK1 increases the expression of ULK1 (unc-51 like kinase 1), which is upstream of the PtdIns3K complex. It also increases the pro-autophagic phosphorylation of ULK1 at Ser555 and the activation of AMPK (AMP-activated protein kinase), which is responsible for that phosphorylation. The inhibition of AMPK by compound C decreases the magnitude of autophagy induction following WNK1 loss; however, it does not prevent autophagy induction. We found that the UVRAG (UV radiation resistance associated gene), which is a component of the PtdIns3K, binds to the N-terminal region of WNK1. Moreover, WNK1 partially colocalizes with UVRAG and this colocalization decreases when autophagy is stimulated in cells. The loss of WNK1 also alters the cellular distribution of UVRAG. The depletion of the downstream target of WNK1, OXSR1/OSR1 (oxidative-stress responsive 1) has no effect on autophagy, whereas the depletion of its relative STK39/SPAK (serine/threonine kinase 39) induces autophagy under nutrient-rich and starved conditions.
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http://dx.doi.org/10.1080/15548627.2017.1286431DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5446055PMC
May 2017

Sucralose activates an ERK1/2-ribosomal protein S6 signaling axis.

FEBS Open Bio 2017 02 18;7(2):174-186. Epub 2017 Jan 18.

Department of Pharmacology UT Southwestern Medical Center Dallas TX USA.

The sweetener sucralose can signal through its GPCR receptor to induce insulin secretion from pancreatic β cells, but the downstream signaling pathways involved are not well-understood. Here we measure responses to sucralose, glucagon-like peptide 1, and amino acids in MIN6 β cells. Our data suggest a signaling axis, whereby sucralose induces calcium and cAMP, activation of ERK1/2, and site-specific phosphorylation of ribosomal protein S6. Interestingly, sucralose acted independently of mTORC1 or ribosomal S6 kinase (RSK). These results suggest that sweeteners like sucralose can influence β-cell responses to secretagogues like glucose through metabolic as well as GPCR-mediated pathways. Future investigation of novel sweet taste receptor signaling pathways in β cells will have implications for diabetes and other emergent fields involving these receptors.
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http://dx.doi.org/10.1002/2211-5463.12172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5292669PMC
February 2017

Chemoattractant concentration-dependent tuning of ERK signaling dynamics in migrating neutrophils.

Sci Signal 2016 12 13;9(458):ra122. Epub 2016 Dec 13.

Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

The directed migration (chemotaxis) of neutrophils toward the bacterial peptide N-formyl-Met-Leu-Phe (fMLP) is a crucial process in immune defense against invading bacteria. While navigating through a gradient of increasing concentrations of fMLP, neutrophils and neutrophil-like HL-60 cells switch from exhibiting directional migration at low fMLP concentrations to exhibiting circuitous migration at high fMLP concentrations. The extracellular signal-regulated kinase (ERK) pathway is implicated in balancing this fMLP concentration-dependent switch in migration modes. We investigated the role and regulation of ERK signaling through single-cell analysis of neutrophil migration in response to different fMLP concentrations over time. We found that ERK exhibited gradated, rather than all-or-none, responses to fMLP concentration. Maximal ERK activation occurred in response to about 100 nM fMLP, and ERK inactivation was promoted by p38. Furthermore, we found that directional migration of neutrophils reached a maximal extent at about 100 nM fMLP and that ERK, but not p38, was required for neutrophil migration. Thus, our data suggest that, in chemotactic neutrophils responding to fMLP, ERK displays gradated activation and p38-dependent inhibition and that these ERK dynamics promote neutrophil migration.
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http://dx.doi.org/10.1126/scisignal.aag0486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5479306PMC
December 2016

Multistep regulation of autophagy by WNK1.

Proc Natl Acad Sci U S A 2016 12 28;113(50):14342-14347. Epub 2016 Nov 28.

Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390;

The with-no-lysine (K) (WNK) kinases are an atypical family of protein kinases that regulate ion transport across cell membranes. Mutations that result in their overexpression cause hypertension-related disorders in humans. Of the four mammalian WNKs, only WNK1 is expressed throughout the body. We report that WNK1 inhibits autophagy, an intracellular degradation pathway implicated in several human diseases. Using small-interfering RNA-mediated WNK1 knockdown, we show autophagosome formation and autophagic flux are accelerated. In cells with reduced WNK1, basal and starvation-induced autophagy is increased. We also show that depletion of WNK1 stimulates focal class III phosphatidylinositol 3-kinase complex (PI3KC3) activity, which is required to induce autophagy. Depletion of WNK1 increases the expression of the PI3KC3 upstream regulator unc-51-like kinase 1 (ULK1), its phosphorylation, and activation of the kinase upstream of ULK1, the AMP-activated protein kinase. In addition, we show that the N-terminal region of WNK1 binds to the UV radiation resistance-associated gene (UVRAG) in vitro and WNK1 partially colocalizes with UVRAG, a component of a PI3KC3 complex. This colocalization decreases upon starvation of cells. Depletion of the SPS/STE20-related proline-alanine-rich kinase, a WNK1-activated enzyme, also induces autophagy in nutrient-replete or -starved conditions, but depletion of the related kinase and WNK1 substrate, oxidative stress responsive 1, does not. These results indicate that WNK1 inhibits autophagy by multiple mechanisms.
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http://dx.doi.org/10.1073/pnas.1617649113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5167150PMC
December 2016

Insulin promoter-driven Gaussia luciferase-based insulin secretion biosensor assay for discovery of β-cell glucose-sensing pathways.

ACS Sens 2016 Oct 12;1(10):1208-1212. Epub 2016 Oct 12.

Department of Phamacology, UT Southwestern Medical Center, Dallas, TX 75235, USA.

High throughput screening of insulin secretion is intractable with current methods. We developed a secreted insulin-luciferase system (Ins-GLuc) in β cells that is rapid, inexpensive, and amenable to 96- and 384-well formats. We treated stable Ins-GLuc-expressing MIN6 cells overnight with 6298 marine natural product fractions. The cells were then washed to remove media and chemicals, followed by stimulation with glucose in the diazoxide paradigm. These conditions allowed the discovery of many insulin secretion suppressors and potentiators. The mechanisms of action of these natural products must be long-lasting given the continuance of secretory phenotypes in the absence of chemical treatment. We anticipate that these natural products and their target pathways will lead to a greater understanding of glucose-stimulated insulin secretion.
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http://dx.doi.org/10.1021/acssensors.6b00433DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5089812PMC
October 2016

Amino Acids Regulate mTORC1 by an Obligate Two-step Mechanism.

J Biol Chem 2016 Oct 1;291(43):22414-22426. Epub 2016 Sep 1.

From the Department of Pharmacology,

The mechanistic target of rapamycin complex 1 (mTORC1) coordinates cell growth with its nutritional, hormonal, energy, and stress status. Amino acids are critical regulators of mTORC1 that permit other inputs to mTORC1 activity. However, the roles of individual amino acids and their interactions in mTORC1 activation are not well understood. Here we demonstrate that activation of mTORC1 by amino acids includes two discrete and separable steps: priming and activation. Sensitizing mTORC1 activation by priming amino acids is a prerequisite for subsequent stimulation of mTORC1 by activating amino acids. Priming is achieved by a group of amino acids that includes l-asparagine, l-glutamine, l-threonine, l-arginine, l-glycine, l-proline, l-serine, l-alanine, and l-glutamic acid. The group of activating amino acids is dominated by l-leucine but also includes l-methionine, l-isoleucine, and l-valine. l-Cysteine predominantly inhibits priming but not the activating step. Priming and activating steps differ in their requirements for amino acid concentration and duration of treatment. Priming and activating amino acids use mechanisms that are distinct both from each other and from growth factor signaling. Neither step requires intact tuberous sclerosis complex of proteins to activate mTORC1. Concerted action of priming and activating amino acids is required to localize mTORC1 to lysosomes and achieve its activation.
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http://dx.doi.org/10.1074/jbc.M116.732511DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5077182PMC
October 2016

Rasip1-Mediated Rho GTPase Signaling Regulates Blood Vessel Tubulogenesis via Nonmuscle Myosin II.

Circ Res 2016 Sep 2;119(7):810-26. Epub 2016 Aug 2.

From the Department of Molecular Biology and Center for Regenerative Science and Medicine (D.M.B., Y.K., K.X., D.B.A., O.C.) and Department of Pharmacology (C.W., M.H.C.), University of Texas Southwestern Medical Center, Dallas; Department of Medical Pharmacology and Physiology, University of Missouri, Columbia (P.R.N., G.E.D.); Department of Biology, University of North Carolina, Chapel Hill (L.A.W.); and Division of Experimental Hematology and Cancer Biology, Children's Hospital Research Foundation, University of Cincinnati, OH (Y.Z.).

Rationale: Vascular tubulogenesis is essential to cardiovascular development. Within initial vascular cords of endothelial cells, apical membranes are established and become cleared of cell-cell junctions, thereby allowing continuous central lumens to open. Rasip1 (Ras-interacting protein 1) is required for apical junction clearance, as well as for regulation of Rho GTPase (enzyme that hydrolyzes GTP) activity. However, it remains unknown how activities of different Rho GTPases are coordinated by Rasip1 to direct tubulogenesis.

Objective: The aim of this study is to determine the mechanisms downstream of Rasip1 that drive vascular tubulogenesis.

Methods And Results: Using conditional mouse mutant models and pharmacological approaches, we dissect GTPase pathways downstream of Rasip1. We show that clearance of endothelial cell apical junctions during vascular tubulogenesis depends on Rasip1, as well as the GTPase Cdc42 (cell division control protein 42 homolog) and the kinase Pak4 (serine/threonine-protein kinase 4). Genetic deletion of Rasip1 or Cdc42, or inhibition of Pak4, all blocks endothelial cell tubulogenesis. By contrast, inactivation of RhoA (Ras homologue gene family member A) signaling leads to vessel overexpansion, implicating actomyosin contractility in control of lumen diameter. Interestingly, blocking activity of NMII (nonmuscle myosin II) either before, or after, lumen morphogenesis results in dramatically different tubulogenesis phenotypes, suggesting time-dependent roles.

Conclusions: Rasip1 controls different pools of GTPases, which in turn regulate different pools of NMII to coordinate junction clearance (remodeling) and actomyosin contractility during vascular tubulogenesis. Rasip1 promotes activity of Cdc42 to activate Pak4, which in turn activates NMII, clearing apical junctions. Once lumens open, Rasip1 suppresses actomyosin contractility via inhibition of RhoA by Arhgap29, allowing controlled expansion of vessel lumens during embryonic growth. These findings elucidate the stepwise processes regulated by Rasip1 through downstream Rho GTPases and NMII.
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http://dx.doi.org/10.1161/CIRCRESAHA.116.309094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5026621PMC
September 2016

ASCL1 and NEUROD1 Reveal Heterogeneity in Pulmonary Neuroendocrine Tumors and Regulate Distinct Genetic Programs.

Cell Rep 2016 08 21;16(5):1259-1272. Epub 2016 Jul 21.

Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. Electronic address:

Small cell lung carcinoma (SCLC) is a high-grade pulmonary neuroendocrine tumor. The transcription factors ASCL1 and NEUROD1 play crucial roles in promoting malignant behavior and survival of human SCLC cell lines. Here, we find that ASCL1 and NEUROD1 identify heterogeneity in SCLC, bind distinct genomic loci, and regulate mostly distinct genes. ASCL1, but not NEUROD1, is present in mouse pulmonary neuroendocrine cells, and only ASCL1 is required in vivo for tumor formation in mouse models of SCLC. ASCL1 targets oncogenic genes including MYCL1, RET, SOX2, and NFIB while NEUROD1 targets MYC. ASCL1 and NEUROD1 regulate different genes that commonly contribute to neuronal function. ASCL1 also regulates multiple genes in the NOTCH pathway including DLL3. Together, ASCL1 and NEUROD1 distinguish heterogeneity in SCLC with distinct genomic landscapes and distinct gene expression programs.
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http://dx.doi.org/10.1016/j.celrep.2016.06.081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4972690PMC
August 2016

Hypertension: the missing WNKs.

Am J Physiol Renal Physiol 2016 07 23;311(1):F16-27. Epub 2016 Mar 23.

Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas; and

The With no Lysine [K] (WNK) family of enzymes are central in the regulation of blood pressure. WNKs have been implicated in hereditary hypertension disorders, mainly through control of the activity and levels of ion cotransporters and channels. Actions of WNKs in the kidney have been heavily investigated, and recent studies have provided insight into not only the regulation of these enzymes but also how mutations in WNKs and their interacting partners contribute to hypertensive disorders. Defining the roles of WNKs in the cardiovascular system will provide clues about additional mechanisms by which WNKs can regulate blood pressure. This review summarizes recent developments in the regulation of the WNK signaling cascade and its role in regulation of blood pressure.
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http://dx.doi.org/10.1152/ajprenal.00358.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4967160PMC
July 2016
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