Old Players with a Newly Defined Function: Fra-1 and c-Fos Support Growth of Human Malignant Breast Tumors by Activating Membrane Biogenesis at the Cytoplasm

Ruben D Motrich,Beatriz L Caputto,Gonzalo M Castro,Klaus Roemer

doi:10.1371/journal.pone.0053211

Ruben D Motrich, Beatriz L Caputto + Show 2 more

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https://doi.org/10.1371/journal.pone.0053211

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Abstract

A shared characteristic of tumor cells is their exacerbated growth. Consequently, tumor cells demand high rates of phospholipid synthesis required for membrane biogenesis to support their growth. c-Fos, in addition to its AP-1 transcription factor activity, is the only protein known up to date that is capable of activating lipid synthesis in normal and brain tumor tissue. For this latter activity, c-Fos associates to the endoplasmic reticulum (ER) through its N-terminal domain and activates phospholipid synthesis, an event that requires it Basic Domain (BD) (aa 139–159). Fra-1, another member of the FOS family of proteins, is over-expressed in human breast cancer cells and its BD is highly homologous to that of c-Fos with two conservative substitutions in its basic amino acids. Consequently, herein we examined if Fra-1 and/or c-Fos participate in growth of breast cancer cells by activating phospholipid synthesis as found previously for c-Fos in brain tumors. We found both Fra-1 and c-Fos over-expressed in >95% of human ductal breast carcinoma biopsies examined contrasting with the very low or undetectable levels in normal tissue. Furthermore, both proteins associate to the ER and activate phospholipid synthesis in cultured MCF7 and MDA-MB231 breast cancer cells and in human breast cancer samples. Stripping tumor membranes of Fra-1 and c-Fos prior to assaying their lipid synthesis capacity in vitro results in non-activated lipid synthesis levels that are restored to their initial activated state by addition of Fra-1 and/or c-Fos to the assays. In MDA-MB231 cells primed to proliferate, blocking Fra-1 and c-Fos with neutralizing antibodies blocks lipid-synthesis activation and cells do not proliferate. Taken together, these results disclose the cytoplasmic activity of Fra-1 and c-Fos as potential targets for controlling growth of breast carcinomas by decreasing the rate of membrane biogenesis required for growth.

Highlights

The fos and jun oncogenes are members of the family of Immediate Early Genes (IEGs) AP-1 transcription factors that are rapidly and transiently expressed in different cell types in response to a myriad of stimuli, such as growth factors, neurotransmitters, etc. [1,2,3]
Upon induction of cells to re-enter growth by feeding with foetal bovine serum (FBS) (+FBS), a marked up-regulation of Fra-1 and c-Fos expression was observed in both MDAMB231 (A) and MCF7 (B) cells
Both MDA-MB231 (Figure 1C, left panel) and MCF7 cells (Figure 1C, right panel) clearly showed higher Fra-1 and c-Fos immuno-reactivity in total homogenate (TH) from growing cells (+FBS) as compared to quiescent (–FBS) cells and both proteins were recovered in the MF

Summary

Introduction

The fos and jun oncogenes are members of the family of Immediate Early Genes (IEGs) AP-1 transcription factors that are rapidly and transiently expressed in different cell types in response to a myriad of stimuli, such as growth factors, neurotransmitters, etc. [1,2,3]. We have shown that in addition to its nuclear AP-1 activity, c-Fos associates to the endoplasmic reticulum (ER) and activates phospholipid synthesis as an additional response to mitogenic stimuli [6]. This cytoplasmic activity of c-Fos has been observed in vivo in light-stimulated retina ganglion and photoreceptor cells [6,7,8,9], in culture in NIH3T3 fibroblasts induced to reenter growth [10], in PC12 cells induced to differentiate [11,12], in actively growing and proliferating T98G glioblastoma multiforme-derived cells [13,14], and in human and mouse tumors from the Peripheral and Central Nervous Systems [15,16]. The mechanism by which c-Fos associates to the ER and activates phospholipid biosynthesis is currently not fully elucidated, it is known that c-Fos physically associates with specific, key enzymes of the pathway of phospholipid synthesis in the ER [17]. c-Fos/ER association is regulated by the phosphorylation state of c-Fostyrosine residues #10 and #30 whereas its activation capacity depends on its BD (20 amino acids spanning from 139–159) [13,14,17]

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