Billadeau, Daniel D.

Rationale: COMMD (copper metabolism MURR1 domain)-containing proteins are a part of the CCC (COMMD–CCDC22 [coiled-coil domain containing 22]–CCDC93 [coiled-coil domain containing 93]) complex facilitating endosomal trafficking of cell surface receptors. Hepatic COMMD1 inactivation decreases CCDC22 and CCDC93 protein levels, impairs the recycling of the LDLR (low-density lipoprotein receptor), and increases plasma low-density lipoprotein cholesterol levels in mice. However, whether any of the other COMMD members function similarly as COMMD1 and whether perturbation in the CCC complex promotes atherogenesis remain unclear. Objective: The main aim of this study is to unravel the contribution of evolutionarily conserved COMMD proteins to plasma lipoprotein levels and atherogenesis. Methods and Results: Using liver-specific Commd1 , Commd6 , or Commd9 knockout mice, we investigated the relation between the COMMD proteins in the regulation of plasma cholesterol levels. Combining biochemical and quantitative targeted proteomic approaches, we found that hepatic COMMD1, COMMD6, or COMMD9 deficiency resulted in massive reduction in the protein levels of all 10 COMMDs. This decrease in COMMD protein levels coincided with destabilizing of the core (CCDC22, CCDC93, and chromosome 16 open reading frame 62 [C16orf62]) of the CCC complex, reduced cell surface levels of LDLR and LRP1 (LDLR-related protein 1), followed by increased plasma low-density lipoprotein cholesterol levels. To assess the direct contribution of the CCC core in the regulation of plasma cholesterol levels, Ccdc22 was deleted in mouse livers via CRISPR/Cas9-mediated somatic gene editing. CCDC22 deficiency also destabilized the complete CCC complex and resulted in elevated plasma low-density lipoprotein cholesterol levels. Finally, we found that hepatic disruption of the CCC complex exacerbates dyslipidemia and atherosclerosis in ApoE3*Leiden mice. Conclusions: Collectively, these findings demonstrate a strong interrelationship between COMMD proteins and the core of the CCC complex in endosomal LDLR trafficking. Hepatic disruption of either of these CCC components causes hypercholesterolemia and exacerbates atherosclerosis. Our results indicate that not only COMMD1 but all other COMMDs and CCC components may be potential targets for modulating plasma lipid levels in humans.

Abstract Dysregulation of the MAPK pathway correlates with progression of pancreatic ductal adenocarcinoma (PDAC) progression. IQ motif containing GTPase-activating protein 1 (IQGAP1) is a MAPK scaffold that directly regulates the activation of RAF, MEK, and ERK. Fructose-1,6-bisphosphatase (FBP1), a key enzyme in gluconeogenesis, is transcriptionally downregulated in various cancers, including PDAC. Here, we demonstrate that FBP1 acts as a negative modulator of the IQGAP1–MAPK signaling axis in PDAC cells. FBP1 binding to the WW domain of IQGAP1 impeded IQGAP1-dependent ERK1/2 phosphorylation (pERK1/2) in a manner independent of FBP1 enzymatic activity. Conversely, decreased FBP1 expression induced pERK1/2 levels in PDAC cell lines and correlated with increased pERK1/2 levels in patient specimens. Treatment with gemcitabine caused undesirable activation of ERK1/2 in PDAC cells, but cotreatment with the FBP1-derived small peptide inhibitor FBP1 E4 overcame gemcitabine-induced ERK activation, thereby increasing the anticancer efficacy of gemcitabine in PDAC. These findings identify a primary mechanism of resistance of PDAC to standard therapy and suggest that the FBP1–IQGAP1–ERK1/2 signaling axis can be targeted for effective treatment of PDAC. Cancer Res; 77(16); 4328–41. ©2017 AACR.

ABSTRACT Proteins of the Wiskott–Aldrich syndrome protein (WASP) family function as nucleation-promoting factors for the ubiquitously expressed Arp2/3 complex, which drives the generation of branched actin filaments. Arp2/3-generated actin regulates diverse cellular processes, including the formation of lamellipodia and filopodia, endocytosis and/or phagocytosis at the plasma membrane, and the generation of cargo-laden vesicles from organelles including the Golgi, endoplasmic reticulum (ER) and the endo-lysosomal network. Recent studies have also identified roles for WASP family members in promoting actin dynamics at the centrosome, influencing nuclear shape and membrane remodeling events leading to the generation of autophagosomes. Interestingly, several WASP family members have also been observed in the nucleus where they directly influence gene expression by serving as molecular platforms for the assembly of epigenetic and transcriptional machinery. In this Cell Science at a Glance article and accompanying poster, we provide an update on the subcellular roles of WHAMM, JMY and WASH (also known as WASHC1), as well as their mechanisms of regulation and emerging functions within the cell.

AbstractAcinar‐to‐ductal metaplasia (ADM) is a reversible epithelial transdifferentiation process that occurs in the pancreas in response to acute inflammation. ADM can rapidly progress towards pre‐malignant pancreatic intraepithelial neoplasia (PanIN) lesions in the presence of mutant KRas and ultimately pancreatic adenocarcinoma (PDAC). In the present work, we elucidate the role and related mechanism of glycogen synthase kinase‐3beta (GSK‐3β) in ADM development using in vitro 3D cultures and genetically engineered mouse models. We show that GSK‐3β promotes TGF‐α‐induced ADM in 3D cultured primary acinar cells, whereas deletion of GSK‐3β attenuates caerulein‐induced ADM formation and PanIN progression in KrasG12D transgenic mice. Furthermore, we demonstrate that GSK‐3β ablation influences ADM formation and PanIN progression by suppressing oncogenic KRas‐driven cell proliferation. Mechanistically, we show that GSK‐3β regulates proliferation by increasing the activation of S6 kinase. Taken together, these results indicate that GSK‐3β participates in early pancreatitis‐induced ADM and thus could be a target for the treatment of chronic pancreatitis and the prevention of PDAC progression. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Abstract Glycogen synthase kinase-3β (GSK-3β), a serine/threonine protein kinase, is a complex regulator of numerous cellular functions. GSK-3β is a unique kinase which is constitutively active in resting and nonstimulated cells. GSK-3β has been implicated in a wide range of diseases including neurodegeneration, inflammation and fibrosis, noninsulin-dependent diabetes mellitus, and cancer. It is a regulator of NF-κB–mediated survival of cancer cells, which provided a rationale for the development of GSK-3 inhibitors targeting malignant tumors. Recent studies, many of them reported over the past decade, have identified GSK-3β as a potential therapeutic target in more than 15 different types of cancer. Whereas only active GSK-3β is expressed in cancer cell nucleus, aberrant nuclear accumulation of GSK-3β has been identified as a hallmark of cancer cells in malignant tumors of different origin. This review focuses on the preclinical and clinical development of GSK-3 inhibitors and the potential therapeutic impact of targeting GSK-3β in human cancer. Clin Cancer Res; 23(8); 1891–7. ©2017 AACR.

Abstract Peripheral T-cell lymphomas (PTCLs) represent a heterogeneous group of T-cell malignancies that generally demonstrate aggressive clinical behavior, often are refractory to standard therapy, and remain significantly understudied. The most common World Health Organization subtype is PTCL, not otherwise specified (NOS), essentially a “wastebasket” category because of inadequate understanding to assign cases to a more specific diagnostic entity. Identification of novel fusion genes has contributed significantly to improving the classification, biologic understanding, and therapeutic targeting of PTCLs. Here, we integrated mate-pair DNA and RNA next-generation sequencing to identify chromosomal rearrangements encoding expressed fusion transcripts in PTCL, NOS. Two of 11 cases had novel fusions involving VAV1, encoding a truncated form of the VAV1 guanine nucleotide exchange factor important in T-cell receptor signaling. Fluorescence in situ hybridization studies identified VAV1 rearrangements in 10 of 148 PTCLs (7%). These were observed exclusively in PTCL, NOS (11%) and anaplastic large cell lymphoma (11%). In vitro, ectopic expression of a VAV1 fusion promoted cell growth and migration in a RAC1-dependent manner. This growth was inhibited by azathioprine, a clinically available RAC1 inhibitor. We also identified novel kinase gene fusions, ITK-FER and IKZF2-ERBB4, as candidate therapeutic targets that show similarities to known recurrent oncogenic ITK-SYK fusions and ERBB4 transcript variants in PTCLs, respectively. Additional novel and potentially clinically relevant fusions also were discovered. Together, these findings identify VAV1 fusions as recurrent and targetable events in PTCLs and highlight the potential for clinical sequencing to guide individualized therapy approaches for this group of aggressive malignancies.

Abstract We aimed to investigate the mechanistic, functional, and therapeutic role of glycogen synthase kinase 3β (GSK-3β) in the regulation and activation of the proinflammatory oncogenic transcription factor nuclear factor of activated T cells (NFATc2) in pancreatic cancer. IHC, qPCR, immunoblotting, immunofluorescence microscopy, and proliferation assays were used to analyze mouse and human tissues and cell lines. Protein–protein interactions and promoter regulation were analyzed by coimmunoprecipitation, DNA pulldown, reporter, and ChIP assays. Preclinical assays were performed using a variety of pancreatic cancer cells lines, xenografts, and a genetically engineered mouse model (GEMM). GSK-3β–dependent SP2 phosphorylation mediates NFATc2 protein stability in the nucleus of pancreatic cancer cells stimulating pancreatic cancer growth. In addition to protein stabilization, GSK-3β also maintains NFATc2 activation through a distinct mechanism involving stabilization of NFATc2–STAT3 complexes independent of SP2 phosphorylation. For NFATc2–STAT3 complex formation, GSK-3β–mediated phosphorylation of STAT3 at Y705 is required to stimulate euchromatin formation of NFAT target promoters, such as cyclin-dependent kinase-6, which promotes tumor growth. Finally, preclinical experiments suggest that targeting the NFATc2–STAT3–GSK-3β module inhibits proliferation and tumor growth and interferes with inflammation-induced pancreatic cancer progression in KrasG12D mice. In conclusion, we describe a novel mechanism by which GSK-3β fine-tunes NFATc2 and STAT3 transcriptional networks to integrate upstream signaling events that govern pancreatic cancer progression and growth. Furthermore, the therapeutic potential of GSK-3β is demonstrated for the first time in a relevant Kras and inflammation-induced GEMM for pancreatic cancer. Mol Cancer Ther; 15(3); 491–502. ©2016 AACR.

Abstract Purpose: Recent studies suggest that SIRT1-activating compounds (STAC) are a promising class of anticancer drugs, although their mechanism of action remains elusive. The main goal of this study is to determine the role of STACs as a potential therapy for pancreatic cancer. In addition, we also explored the mechanism by which these compounds affect pancreatic cancer. Experimental design: Using in vitro (cell culture experiments) and in vivo (xenograft experiments) approaches, we studied the role of SIRT1 agonists (STAC) in human pancreatic cancer cell viability and growth. Results: We show that SIRT1 is highly expressed in pancreatic cancer cells and that the STACs SRT1720, SRT1460, and SRT3025 inhibited cell growth and survival of pancreatic cancer cells. STACs enhanced the sensitivity of pancreatic cells to gemcitabine and paclitaxel, indicating that these drugs could be used in combination with other chemotherapy drugs. We also show that STACs were very effective in inhibiting tumor xenograft growth. In mechanistic studies, we observed that STACs activated a SIRT1 lysosomal–dependent cell death. Furthermore, the effect of STACs on cell viability was also dependent on the expression of the endogenous SIRT1 inhibitor DBC1. Conclusions: Taken together, our results reveal an essential role for SIRT1 and lysosomes in the death pathway regulated by STACs in pancreatic cancer cells. Clin Cancer Res; 22(10); 2496–507. ©2015 AACR.

AbstractThe histone deacetylases (HDACs) occur in 11 different isoforms, and these enzymes regulate the activity of a large number of proteins involved in cancer initiation and progression. The discovery of isoform‐selective HDAC inhibitors (HDACIs) is desirable, as it is likely that such compounds would avoid some of the undesirable side effects found with the first‐generation inhibitors. A series of HDACIs previously reported by us were found to display some selectivity for HDAC6 and to induce cell‐cycle arrest and apoptosis in pancreatic cancer cells. In the present work, we show that structural modification of these isoxazole‐based inhibitors leads to high potency and selectivity for HDAC6 over HDAC1–3 and HDAC10, while unexpectedly abolishing their ability to block cell growth. Three inhibitors with lower HDAC6 selectivity inhibit the growth of cell lines BxPC3 and L3.6pl, and they only induce apoptosis in L3.6pl cells. We conclude that HDAC6 inhibition alone is insufficient for disruption of cell growth, and that some degree of class 1 HDAC inhibition is required. Moreover, the highly selective HDAC6Is reported herein that are weakly cytotoxic may find use in cancer immune system reactivation.