The Bca2 And Ampk Paradigm: Unraveling The Cancer Connection

THE BCA2 & AMPK PARADIGM: UNRAVELING THE CANCER CONNECTION By DANIELA (BUAC) VENTRO December 2013 Advisor: Dr. Q Ping Dou and Angelika M. Burger (deceased) Major: Cancer Biology Degree: Doctor of Philosophy Adenosine monophosphate-activated kinase (AMPK), a master regulator of cellular energy homeostasis, has emerged as a promising molecular target in the prevention of breast cancer, and phase II and III clinical trials using the FDA-approved, AMPK activating, anti-diabetic drug metformin are promising in this regard, but the question of why metformin is protective for some women but not others still remains. Breast Cancer Associated Gene 2 (BCA2/Rabring7/RNF115), a novel RING-finger ubiquitin E3 ligase, is overexpressed in \u3e50% of breast tumors. Herein, I hypothesized that BCA2 is an endogenous inhibitor of AMPK activation in breast cancer cells and that BCA2 inhibition would therefore increase the efficacy of metformin. My hypothesis is strongly supported by the finding that BCA2 overexpression inhibited both basal and inducible Thr172 phosphorylation/activation of AMPK while BCA2-specific siRNA enhanced pAMPK. Furthermore, the AMPK-suppressive function of BCA2 requires its E3 ligase-specific RING domain, suggesting that BCA2 targets a critical protein controlling (de)phosphorylation of pAMPKfor degradation. A large scale proteomics analysis has revealed that PP2A and ITPR3 may be promising leads in this regard and should be further investigated in relation to AMPK activation by specifically CaMKK. Activation of AMPK by metformin not only triggered growth a inhibitory signal, but also increased BCA2 protein (but not mRNA) levels, which correlated positively with activation of AKT and could be curbed by an AMPK inhibitor, suggesting a potential feedback mechanism from AMPK to pAkt to BCA2. Finally, BCA2 siRNA, or inhibition of its upstream stabilizing kinase AKT, increased the growth-inhibitory effect of metformin in multiple breast cancer cell lines, supporting the conclusion that BCA2 weakens metformin\u27s efficacy in breast cancer cells. My data therefore suggests that metformin in combination with a BCA2 inhibitor may be a more effective breast cancer treatment strategy than metformin alone. On this account, a first generation of specific BCA2 inhibitors, as well as DSF inhibitors, is in existence and all compounds have been evaluated for their cancer cell anti-proliferative effects and should be further tested in combination with metformin. The studies performed in this dissertation provide new grounds for the development of BCA2 as a novel anti-cancer drug target, and in addition, provide awareness for the potential limitations metformin use in the clinic may have

N-cadherin prevents the premature differentiation of anterior heart field progenitors in the pharyngeal mesodermal microenvironment

The cardiac progenitor cells (CPCs) in the anterior heart field (AHF) are located in the pharyngeal mesoderm (PM), where they expand, migrate and eventually differentiate into major cell types found in the heart, including cardiomyocytes. The mechanisms by which these progenitors are able to expand within the PM microenvironment without premature differentiation remain largely unknown. Through in silico data mining, genetic loss-of-function studies, and in vivo genetic rescue studies, we identified N-cadherin and interaction with canonical Wnt signals as a critical component of the microenvironment that facilitates the expansion of AHF-CPCs in the PM. CPCs in N-cadherin mutant embryos were observed to be less proliferative and undergo premature differentiation in the PM. Notably, the phenotype of N-cadherin deficiency could be partially rescued by activating Wnt signaling, suggesting a delicate functional interaction between the adhesion role of N-cadherin and Wnt signaling in the early PM microenvironment. This study suggests a new mechanism for the early renewal of AHF progenitors where N-cadherin provides additional adhesion for progenitor cells in the PM, thereby allowing Wnt paracrine signals to expand the cells without premature differentiation

Link between Adverse Childhood Experiences and Five Factor Model Traits among Filipinos

The relationship between adverse childhood experiences (ACEs) and personality pathology is a growing area of research. Problems with the categorical model of personality disorders have led researchers to explore the relationship between dimensional models of personality and ACEs. Seven hundred seventeen Filipinos, aged between 18 and 87, completed the ACE-IQ and NEO-FFI-3. Results revealed all Five Factor Model (FFM) traits were influenced by ACEs. In general, ACEs increased neuroticism (decreased emotional stability), decreased extraversion (increased introversion), decreased agreeableness (increased antagonism), and decreased conscientiousness (increased disinhibition). For openness, however, the relationship was complex. Some ACEs were positively correlated with openness, while others were negatively correlated, leading to no significant correlation between openness and total ACE-IQ score. ACEs thus affect the total personality, including openness. Understanding the relationship between ACEs and personality pathology, however, may involve going beyond the ACE-IQ total score in order to examine the influence of particular ACEs. In our study, 12 of 13 ACE categories were significantly correlated with at least one FFM trait, the exception being community violence

Investigation of the key chemical structures involved in the anticancer activity of disulfiram in A549 non-small cell lung cancer cell line

© 2018 The Author(s). Background: Disulfiram (DS), an antialcoholism medicine, demonstrated strong anticancer activity in the laboratory but did not show promising results in clinical trials. The anticancer activity of DS is copper dependent. The reaction of DS and copper generates reactive oxygen species (ROS). After oral administration in the clinic, DS is enriched and quickly metabolised in the liver. The associated change of chemical structure may make the metabolites of DS lose its copper-chelating ability and disable their anticancer activity. The anticancer chemical structure of DS is still largely unknown. Elucidation of the relationship between the key chemical structure of DS and its anticancer activity will enable us to modify DS and speed its translation into cancer therapeutics. Methods: The cytotoxicity, extracellular ROS activity, apoptotic effect of DS, DDC and their analogues on cancer cells and cancer stem cells were examined in vitro by MTT assay, western blot, extracellular ROS assay and sphere-reforming assay. Results: Intact thiol groups are essential for the in vitro cytotoxicity of DS. S-methylated diethyldithiocarbamate (S-Me-DDC), one of the major metabolites of DS in liver, completely lost its in vitro anticancer activity. In vitro cytotoxicity of DS was also abolished when its thiuram structure was destroyed. In contrast, modification of the ethyl groups in DS had no significant influence on its anticancer activity. Conclusions: The thiol groups and thiuram structure are indispensable for the anticancer activity of DS. The liver enrichment and metabolism may be the major obstruction for application of DS in cancer treatment. A delivery system to protect the thiol groups and development of novel soluble copper-DDC compound may pave the path for translation of DS into cancer therapeutics.This work was supported by grant from British Lung Foundation (RG14–8) and Innovate UK (104022).Published versio

Endothelin-1 supports clonal derivation and expansion of cardiovascular progenitors derived from human embryonic stem cells

Coronary arteriogenesis is a central step in cardiogenesis, requiring coordinated generation and integration of endothelial cell and vascular smooth muscle cells. At present, it is unclear whether the cell fate programme of cardiac progenitors to generate complex muscular or vascular structures is entirely cell autonomous. Here we demonstrate the intrinsic ability of vascular progenitors to develop and self-organize into cardiac tissues by clonally isolating and expanding second heart field cardiovascular progenitors using WNT3A and endothelin-1 (EDN1) human recombinant proteins. Progenitor clones undergo long-term expansion and differentiate primarily into endothelial and smooth muscle cell lineages in vitro, and contribute extensively to coronary-like vessels in vivo, forming a functional human–mouse chimeric circulatory system. Our study identifies EDN1 as a key factor towards the generation and clonal derivation of ISL1+ vascular intermediates, and demonstrates the intrinsic cell-autonomous nature of these progenitors to differentiate and self-organize into functional vasculatures in vivo

A dual role for Sox10 in the maintenance of the postnatal melanocyte lineage and the differentiation of melanocyte stem cell progenitors

During embryogenesis, the transcription factor, Sox10, drives the survival and differentiation of the melanocyte lineage. However, the role that Sox10 plays in postnatal melanocytes is not established. We show in vivo that melanocyte stem cells (McSCs) and more differentiated melanocytes express SOX10 but that McSCs remain undifferentiated. Sox10 knockout (Sox10(fl); Tg(Tyr::CreER)) results in loss of both McSCs and differentiated melanocytes, while overexpression of Sox10 (Tg(DctSox10)) causes premature differentiation and loss of McSCs, leading to hair graying. This suggests that levels of SOX10 are key to normal McSC function and Sox10 must be downregulated for McSC establishment and maintenance. We examined whether the mechanism of Tg(DctSox10) hair graying is through increased expression of Mitf, a target of SOX10, by asking if haploinsufficiency for Mitf (Mitf(vga9) ) can rescue hair graying in Tg(DctSox10) animals. Surprisingly, Mitf(vga9) does not mitigate but exacerbates Tg(DctSox10) hair graying suggesting that MITF participates in the negative regulation of Sox10 in McSCs. These observations demonstrate that while SOX10 is necessary to maintain the postnatal melanocyte lineage it is simultaneously prevented from driving differentiation in the McSCs. This data illustrates how tissue-specific stem cells can arise from lineage-specified precursors through the regulation of the very transcription factors important in defining that lineage

Regulation of Metformin Response by Breast Cancer Associated Gene 2

Adenosine monophosphate-activated protein kinase (AMPK), a master regulator of cellular energy homeostasis, has emerged as a promising molecular target in the prevention of breast cancer. Clinical trials using the United States Food and Drug Administration (FDA)-approved, AMPK-activating, antidiabetic drug metformin are promising in this regard, but the question of why metformin is protective for some women but not others still remains. Breast cancer associated gene 2 (BCA2/Rabring7/RNF115), a novel Really Interesting New Gene (RING) finger ubiquitin E3 ligase, is overexpressed in >50% of breast tumors. Herein, we report that BCA2 is an endogenous inhibitor of AMPK activation in breast cancer cells and that BCA2 inhibition increases the efficacy of metformin. BCA2 overexpression inhibited both basal and inducible Thr172 phosphorylation/activation of AMPKα1, while BCA2-specific small interfering RNA (siRNA) enhanced phosphorylated AMPKα1 (pAMPKα1). The AMPK-suppressive function of BCA2 requires its E3 ligase-specific RING domain, suggesting that BCA2 targets some protein controlling (de)phosphorylation of AMPKα1 for degradation. Activation of AMPK by metformin triggered a growth inhibitory signal but also increased BCA2 protein levels, which correlated with AKT activation and could be curbed by an AMPK inhibitor, suggesting a potential feedback mechanism from pAMPKα1 to pAkt to BCA2. Finally, BCA2 siRNA, or inhibition of its upstream stabilizing kinase AKT, increased the growth inhibitory effect of metformin in multiple breast cancer cell lines, supporting the conclusion that BCA2 weakens metformin's efficacy. Our data suggest that metformin in combination with a BCA2 inhibitor may be a more effective breast cancer treatment strategy than metformin alone