Publication Date

2014-08-01

Availability

Embargoed

Embargo Period

2016-08-02

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Biochemistry and Molecular Biology (Medicine)

Date of Defense

2014-05-28

First Committee Member

Ralf Landgraf

Second Committee Member

Zafar Nawaz

Third Committee Member

Antonio Barrientos

Fourth Committee Member

Murray Deutscher

Fifth Committee Member

Bal Lokeshwar

Abstract

Dysregulation of ERBBs signaling has been known to be responsible for several types of tumors including breast, ovary, lung, pancreas, prostate and more. Functions of ERBB receptors are mainly known from its overexpression in cancers. Apart from elevated levels of ERBB receptors, low levels of ERBB signaling also play a crucial role in normal development and maintenance of mammalian organs. However, the role of signaling by the low “normal” levels of ERBB receptors is poorly understood. Altered ERBB signaling has been implicated with several cardiac defects. One of the severe side effects of ERBB2-targeted therapy in breast cancer patients is cardiac toxicity. Studies suggest that the inhibition of ERBB signaling causes mitochondrial defects such as uncoupling, metabolic reprogramming and altered ROS levels. Hitherto the role of ERBB signaling in regulating mitochondrial activity and metabolic reprograming has remained largely unknown. We carried out a 2-D difference gel electrophoresis based proteomics study (DIGE) of early post-translational protein modifications in MCF7 breast cancer cells in response to ligand induced ERBB2/ERBB3 (or ERBB2/3) activation. The aim of our study was to evaluate the cellular changes due to the normal ERBB2/3 immediate signaling response. This study identified heterogeneous nuclear ribonucleoprotein K (HNRNPK) as a target for rapid tyrosine phosphorylation after ERBB2/3 activation by neuregulin β1. Upregulation of HNRNPK protein coupled with its cytoplasmic accumulation has been found to be associated with its oncogenic role in several types of cancer. Previous studies have shown that HNRNPK levels are reduced in breast cancer cells after prolonged treatment with EGFR or ERBB2 directed therapeutic antibodies. However, while molecular mechanistic data exist on HNRNPK from a variety of model systems, we currently lack a coherent mechanistic framework of the crosstalk between ERBB2 and HNRNPK signaling in breast cancer. Following our DIGE analysis, we found that ligand induced and ERBB2/3 mediated tyrosine phosphorylation of HNRNPK in MCF7 cells is carried out by the members of the SRC kinase family. Following ligand dependent stimulation, activation of HNRNPK coincides with a cytoplasmic relocalization of a relatively small portion of the predominantly nuclear HNRNPK pool. This part of my studies aimed at elucidating the interplay of HNRNPK and constitutive ERBB2 signaling in breast cancer cells and its role in cellular transformation. HNRNPK is involved in several processes such as chromatin remodeling, transcription, translational control and many more. HNRNPK is an mRNA shuttling protein and the founding member of KH-domain containing protein family. The three KH domains of HNRNPK are involved in the binding to mRNA targets. HNRNPK has been shown to enhance, by direct binding, the translation of crucial mRNAs such as SRC, MYC and UCP2. The post-translational modifications of HNRNPK have been shown to regulate its function of binding to mRNA and modulating translation. However, the role of ERBB2/3 signaling mediated post-translational modifications of HNRNPK in regulating the translation of mRNA targets is largely unknown. Also the individual and combinatorial contribution of each KH domain of HNRNPK in identification and binding of RNA target is poorly understood. To investigate the spectrum of RNA targets of HNRNPK, we utilized different modifications (mediated by ERBB2/3 signaling) and truncations of HNRNPK to isolate RNA (in vivo and in vitro) followed by Deep (RNA-seq) sequencing. Analysis of RNA-seq data revealed that we were able to pull down a significant amount of RNA targets from all experimental sets, and ERBB2/3 mediated phosphorylation of HNRNPK reduces the binding of HNRNPK to its RNA targets. But the regulatory mechanism involved in the regulation of different mRNA by ERBB2/3 signaling is not known. Among the different RNA targets, we found a significant number of RNAs associated with mitochondrial functions. One of the crucial targets we obtained was UCP2. UCP2, uncoupling protein 2, belongs to the family of mitochondrial uncoupling proteins. UCP2 is known to uncouple the mitochondrial electrochemical gradient from ATP production through a leakage of protons, has been associated with several disease conditions including several types of tumors. Overexpression of UCP2 is capable of creating mitochondrial uncoupling, and it is frequently interpreted as a compensatory response to oxidative stress. Elevated levels of UCP2 have been proposed to alter the usage of carbon source and hence it is also referred as “glucose sparing switch”. In most tissues, the short-lived UCP2 protein is only present at very low levels, but it has been genetically linked to obesity, type II diabetes and cardiac risk. However, the forces that drive changes in the UCP2 expression in cancers as well as its “normal” physiological function remain unclear. In our studies we found that the activation of ERBB2/3 signaling by neuregulin β1 immediately downregulates UCP2 through MAPK signaling. Also the neuregulin β1 dependent ERBB2/3 acute signaling decreases the efficiency of mitochondria by reducing mitochondrial coupling and lowering mitochondrial membrane potential. Normal cells use glucose metabolism and oxidative phosphorylation to meet their energy requirements, whereas cancer cells predominantly use the low energy yielding process of aerobic glycolysis (i.e. glucose to pyruvate) for formation of biosynthetic building blocks. This phenomenon in cancer is known as the Warburg effect in which cancer cells increase the uptake and utilization of glucose by glycolysis even in the presence of oxygen. Our study confirms the role of UCP2 in cancer as a metabolic regulator mainly by providing protection against high glucose. And under the regulation of ERBB signaling, the elevated levels of UCP2 function as a bonafide uncoupler along with encouraging glucose uptake in cancer cells. We show that normal ERBB2/3 signaling can induce mitochondrial changes, and the elevated levels of ERBB2 can utilize already present components to regulate the activity of mitochondria. Our data provide insights into the cellular transformation and associated changes in mitochondrial properties found frequently in ERBB2 overexpressing cancers, as well as the effects of ERBB2 directed therapeutics on non-overexpressing cells. These studies indicate that ERBB receptor signaling plays a very crucial role in regulating the mitochondrial activity and the metabolic state of a cell, as well as fine tuning the metabolic activity of the cell under the influence of microenvironment.

Keywords

ERBB; UCP2; Cancer; Mitochondria; MAPK; OXPHOS

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