CPEB2 in mammary gland homeostasis and breast cancer

Abstract

The mammary gland develops postnatally and is remodeled, during each estrous cycle and pregnancy, through very dynamic expansions and involutions of its polarized epithelial tree. Moreover, the mammary gland is hierarchically organized, where the two main epithelial populations may arise from a common bipotent mammary stem cell (MaSC). The principal epithelial cell types in the mammary gland are luminal cells and myoepithelial cells (also named basal cells), which are found surrounding luminal cells and in contact with the extracellular matrix. Further, the luminal compartment has two main lineages: the alveolar lineage in charge of producing milk during lactation, and the ductal lineage that include the cells that express receptors of the ovarian hormones like estrogen and progesterone. Interestingly, each of these luminal lineages is sustained by their own progenitors. Thus, the mammary gland requires a tightly regulated balance between proliferation and differentiation, which is disrupted in breast tumors. Distinct transcriptional circuits orchestrating such finely tuned processes have been described; however, how translational control contributes to mammary gland homeostasis and tumorigenesis remains largely unexplored.

The CPEB-family (Cytoplasmic Polyadenylation Element Binding Proteins) of RNA- binding proteins (RBPs) is composed of four members in vertebrates (CPEB1-4); differentially, in Drosophila Melanogaster, there are two CPEB orthologs (Orb and Orb2). Within the CPEB-family, there are two subfamilies based on sequence identity of the RNA recognition motifs: CPEB1 and CPEB2-4. CPEB proteins share the same RNA- binding C-terminal domain (CTD), while they differ on their unstructured regulatory N- terminal domain (NTD). This fact has two significant implications: CPEBs can bind overlapping target mRNAs by recognizing CPEs in the 3’UTR; however, they are regulated by different post-transcriptional modifications at the NTD that will be triggered by distinct signaling pathways. Therefore, CPEB proteins could compete against one another for the binding, act sequentially on the same target mRNA or even

compensate the loss of one member of the CPEB-family. CPEBs regulated cytoplasmic polyadenylation by recognizing a motif called Cytoplasmic Polyadenylation Element (CPE) present in the 3’UTR of certain mRNAs. Remarkably, CPEBs can potentially regulate up to 25% of the genome and they can control translation of CPEB-bound mRNAs in time and subcellular space. Furthermore, CPEB proteins play pivotal roles in cell proliferation and lineage-specification. Therefore, the study of the CPEBs in a hierarchically organized tissue undergoing a lot of proliferation and remodeling like the mammary epithelia may shed light on novel functions of this family of RBPs. The main goal of this study is to analyze in vivo the contribution of the four members of the CPEB-family of RNA-binding proteins in mammary epithelial morphodynamics.

Here we present a systematic study of the four members of the CPEB family (CPEB1-4) in the context of the adult mammary gland in vivo, using Knock-out (KO) models for all four CPEBs. By mammary wholemounts and flow cytometry analysis, we discovered that the lack of CPEB2 resulted in defects in mammary gland branching and lineage specification. Interestingly, CPEB2 depletion also had consequences for breast tumorigenesis. Moreover, were able to identify the target mRNAs bound by CPEB2 in mammary epithelial cells and to establish a molecular mechanism by which CPEB2 regulates mammary gland homeostasis and breast cancer.

Altogether, this work unravels a novel translational mechanism regulating cell fate in the mammary gland and breast tumor development.

La glándula mamaria es el único órgano que se desarrolla principalmente después del nacimiento y, además, se remodela durante cada ciclo menstrual y embarazo a través de expansiones e involuciones muy dinámicas del epitelio polarizado que la constituye. Por lo tanto, la glándula mamaria requiere un equilibrio finamente regulado entre proliferación y diferenciación. Este equilibro se encuentra perturbado en casos de cáncer de mama. De qué manera el control tradicional contribuye a la homeostasis y tumorigenesis de la glándula mamaria es un terreno por explorar. La familia de proteínas CPEBs (Cytoplasmic Polyadenylation Element Binding) incluye cuatro proteínas de unión al RNA mensajero que regulan, de manera temporal y espacial, la traducción y la localización sub-celular de los mRNAs que unen. Las CPEBs podrían regular hasta el 25% del genoma.

Aquí presentamos un estudio sistemático de los cuatro miembro de la familia de las CPEBs (CPEB1-4) en el contexto de la glándula mamaria adulta, utilizando modelos knock-out (KO) en ratón. Durante esta investigación hemos descubierto que la falta de CPEB2 resulta en defectos en las ramificaciones de la glándula mamaria, y también en diferenciación. De manera muy relevante, la depleción de CPEB2 tiene consecuencias en cáncer de mama.

En conclusión, es trabajo descifra un nuevo mecanismo a nivel de traducción responsable de la regulación de la homeostasis y el desarrollo de tumores en la glándula mamaria.