Mary G. Johlfs

Protein Kinase G Type Iα Activity in Human Ovarian Cancer Cells Significantly Contributes to Enhanced Src Activation and DNA Synthesis/Cell Proliferation

Previously, we showed that basal activity of nitric oxide (NO)/cyclic GMP (cGMP)/protein kinase G (PKG) signaling pathway protects against spontaneous apoptosis and confers resistance to cisplatin-induced apoptosis in human ovarian cancer cells. The present study determines whether basal PKG kinase activity regulates Src family kinase (SFK) activity and proliferation in these cells. PKG-Iα was identified as predominant isoform in both OV2008 (cisplatin-sensitive, wild-type p53) and A2780cp (cisplatin-resistant, mutated p53) ovarian cancer cells. In both cell lines, ODQ (inhibitor of endogenous NO-induced cGMP biosynthesis), DT-2 (highly specific inhibitor of PKG-Iα kinase activity), and PKG-Iα knockdown (using small interfering RNA) caused concentration-dependent inhibition of DNA synthesis (assessed by bromodeoxyuridine incorporation), indicating an important role of basal cGMP/PKG-Iα kinase activity in promoting cell proliferation. DNA synthesis in OV2008 cells was dependent on SFK activity, determined using highly selective SFK inhibitor, 4-(4′-phenoxyanilino)-6,7-dimethoxyquinazoline (SKI-1). Studies using DT-2 and PKG-Iα small interfering RNA revealed that SFK activity was dependent on PKG-Iα kinase activity. Furthermore, SFK activity contributed to endogenous tyrosine phosphorylation of PKG-Iα in OV2008 and A2780cp cells. In vitro coincubation of recombinant human c-Src and PKG-Iα resulted in c-Src–mediated tyrosine phosphorylation of PKG-Iα and enhanced c-Src autophosphorylation/activation, suggesting that human c-Src directly tyrosine phosphorylates PKG-Iα and the c-Src/PKG-Iα interaction enhances Src kinase activity. Epidermal growth factor–induced stimulation of SFK activity in OV2008 cells increased PKG-Iα kinase activity (indicated by Ser239 phosphorylation of the PKG substrate vasodilator-stimulated phosphoprotein), which was blocked by both SKI-1 and SU6656. The data suggest an important role of Src/PKG-Iα interaction in promoting DNA synthesis/cell proliferation in human ovarian cancer cells. The NO/cGMP/PKG-Iα signaling pathway may provide a novel therapeutic target for disrupting ovarian cancer cell proliferation. Mol Cancer Res; 8(4); 578–91. ©2010 AACR.

Wnt interaction and extracellular release of prominin-1/CD133 in human malignant melanoma cells

Prominin-1 (CD133) is the first identified gene of a novel class of pentaspan membrane glycoproteins. It is expressed by various epithelial and non-epithelial cells, and notably by stem and cancer stem cells. In non-cancerous cells such as neuro-epithelial and hematopoietic stem cells, prominin-1 is selectively concentrated in plasma membrane protrusions, and released into the extracellular milieu in association with small vesicles. Previously, we demonstrated that prominin-1 contributes to melanoma cells pro-metastatic properties and suggested that it may constitute a molecular target to prevent prominin-1-expressing melanomas from colonizing and growing in lymph nodes and distant organs. Here, we report that three distinct pools of prominin-1 co-exist in cultures of human FEMX-I metastatic melanoma. Morphologically, in addition to the plasma membrane localization, prominin-1 is found within the intracellular compartments, (e.g., Golgi apparatus) and in association with extracellular membrane vesicles. The latter prominin-1-positive structures appeared in three sizes (small, ≤40 nm; intermediates ~40-80 nm, and large, >80 nm). Functionally, the down-regulation of prominin-1 in FEMX-I cells resulted in a significant reduction of number of lipid droplets as observed by coherent anti-Stokes Raman scattering image analysis and Oil red O staining, and surprisingly in a decrease in the nuclear localization of beta-catenin, a surrogate marker of Wnt activation. Moreover, the T-cell factor/lymphoid enhancer factor (TCF/LEF) promoter activity was 2 to 4 times higher in parental than in prominin-1-knockdown cells. Collectively, our results point to Wnt signaling and/or release of prominin-1-containing membrane vesicles as mediators of the pro-metastatic activity of prominin-1 in FEMX-I melanoma.

Imaging Immune and Metabolic Cells of Visceral Adipose Tissues with Multimodal Nonlinear Optical Microscopy

Visceral adipose tissue (VAT) inflammation is recognized as a mechanism by which obesity is associated with metabolic diseases. The communication between adipose tissue macrophages (ATMs) and adipocytes is important to understanding the interaction between immunity and energy metabolism and its roles in obesity-induced diseases. Yet visualizing adipocytes and macrophages in complex tissues is challenging to standard imaging methods. Here, we describe the use of a multimodal nonlinear optical (NLO) microscope to characterize the composition of VATs of lean and obese mice including adipocytes, macrophages, and collagen fibrils in a label-free manner. We show that lipid metabolism processes such as lipid droplet formation, lipid droplet microvesiculation, and free fatty acids trafficking can be dynamically monitored in macrophages and adipocytes. With its versatility, NLO microscopy should be a powerful imaging tool to complement molecular characterization of the immunity-metabolism interface.

Nitric Oxide/Protein Kinase G-Iα Promotes c-Src Activation, Proliferation and Chemoresistance in Ovarian Cancer

Ronald R. Fiscus1,2, Elaine L. Leung1,2,3, Janica C. Wong1,2,4 and Mary G. Johlfs1,2 1Center for Diabetes and Obesity Prevention, Treatment, Research and Education, and the College of Pharmacy, Roseman University of Health Sciences (Formerly University of Southern Nevada), Henderson, Nevada, 2Cancer Molecular Biology Section, Nevada Cancer Institute, Las Vegas, Nevada, 3State Key Laboratory for Quality Research in Chinese Medicines, Macau University of Science and Technology, Taipa, Macau Special Administrative Region, and Shum Yiu Foon Shum Bik Chuen Memorial Centre for Cancer and Inflammation Research, School of Chinese Medicine, Hong Kong Baptist University, and Department of Pathology, Faculty of Medicine, The University of Hong Kong, Hong Kong Special Administrative Region, 4Department of Chemistry, University of Nevada Las Vegas, Las Vegas, Nevada, 1,2,4USA 3China

Capillary Isoelectric Focusing Immunoassay for Fat Cell Differentiation Proteomics

Profiling cellular proteome is critical to understanding signal integration during cell fate determination. In this study, the capability of capillary isoelectric focusing (cIEF) immunoassays to detect post-translational modifications (PTM) of protein isoforms is demonstrated. cIEF immunoassays exhibit protein detection sensitivity at up to 5 orders of magnitude higher than traditional methods. This detection ultra-sensitivity permits proteomic profiling of several nanograms of tissue samples. cIEF immunoassays are employed to simultaneously profile three protein kinases during fat cell differentiation: cGMP-dependent protein kinase type I (PKG-I) of the nitric oxide (NO) signaling pathway, protein kinase B (Akt) of the insulin signaling pathway, and extracellular signal-regulated kinase (ERK) of the mitogen-activated protein kinase (MAPK) signaling pathway. Interestingly, a switch in the expression level of PKG- isoforms is observed during fat cell differentiation. While both PKG-Iα and PKG-Iβ isoforms are present in preadipocytes, only PKG-Iβ isoform is expressed in adipocytes. On the other hand, the phosphorylation level increases for Akt while decreases for ERK1 and ERK2 following the maturation of preadipocytes into adipocytes. Taken together, cIEF immunoassay provides a highly sensitive means to study fat cell differentiation proteomics. cIEF immunoassay should be a powerful proteomics tool to study complex protein signal integration in biological systems.

Protein kinase G type-I phosphorylates c-Src at serine-17 and promotes cell survival, proliferation and attachment in human mesothelioma and non-small cell lung cancer cells.

Background Previously, we demonstrated that protein kinase G type-Ia (PKG-Ia) plays an important role in promoting cell survival in neural cells (N1E-115 neuroblastoma and NG10815 neuroblastoma-glioma hybrid cells) and significantly contributes to the serine-155 phosphorylation of BAD, an apoptosis-regulating protein [1]. We also found that PKGIa promotes cell survival and proliferation in mouse OP9 bone marrow stromal cells [2] and human ovarian cancer cells [3] [determined by using both pharmacological inhibitors (ODQ, DT-2 and DT-3) and gene knockdown (siRNA) to reduce PKG-Ia/b activity]. In the case of ovarian cancer cells, which predominantly express the PKG-Ia isoform, the pro-growth and pro-survival effects involved a novel interaction between PKG-Ia and the oncogenic protein c-Src [3]. For example, intracellular activation of PKG-Ia, assessed by VASP serine-239 phosphorylation, was dependent on c-Src-catalyzed tyrosine phosphorylation of PKG-Ia (i.e. VASP phosphorylation was blocked by Src inhibitors, SKI-1 or SU6656) and the intracellular activation of c-Src was (somehow) dependent on the kinase activity and expression levels of PKG-Ia (i.e. c-Src activation was decreased by DT-2 or siRNA-induced knockdown of PKG-Ia).

Abstract B01: Phosphorylation of pro-oncogenic c-Src at serine-17 by protein kinase G-Iα promotes chemoresistance/cell proliferation in human non-small cell lung cancer cells.

We previously showed that PKG-Iα promotes cell survival in neural cells (N1E-115 neuroblastoma and NG108-15 neuroblastoma-glioma hybrid cells) and significantly contributes to serine-155 phosphorylation of BAD, an apoptosis-regulating protein (Johlfs and Fiscus 2010). We also found, by using both gene knockdown and pharmacological inhibitors (ODQ, DT-2 and DT-3) to decrease PKG-Iα kinase activity, that PKG-Iα promotes cell proliferation and chemoresistance in human ovarian cancer cells, involving a novel interaction between PKG-Iα and the oncogenic protein c-Src (Fiscus et al 2012, Leung et al 2010). PKG-Iα activity was dependent on c-Src-catalyzed tyrosine phosphorylation of PKG-Iα; and, reciprocally, c-Src activation was dependent on expression and kinase activity of PKG-Iα;. We hypothesized that activation of c-Src involves PKG-Iα-catalyzed phosphorylation at serine-17 of c-Src, because amino acids around serine-17 provide consensus sequence for PKG-I, and tαhat this plays an important role in proliferation and chemoresistance. As c-Src has been proposed to promote cell proliferation and chemoresistance in NSCLC cells, the present study aimed to: 1) determine if c-Src increases PKG-Iα kinase activity in NSCLC cells using our newly-developed near-infrared-fluorescence (NIRF)-kinase assay, 2) determine if serine-17-phosphorylation of c-Src, in intact cancer cells, is dependent on PKG-Iα expression and kinase activity (using siRNA gene knockdown and pharmacological inhibitors), and 3) determine if inhibition/knockdown of c-Src and PKG-Iα alters chemoresistance and proliferation of NSCLC cells. Protein expression of c-Src and PKG-Iα was determined by conventional Western blot analysis and new ultrasensitive capillary-electrophoresis-based NanoPro100/1000 (ProteinSimple, Santa Clara, CA, USA). NanoPro100/1000 allows clear separation and identification of different c-Src and PKG-Iαphospho-forms (a novel way of identifying the activation of c-Src and PKG-Iα). We found PKG-Iα was able to directly catalyze phosphorylation of serine-17 of c-Src, increasing autophosphorylation of c-Src at tyrosine-419, the activation site of c-Src. Inhibition of PKG-Iα kinase activity using DT-2 or silencing of PKG-Iα expression using siRNA dramatically reduced the intracellular phosphorylation of c-Src at serine-17. Both kinase-activity inhibition and knockdown of PKG-Iα caused significant increases in apoptosis, synergistically enhancing apoptosis induced by the chemotherapeutic agent cisplatin, and dramatically decreased cell proliferation/colony formation in NSCLC cells. This novel reciprocal relationship between c-Src and PKG-Iα provides a new target for the development of new anti-cancer therapeutic agents. Fiscus RR, Leung EL, Wong JC, Johlfs MG (2012). Nitric oxide/protein kinase G-Ialpha promote c-Src activation, proliferation and chemoresistance in ovarian cancer. In: Farghaly S (ed). Ovarian Cancer - Basic Science Perspective. Intech Open Access Publisher. pp 315-334. Johlfs MG, Fiscus RR (2010). Protein kinase G type-Ialpha phosphorylates the apoptosis-regulating protein Bad at serine 155 and protects against apoptosis in N1E-115 cells. Neurochem Int 56: 546-553. Leung EL, Wong JC, Johlfs MG, Tsang BK, Fiscus RR (2010). Protein kinase G type Ialpha activity in human ovarian cancer cells significantly contributes to enhanced Src activation and DNA synthesis/cell proliferation. Mol Cancer Res 8: 578-591. Citation Format: Mary G. Johlfs, Ronald R. Fiscus, Priyatham Gorjala, Renee Coffman, Harry Rosenberg. Phosphorylation of pro-oncogenic c-Src at serine-17 by protein kinase G-Iα promotes chemoresistance/cell proliferation in human non-small cell lung cancer cells. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B01.

Abstract A29: Advanced nano-proteomics based on ultrasensitive CE toquantitate protein expression/phosphorylation levels: Discovery of novel proteins involved in chemoresistance/invasion of lung cancer cells.

Our laboratory has shown that resistance to chemotherapeutic agent cisplatin (chemoresistance) in non-small cell lung cancer (NSCLC) cells involves exaggerated phosphorylation of transcription factor CREB and high-level expression of pro-survival/anti-apoptotic proteins c-IAP1, livin, Mcl-1 and survivin, resulting from hyperactivation of a novel serine/threonine kinase, protein kinase G-Iα (PKG-Iα) (Wong et al. 2012). PKG-Iα also promotes cell proliferation/DNA synthesis and migration/invasion in both NSCLC and ovarian cancer cells (Fiscus et al., 2012; Wong et al. 2012). Because of low expression levels, these proteins are difficult to accurately quantify by conventional Western blotting. To solve this problem, we are developing new applications for NanoPro 100 and NanoPro 1000 (ProteinSimple), state-of-the-art instruments based on advanced “nano-proteomics” ultilizing ultrasensitive capillary electrophoresis (CE)-based technologies (Fiscus and Johlfs, 2012; Fiscus et al., 2012). The NanoPro 100/NanoPro 1000 are capable of accurately quantifying protein expression/phosphorylation levels with sensitivities >100-times and >500-times, respectively, that of conventional Western blots, thus allowing, for the first time, accurate measurements of lower-abundance proteins (e.g. PKG-Iα, survivin and other pro-survival proteins). Extremely small samples of Wong JC, Bathina M and RR Fiscus (2012) Cyclic GMP/protein kinase G type-Iα (PKG-Iα signaling pathway promotes CREB phosphorylation and maintains higher c-IAP1, livin, survivin and Mcl-1 expression and the inhibition of PKG-Iα kinase activity synergizes with cisplatin in non-small cell lung cancer cells. J Cell Biochem. 113: 3587-3598. Fiscus RR, Leung EL, Wong JC and MG Johlfs (2012) Nitric Oxide/Protein Kinase G-IαPromotes c-Src Activation, Proliferation and Chemoresistance in Ovarian Cancer. In: Farghaly, S. (ed). Ovarian Cancer - Basic Science Perspective. Intech Open Access Publisher. pp 315-334. Fiscus RR and MG Johlfs (2012) Protein Kinase G (PKG): Involvement in Promoting Neural Cell Survival, Proliferation, Synaptogenesis and Synaptic Plasticity and the Use of New Ultrasensitive Capillary-Electrophoresis-based Methodologies for Measuring PKG Expression and Molecular Actions. In: Mukai H (ed). Protein Kinase Technologies, in NEUROMETHODS series, Springer. pp 319-348. Citation Format: Ronald R. Fiscus, Mary G. Johlfs, Janica C. Wong, Renee Coffman, Harry Rosenberg. Advanced nano-proteomics based on ultrasensitive CE toquantitate protein expression/phosphorylation levels: Discovery of novel proteins involved in chemoresistance/invasion of lung cancer cells. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr A29.

Abstract 4558: Resveratrol alters the kinase activity of PKG-Iα and Src family in A2780cp cells (ovarian cancer cell line with mutated p53) resulting in growth inhibition

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Epithelial ovarian cancer is a leading cause of death in gynecological cancer patients. One major hurdle in treating ovarian cancer is drug resistance to commonly-used chemotherapy like cisplatin. Resveratrol (3,5,4′-trihydroxy-trans-stilbene), a natural polyphenol found in foods/drinks like grapes, berries, peanuts and red wine, can inhibit tumor growth. Our lab has previously shown that resveratrol down-regulates expression of protein kinase G type-I (PKG-I) in cancer cells, which correlated with decreased cell proliferation and increased apoptosis. For the present study, we used A2780cp cells, which are cisplatin-resistant epithelial ovarian cancer cells with mutated p53. Our data show that resveratrol did not induce apoptosis even at a higher concentration of 500 µM. However, resveratrol caused concentration-dependent decreases (down to 50% of control levels at 100 µM) of both DNA synthesis and cell adhesion. We also observed a concentration-dependent increase in protein expression levels of PKG-I to 3 times of control levels at 50 µM resveratrol, but a drop at 100 µM resveratrol. Though total Src remained fairly unchanged, pSrcY416 (phosphorylation at tyrosine-416) and pSrcS17 (phosphorylation at serine-17), indicators of Src activation, decreased at 50 and 100 µM resveratrol [assessed by ultrasensitive quantitative NanoPro 1000 (ProteinSimple), a new state-of-the-art capillary electrophoresis-based protein analysis system]. Decreased pSrcY416 levels (44% of control levels at 100 µM resveratrol) suggest that the kinase activity of Src family kinases are decreased at the higher concentrations of resveratrol that cause inhibition of ovarian cancer cell proliferation. These results suggest that resveratrol can be effective in decreasing cell proliferation in chemoresistant ovarian cancer cells and that the mechanism of action of resveratrol may involve a down-regulation of the protein expression levels of PKG-I and the typrosine-kinase activity of Src family of kinases. Citation Format: Priyatham Gorjala, Janica C. Wong, Benjamin F.b Constantino, Mary G. Johlfs, Renee Coffman, Harry Rosenberg, Ronald Fiscus. Resveratrol alters the kinase activity of PKG-Iα and Src family in A2780cp cells (ovarian cancer cell line with mutated p53) resulting in growth inhibition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4558. doi:10.1158/1538-7445.AM2014-4558