Walid T. Khaled

Mammary development in the embryo and adult: a journey of morphogenesis and commitment

Mammary gland development occurs through distinctive stages throughout embryonic and pubertal development and reproductive life. At each stage, different signals are required to induce changes in both the epithelium and the surrounding mesenchyme/stroma. Recent studies have provided new insights into the origin, specification and fate of mammary stem and progenitor cells and into how the differentiated lineages that comprise the functional mammary gland are determined. The development of new tools and culture techniques has also enabled the factors that influence branching morphogenesis in the embryonic and pubertal gland to be identified. A surprising recent discovery has been that mammary epithelial cells commit to differentiated lineages using the same signalling pathways that regulate lineage determination in T helper cells.

The IL-4/IL-13/Stat6 signalling pathway promotes luminal mammary epithelial cell development

Naïve T helper cells differentiate into Th1 and Th2 subsets, which have unique cytokine signatures, activators and transcriptional targets. The Th1/Th2 cytokine milieu is a key paradigm in lineage commitment, and IL-4 (Il4), IL-13 (Il13) and Stat6 are important mediators of Th2 development. We show here, for the first time, that this paradigm applies also to mammary epithelial cells, which undergo a switch from Th1 to Th2 cytokine production upon the induction of differentiation. Thus, the Th1 cytokines IL-12 (Il12), interferon gamma (INFγ; also known as Ifng) and Tnfα are downregulated concomitantly with the upregulation of the Th2 cytokines IL-4, IL-13 and IL-5 (Il5) as epithelial cells commit to the luminal lineage. Moreover, we show that Th2 cytokines play a crucial role in mammary gland development in vivo, because differentiation and alveolar morphogenesis are reduced in both Stat6 and IL-4/IL-13 doubly deficient mice during pregnancy. This unexpected discovery demonstrates a role for immune cell cytokines in epithelial cell fate and function, and adds an unexpected tier of complexity to the previously held paradigm that steroid and peptide hormones are the primary regulators of mammary gland development.

PML depletion disrupts normal mammary gland development and skews the composition of the mammary luminal cell progenitor pool

Nuclear domains of promyelocytic leukemia protein (PML) are known to act as signaling nodes in many cellular processes. Although the impact of PML expression in driving cell fate decisions for injured cells is well established, the function of PML in the context of tissue development is less well understood. Here, the in vivo role of PML in developmental processes in the murine mammary gland has been investigated. Data are presented showing that expression of PML is tightly regulated by three members of the Stat family of transcription factors that orchestrate the functional development of the mammary secretory epithelium during pregnancy. Developmental phenotypes were also discovered in the virgin and pregnant Pml null mouse, typified by aberrant differentiation of mammary epithelia with reduced ductal and alveolar development. PML depletion was also found to disturb the balance of two distinct luminal progenitor populations. Overall, it is shown that PML is required for cell lineage determination in bi-potent luminal progenitor cells and that the precise regulation of PML expression is required for functional differentiation of alveolar cells.

BCL11A is a triple-negative breast cancer gene with critical functions in stem and progenitor cells

Triple-negative breast cancer (TNBC) has poor prognostic outcome compared with other types of breast cancer. The molecular and cellular mechanisms underlying TNBC pathology are not fully understood. Here, we report that the transcription factor BCL11A is overexpressed in TNBC including basal-like breast cancer (BLBC) and that its genomic locus is amplified in up to 38% of BLBC tumours. Exogenous BCL11A overexpression promotes tumour formation, whereas its knockdown in TNBC cell lines suppresses their tumourigenic potential in xenograft models. In the DMBA-induced tumour model, Bcl11a deletion substantially decreases tumour formation, even in p53-null cells and inactivation of Bcl11a in established tumours causes their regression. At the cellular level, Bcl11a deletion causes a reduction in the number of mammary epithelial stem and progenitor cells. Thus, BCL11A has an important role in TNBC and normal mammary epithelial cells. This study highlights the importance of further investigation of BCL11A in TNBC-targeted therapies.

Cytokine signalling in mammary gland development

Mammary gland development occurs in three distinct stages during the lifetime of the female mammal: in embryonic, pubertal and reproductive life. At each of these developmental stages, different signalling molecules induce changes in both the epithelium and the surrounding stroma. However, it is during pregnancy that the most dramatic changes occur, resulting in a massive increase in the number of epithelial cells and in their function. Pregnancy initiates the development of a new epithelial lineage, the alveolar cells, which form the milk-producing lobuloalveolar structures. These cells become redundant at the end of lactation and are removed in an exquisitely controlled process of tissue remodelling coupled with extensive cell death. All of these events require not only steroid hormones but also sequential signalling by cytokines. A recent surprising discovery was that the signalling pathways and cytokines that regulate lineage determination in T helper cells are also involved in mammary gland development during pregnancy.

Cancer mouse models: Past, present and future

The development and advances in gene targeting technology over the past three decades has facilitated the generation of cancer mouse models that recapitulate features of human malignancies. These models have been and still remain instrumental in revealing the complexities of human cancer biology. However, they will need to evolve in the post-genomic era of cancer research. In this review we will highlight some of the key developments over the past decades and will discuss the new possibilities of cancer mouse models in the light of emerging powerful gene manipulating tools.

The Stat6-regulated KRAB domain zinc finger protein Zfp157 regulates the balance of lineages in mammary glands and compensates for loss of Gata-3

Lineage commitment studies in mammary glands have focused on identifying cell populations that display stem or progenitor properties. However, the mechanisms that control cell fate have been incompletely explored. Herein we show that zinc finger protein 157 (Zfp157) is required to establish the balance between luminal alveolar pStat5- and Gata-3-expressing cells in the murine mammary gland. Using mice in which the zfp157 gene was disrupted, we found that alveologenesis was accelerated concomitantly with a dramatic skewing of the proportion of pStat5-expressing cells relative to Gata-3⁺ cells. This suppression of the Gata-3⁺ lineage was associated with increased expression of the inhibitor of helix-loop-helix protein Id2. Surprisingly, Gata-3 becomes dispensable in the absence of Zfp157, as mice deficient for both Zfp157 and Gata-3 lactate normally, although the glands display a mild epithelial dysplasia. These data suggest that the luminal alveolar compartment of the mammary gland is comprised of a number of distinct cell populations that, although interdependant, exhibit considerable cell fate plasticity.

Differentiation dynamics of mammary epithelial cells revealed by single-cell RNA sequencing

Characterising the hierarchy of mammary epithelial cells (MECs) and how they are regulated during adult development is important for understanding how breast cancer arises. Here we report the use of single-cell RNA sequencing to determine the gene expression profile of MECs across four developmental stages; nulliparous, mid gestation, lactation and post involution. Our analysis of 23,184 cells identifies 15 clusters, few of which could be fully characterised by a single marker gene. We argue instead that the epithelial cells—especially in the luminal compartment—should rather be conceptualised as being part of a continuous spectrum of differentiation. Furthermore, our data support the existence of a common luminal progenitor cell giving rise to intermediate, restricted alveolar and hormone-sensing progenitors. This luminal progenitor compartment undergoes transcriptional changes in response to a full pregnancy, lactation and involution. In summary, our results provide a global, unbiased view of adult mammary gland development.There is a need to understand how mammary epithelial cells respond to changes at various developmental stages. Here, the authors use single-cell RNA sequencing of mammary epithelial cells at different adult developmental stages, identifying different cell types and charting their developmental trajectory.

Hypogonadal Mouse, a Model to Study the Effects of the Endogenous Lack of Gonadotropins on Apoptosis

Abstract Testicular apoptosis is involved in the regulation of germ cell numbers, allowing optimal sperm production. Apoptosis has been described to occur in response to the absence of hormonal stimulation of the testis. Here we investigate the effect of the physiological lack of gonadotropins from birth using the hypogonadal (homozygous for the mutant allele Gnrh1hpg) mouse as a model. We pursued a concerted strategy using microarray analysis and RT-PCR to assess transcript levels, TUNEL to quantify the incidence of apoptosis, and Western blotting to assess the respective contribution of the extrinsic and intrinsic apoptotic pathways. Our results indicate a large increase in apoptosis of both somatic and germ cell compartments in the hpg testis, affecting Sertoli cells as well as germ cells of all ages. We confirmed our observations of Sertoli cell apoptosis using anti-Mullerian inhibiting substance staining and staining for cleaved fodrin alpha. In the somatic compartment, apoptosis is primarily regulated via the membrane receptor (extrinsic) apoptotic pathway, while in the germ cell compartment, regulation occurs via both the mitochondrial (intrinsic) and membrane receptor (extrinsic) apoptotic pathways, the latter potentially in a stage-specific manner. This study is the first report of spermatogonial apoptosis in response to gonadotropin deficiency as well as the first report of Sertoli cell apoptosis in response to gonadotropin deficiency in the mouse.

Naked Mole-Rat Cells are Susceptible to Malignant Transformation by SV40LT and Oncogenic Ras

The Naked Mole-Rat, Hetercephalus glaber, is a mouse-sized subterranean rodent native to East Africa. Research on NMRs is intensifying in an effort to gain leverage from their unusual physiology, long-life span and cancer resistance. Few studies have attempted to explain the reasons behind NMRs cancer resistance, but most prominently Tian et al. reported that NMR cells produce high-molecular weight hyaluronan as a potential cause for the NMR’s cancer resistance. Tian et al. have shown that NMR cells are resistant to transformation by SV40 Large T Antigen (SV40LT) and oncogenic HRAS (HRASG12V), a combination of oncogenes sufficient to transform mouse and rat fibroblasts. We have developed a single lentiviral vector to deliver both these oncogenes and generated multiple cell lines from five different tissues and nine different NMRs, and report here that contrary to Tian et al.’s observation, NMR cells are susceptible to oncogenic transformation by SV40LT and HRAS. Our data thus point to a non-cell autonomous mechanism underlying the remarkable cancer resistance of NMRs. Identifying these non-cell autonomous mechanisms could have implications on our understanding of human cancer development.