Inhibition of GSK-3 induces differentiation and impaired glucose metabolism in renal cancer

Glycogen synthase kinase-3 (GSK-3), a constitutively active serine/threonine kinase, is a key regulator of numerous cellular processes ranging from glycogen metabolism to cell cycle regulation and proliferation. Consistent with its involvement in many pathways, it has also been implicated in the pathogenesis of various human diseases including Type II diabetes, Alzheimer's disease, bipolar disorder, inflammation and cancer. Consequently it is recognized as an attractive target for the development of new drugs. In the present study, we investigated the effect of both pharmacological and genetic inhibition of GSK-3 in two different renal cancer cell lines. We have shown potent anti-proliferative activity of 9-ING-41, a maleimide-based GSK-3 inhibitor. The anti-proliferative activity is most likely caused by G0-G1 and G2-M phase arrest as evident from cell cycle analysis. We have established that inhibition of GSK-3 imparted a differentiated phenotype in renal cancer cells. We have also shown that GSK-3 inhibition induced autophagy, likely as a result of imbalanced energy homeostasis caused by impaired glucose metabolism. Additionally, we have demonstrated the antitumor activity of 9-ING-41 in two different subcutaneous xenograft RCC tumor models. To our knowledge, this is the first report describing autophagy induction due to GSK-3 inhibition in renal cancer cells

Adrenomedullin is up-regulated in patients with pancreatic cancer and causes insulin resistance in β cells and mice

New-onset diabetes in patients with pancreatic cancer is likely to be a paraneoplastic phenomenon caused by tumor-secreted products. We aimed to identify the diabetogenic secretory product(s) of pancreatic cancer. Methods: Using microarray analysis, we identified adrenomedullin as a potential mediator of diabetes in patients with pancreatic cancer. Adrenomedullin was up-regulated in pancreatic cancer cell lines, in which supernatants reduced insulin signaling in beta cell lines. We performed quantitative reverse-transcriptase polymerase chain reaction and immunohistochemistry on human pancreatic cancer and healthy pancreatic tissues (controls) to determine expression of adrenomedullin messenger RNA and protein, respectively. We studied the effects of adrenomedullin on insulin secretion by beta cell lines and whole islets from mice and on glucose tolerance in pancreatic xenografts in mice. We measured plasma levels of adrenomedullin in patients with pancreatic cancer, patients with type 2 diabetes mellitus, and individuals with normal fasting glucose levels (controls). Results: Levels of adrenomedullin messenger RNA and protein were increased in human pancreatic cancer samples compared with controls. Adrenomedullin and conditioned media from pancreatic cell lines inhibited glucose-stimulated insulin secretion from beta cell lines and islets isolated from mice; the effects of conditioned media from pancreatic cancer cells were reduced by small hairpin RNA-mediated knockdown of adrenomedullin. Conversely, overexpression of adrenomedullin in mice with pancreatic cancer led to glucose intolerance. Mean plasma levels of adrenomedullin (femtomoles per liter) were higher in patients with pancreatic cancer compared with patients with diabetes or controls. Levels of adrenomedullin were higher in patients with pancreatic cancer who developed diabetes compared those who did not. Conclusions: Adrenomedullin is up-regulated in patients with pancreatic cancer and causes insulin resistance in β cells and mice.Fil: Aggarwal, Gaurav. Mayo Clinic College of Medicine; Estados UnidosFil: Ramachandran, Vijaya. University of Texas Health Science Center at Houston. University of Texas Md Anderson Cancer Center; Estados UnidosFil: Javeed, Naureen. Mayo Clinic College of Medicine; Estados UnidosFil: Arumugam, Thiruvengadam. University of Texas Health Science Center at Houston. University of Texas Md Anderson Cancer Center; Estados UnidosFil: Dutta, Shamit. Mayo Clinic College of Medicine; Estados UnidosFil: Klee, George G.. Mayo Clinic College of Medicine; Estados UnidosFil: Klee, Eric W.. Mayo Clinic College of Medicine; Estados UnidosFil: Smyrk, Thomas C.. Mayo Clinic College of Medicine; Estados UnidosFil: Bamlet, William. Mayo Clinic College of Medicine; Estados UnidosFil: Han, Jing Jing. Mayo Clinic College of Medicine; Estados UnidosFil: Rumie Vittar, Natalia Belen. Mayo Clinic College of Medicine; Estados Unidos. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Biología Molecular. Sección Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: De Andrade, Mariza. Mayo Clinic College of Medicine; Estados UnidosFil: Mukhopadhyay, Debabrata. Mayo Clinic College of Medicine; Estados UnidosFil: Petersen, Gloria M.. Mayo Clinic College of Medicine; Estados UnidosFil: Fernandez Zapico, Martin Ernesto. Mayo Clinic College of Medicine; Estados UnidosFil: Logsdon, Craig D.. University of Texas Health Science Center at Houston. University of Texas Md Anderson Cancer Center; Estados UnidosFil: Chari, Suresh T.. Mayo Clinic College of Medicine; Estados Unido

Role of PLEXIND1/TGFβ signaling axis in pancreatic ductal adenocarcinoma progression correlates with the mutational status of KRAS

PLEXIND1 is upregulated in several cancers, including pancreatic ductal adenocarcinoma (PDAC). It is an established mediator of semaphorin signaling, and neuropilins are its known coreceptors. Herein, we report data to support the proposal that PLEXIND1 acts as a transforming growth factor beta (TGFβ) coreceptor, modulating cell growth through SMAD3 signaling. Our findings demonstrate that PLEXIND1 plays a pro-tumorigenic role in PDAC cells with oncogenic KRAS (KRASmut). We show in KRASmut PDAC cell lines (PANC-1, AsPC-1,4535) PLEXIND1 downregulation results in decreased cell viability (in vitro) and reduced tumor growth (in vivo). Conversely, PLEXIND1 acts as a tumor suppressor in the PDAC cell line (BxPC-3) with wild-type KRAS (KRASwt), as its reduced expression results in higher cell viability (in-vitro) and tumor growth (in vivo). Additionally, we demonstrate that PLEXIND1-mediated interactions can be selectively disrupted using a peptide based on its C-terminal sequence (a PDZ domain-binding motif), an outcome that may possess significant therapeutic implications. To our knowledge, this is the first report showing that (1) PLEXIND1 acts as a TGFβ coreceptor and mediates SMAD3 signaling, and (2) differential roles of PLEXIND1 in PDAC cell lines correlate with KRASmut and KRASwt status

The crosstalk between neuropilin-1 and tumor necrosis factor-α in endothelial cells

Tumor necrosis factor-α (TNFα) is a master cytokine which induces expression of chemokines and adhesion molecules, such as intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), in endothelial cells to initiate the vascular inflammatory response. In this study, we identified neuropilin-1 (NRP1), a co-receptor of several structurally diverse ligands, as a modulator of TNFα-induced inflammatory response of endothelial cells. NRP1 shRNA expression suppressed TNFα-stimulated leukocyte adhesion and expression of ICAM-1 and VCAM-1 in human umbilical vein endothelial cells (HUVECs). Likewise, it reduced TNFα-induced phosphorylation of MAPK p38 but did not significantly affect other TNF-induced signaling pathways, such as the classical NFκB and the AKT pathway. Immunofluorescent staining demonstrated co-localization of NRP1 with the two receptors of TNF, TNFR1 and TNFR2. Co-immunoprecipitation further confirmed that NRP1 was in the same protein complex or membrane compartment as TNFR1 and TNFR2, respectively. Modulation of NRP1 expression, however, neither affected TNFR levels in the cell membrane nor the receptor binding affinities of TNFα. Although a direct interface between NRP1 and TNFα/TNFR1 appeared possible from a protein docking model, a direct interaction was not supported by binding assays in cell-free microplates and cultured cells. Furthermore, TNFα was shown to downregulate NRP1 in a time-dependent manner through TNFR1-NFκB pathway in HUVECs. Taken together, our study reveals a novel reciprocal crosstalk between NRP1 and TNFα in vascular endothelial cells

Designing Nanoconjugates to Effectively Target Pancreatic Cancer Cells In Vitro and In Vivo

Pancreatic cancer is the fourth leading cause of cancer related deaths in America. Monoclonal antibodies are a viable treatment option for inhibiting cancer growth. Tumor specific drug delivery could be achieved utilizing these monoclonal antibodies as targeting agents. This type of designer therapeutic is evolving and with the use of gold nanoparticles it is a promising approach to selectively deliver chemotherapeutics to malignant cells. Gold nanoparticles (GNPs) are showing extreme promise in current medicinal research. GNPs have been shown to non-invasively kill tumor cells by hyperthermia using radiofrequency. They have also been implemented as early detection agents due to their unique X-ray contrast properties; success was revealed with clear delineation of blood capillaries in a preclinical model by CT (computer tomography). The fundamental parameters for intelligent design of nanoconjugates are on the forefront. The goal of this study is to define the necessary design parameters to successfully target pancreatic cancer cells.The nanoconjugates described in this study were characterized with various physico-chemical techniques. We demonstrate that the number of cetuximab molecules (targeting agent) on a GNP, the hydrodynamic size of the nanoconjugates, available reactive surface area and the ability of the nanoconjugates to sequester EGFR (epidermal growth factor receptor), all play critical roles in effectively targeting tumor cells in vitro and in vivo in an orthotopic model of pancreatic cancer.Our results suggest the specific targeting of tumor cells depends on a number of crucial components 1) targeting agent to nanoparticle ratio 2) availability of reactive surface area on the nanoparticle 3) ability of the nanoconjugate to bind the target and 4) hydrodynamic diameter of the nanoconjugate. We believe this study will help define the design parameters for formulating better strategies for specifically targeting tumors with nanoparticle conjugates

Neuropilin-1 Modulates p53/Caspases Axis to Promote Endothelial Cell Survival

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), one of the crucial pro-angiogenic factors, functions as a potent inhibitor of endothelial cell (EC) apoptosis. Previous progress has been made towards delineating the VPF/VEGF survival signaling downstream of the activation of VEGFR-2. Here, we seek to define the function of NRP-1 in VPF/VEGF-induced survival signaling in EC and to elucidate the concomitant molecular signaling events that are pivotal for our understanding of the signaling of VPF/VEGF. Utilizing two different in vitro cell culture systems and an in vivo zebrafish model, we demonstrate that NRP-1 mediates VPF/VEGF-induced EC survival independent of VEGFR-2. Furthermore, we show here a novel mechanism for NRP-1-specific control of the anti-apoptotic pathway in EC through involvement of the NRP-1-interacting protein (NIP/GIPC) in the activation of PI-3K/Akt and subsequent inactivation of p53 pathways and FoxOs, as well as activation of p21. This study, by elucidating the mechanisms that govern VPF/VEGF-induced EC survival signaling via NRP-1, contributes to a better understanding of molecular mechanisms of cardiovascular development and disease and widens the possibilities for better therapeutic targets

Dopamine regulates phosphorylation of VEGF receptor 2 by engaging Src-homology-2-domain-containing protein tyrosine phosphatase 2

Vascular endothelial growth factor (VEGF)-induced receptor phosphorylation is the crucial step for initiating downstream signaling pathways that lead to angiogenesis or related pathophysiological outcomes. Our previous studies have shown that the neurotransmitter dopamine could inhibit VEGF-induced phosphorylation of VEGF receptor 2 (VEGFR-2), endothelial cell proliferation, migration, microvascular permeability, and thus, angiogenesis. In this study, we address the mechanism by which VEGFR-2 phosphorylation is regulated by dopamine. Here, we demonstrate that D2 dopamine receptor (D2DR) colocalizes with VEGFR-2 at the cell surface. Dopamine pretreatment increases the translocation and colocalization of Src-homology-2-domain-containing protein tyrosine phosphatase (SHP-2) with D2DR at the cell surface. Dopamine administration leads to increased VEGF-induced phosphorylation of SHP-2 and this increased phosphorylation parallels the increased phosphatase activity of SHP-2. Active SHP-2 then dephosphorylates VEGFR-2 at Y951, Y996 and Y1059, but not Y1175. We also observe that SHP-2 knockdown impairs the dopamine-regulated inhibition of VEGF-induced phosphorylation of VEGFR-2 and, subsequently, Src phosphorylation and migration. Our data establish a novel role for SHP-2 phosphatase in the dopamine-mediated regulation of VEGFR-2 phosphorylation

Genetic status of KRAS modulates the role of Neuropilin-1 in tumorigenesis

Abstract Neuropilin-1 (NRP1), a non–tyrosine kinase receptor, is overexpressed in many cancers including pancreatic and lung cancers. Inhibition of NRP1 expression, however, has differing pro-tumor vs. anti-tumor effects, depending on the cancer types. To understand the differential role of NRP1 in tumorigenesis process, we utilized cells from two different cancer types, pancreatic and lung, each containing either wild type KRAS (KRAS wt) or mutant KRAS (KRAS mt). Inhibition of NRP1 expression by shRNA in both pancreatic and lung cancer cells containing dominant active KRAS mt caused increased cell viability and tumor growth. On the contrary, inhibition of NRP1, in the tumor cells containing KRAS wt showed decreased tumor growth. Importantly, concurrent inhibition of KRAS mt and NRP1 in the tumor cells reverses the increased viability and leads to tumor inhibition. We found that NRP1 shRNA expressing KRAS mt tumor cells caused increased cell viability by decreasing SMAD2 phosphorylation. Our findings demonstrate that the effects of NRP1 knockdown in cancer cells are dependent on the genetic status of KRAS