Contributions of Yap and Taz dysfunction to breast cancer initiation, progression, and aging-related susceptibility

1 INTRODUCTION

Yap and Taz co-transcription factors initiate new transcriptional programs in a variety of cellular contexts (Figure 1). Subsequently, we will refer to both transcription factors as “Yap/Taz” when the described conditions are known to apply to both. The Yap/Taz ortholog Yorkie was originally discovered in the fruit fly, downstream of the Hippo signaling pathway, where it causes cell proliferation as a part of normal tissue size control during development.¹ Since then, Yap and Taz have both been shown to be activated in a variety of contexts including: perturbed cell polarity, extracellular stiffness, low cell density, wound healing, regeneration, embryonic development, and stem cell maintenance (Figure 1).^2-15 In addition, Yap/Taz can be activated by other cell signaling events including signaling through Wnts, glucocorticoid receptors, G-protein coupled receptors, and PI3K signaling.^16-22 Yap/Taz induced transcription causes many different cellular phenotypes, such as cell growth, apoptosis, stem cell renewal, and cellular differentiation (Figure 1).^12-15 With their ability to activate proliferation it is no surprise that Yap/Taz are upregulated in many diseases, including cancers (Figure 1). Some of the cancers in which Yap/Taz are involved include: breast, lung, colorectal, liver, gastric, and pancreatic.^{12, 23, 24} In most of these contexts Yap/Taz are implicated in tumor growth and metastasis. However, in some cases Yap/Taz also are involved in drug resistance and cancer stem cell phenotypes.¹²

The goal of this review is to take a focused perspective on the function of Yap and Taz in normal breast and breast cancer tissues. This review will be broken into three parts: (i) the role of Yap/Taz in a normal breast context, (ii) the role of Yap/Taz in a breast cancer context, and (iii) the differential roles that Yap/Taz may play in separate breast epithelial cell types to promote breast cancer during the earliest stages of progression.

2 YAP AND TAZ IN NORMAL BREAST TISSUE

At first glance, the mammary gland is comprised of two main cell layers. The outer, or basal-located, layer contains contractile myoepithelial cells whereas the inner cell layer is composed of secretory luminal epithelial cells (Figure 2A).²⁵ Most human studies suggest Yap/Taz functions in the basal cell layer of breast epithelium because Yap/Taz protein is primarily localized in the nuclei of myoepithelial cells (Figure 2B; Table 1).^{4, 26-34} Furthermore, genes that typically change in response to Yap/Taz upregulation (ie, Yap/Taz transcriptional signatures) are detected in the RNA expression profiles of myoepithelial cells and mammary stem cells.^{32, 35} In general, it is thought that Yap/Taz normally functions to maintain the stem cell population during high rates of proliferation in non-homeostatic conditions such as wound healing, regeneration, and cancer.¹⁵ The mammary gland goes through many cycles of heavy proliferation throughout a woman's lifetime, including early development, puberty, menstruation, and pregnancy.^{36, 37} Therefore, it is reasonable to speculate that Yap/Taz may function similarly in mammary epithelium to maintain a stem cell fate during periods of high proliferation.

Studies in mice suggest Yap/Taz functions dynamically in mammary epithelium depending on contexts that require high rates of proliferation (Figure 2D-F and Table 1). For example, Taz protein accumulates in the nuclei of myoepithelial cells during puberty and is necessary for the maintenance of the myoepithelial cell lineage throughout this period of massive tissue re-structuring and growth (Figure 2D).³² The localization of Yap protein changes drastically surrounding pregnancy, a period in which mammary tissue is heavily remodeled. Prior to pregnancy, Yap accumulates in the nuclei of myoepithelial cells (Figure 2D). During pregnancy, Yap additionally localizes in the nuclei of luminal cells and functions to promote the survival of progenitor cells (Figure 2E). And after pregnancy, Yap expression is significantly decreased in both cell types (Figure 2F).³⁸ Although, Yap/Taz accumulation has not been looked at during pregnancy in humans it is feasible that they perform similar functions. These studies in mammary epithelial tissues of humans and mice support the hypothesis that Yap/Taz generally functions to maintain a progenitor cell fate during periods of high proliferation. However, it is not clear what are the precise molecular changes that Yap/Taz induce during these periods and if they vary during different proliferative events (i.e. development, puberty, menstruation, and pregnancy). Moreover, a number of different myoepithelial, luminal, and progenitor cell subtypes have been identified in the breast epithelium,^39-42 and it is important to consider that Yap/Taz may confer distinct functions in these different epithelial cell subtypes.

3 YAP AND TAZ IN BREAST CANCER

Many studies have examined the expression of Yap/Taz in different in vivo breast cancer contexts to determine whether and how Yap/Taz contributes to breast cancer. Here, we summarize the results of such studies that have looked at Yap/Taz mRNA expression, protein expression, or transcriptional signatures in specific breast cancer subtypes or phenotypes (Figure 3; Tables 2-4). Taz expression positively correlates with the triple-negative subtype and cancer stem cell phenotype (Figure 3K,O,Q).^{35, 43-45} Consistent with involvement in the triple-negative subtype, it is estimated that Taz gene amplifications occur in 6.5-44% of basal tumors (Table 9).^{2, 4, 32, 43} Yap/Taz expression was significantly associated, mainly a negative correlation, with breast cancer survival in a number of studies and is, therefore, highly likely to be involved in breast cancer progression (Figure 3F,P).^{4, 24, 32, 33, 35, 43-48} However, our review of literature indicates that more studies need to be done to define the precise environments in which Yap/Taz contributes to breast cancer. One common theme we see in our analysis is the seemingly contradictory evidence about Yap/Taz involvement in any particular breast cancer subtype or phenotype. For example, different studies conclude that Yap is upregulated, downregulated, or does not change in association with the luminal subtype (Figure 3C).^{26, 27, 33, 46-48} This suggests either that (i) some of the methods used in these studies do not accurately reflect Yap/Taz activation and/or (ii) that Yap/Taz play differential roles within particular breast cancer subtypes or phenotypes and more work is necessary to decipher the precise contexts in which Yap/Taz actually acts.

In support of the first scenario, we note that these studies used drastically different methods (eg, antibodies, concentrations, and scoring methods) to come to their conclusions (Table 4). Yap/Taz expression itself also does not necessarily translate to increased transcriptional function. We suggest future studies use methods that more accurately measure Yap/Taz activation such as: nuclear localization and/or transcriptional signatures. Indeed, emerging studies that analyze Yap/Taz transcriptional signatures have uncovered some contexts in which Yap/Taz contribute to breast cancer: high tumor grade, cells that have mutated p53 activity, and cells that have high glucocorticoid receptor activity.^{4, 18, 24, 49}

In support of the second scenario, we note there are some contexts in which Yap/Taz function has not been thoroughly assessed. We suggest Yap/Taz may act at an earlier time point during cancer progression; these studies primarily looked at Yap/Taz expression in cancers that have already formed. To this end, Yap/Taz function should be compared between young normal epithelial tissues and tissues that have an increased risk to become cancerous including: BRCA mutant, aged, hyperplasia, and DCIS tissues. In addition, Yap/Taz may have diverse roles that contribute to breast cancer in different epithelial cell types. For example, Yap/Taz may be upregulated or downregulated differentially in myoepithelial, progenitor, and luminal cells and cause different phenotypes that collectively lead to an increase in breast cancer development. We propose that future studies in diverse breast epithelial cell types and at different points in breast cancer progression, using methods that accurately measure Yap/Taz function, will improve our knowledge of when Yap/Taz acts during breast cancer development.

Review of the literature in vitro further suggests Yap/Taz are involved in breast cancer (Figure 4). Cell culture studies showed that Yap/Taz contribute to breast cancer-relevant cell culture phenotypes including: proliferation, migration, anchorage-independent growth, tumor growth, and metastasis (Figure 4A,C) (Table 5, 6).^{2-4, 18, 24, 28, 32, 34, 35, 38, 44, 49-51} These studies also found that Yap/Taz induced breast cancer stem cell phenotypes, such as mammosphere initiation, tumor initiation, and chemo-resistance (Figure 4B,D; Table 7).^{2, 4, 18, 24, 32, 35, 44} In addition, Yap/Taz were generally found to be upregulated in basal/triple-negative cell lines compared to cell lines that represent Her2+ or luminal breast cancer subtypes (Table 8).^{32, 35, 43, 52} However, in our analysis of this literature, we notice that a majority of the studies, especially the ones that analyze Taz function and a cancer stem cell phenotype, look at a limited subset of breast epithelial cell lines (mainly MCF10A, which were derived from human fibrocystic breast tissue; Tables 5-7). Also, some limitations exist by only looking at Yap/Taz function in cell lines. Cell lines, by definition, are already immortal, and therefore exclude the analysis of Yap/Taz function during the earliest stages of breast cancer progression, prior to immortalization. Evidence suggests Yap/Taz function differentially depending on immortalization. For example, mechanically triggered Yap/Taz activity in human mammary epithelial cells (HMEC) from postmenopausal women requires exposure to super-physiological matrix stiffness (>3 GPa) and results in lineage-specific proliferation patterns, whereas the isogenic immortalized HMEC cell lines trigger Yap/Taz at physiological stiffness (1000-2500 Pa) and cause proliferation in all lineages.³¹ In addition, cell lines likely do not represent the majority of epithelial cell types in which Yap/Taz could function. We reiterate that future cell culture studies in diverse epithelial cell types and during earlier time points during breast cancer progression would greatly expand our knowledge of Yap/Taz involvement in breast cancer.

Indeed, many studies seemingly contradict each other in regard to Yap/Taz involvement in breast cancer. Yap/Taz expression has been concluded to correlate negatively, positively, and not at all with the same breast cancer subtype, depending on the study (Figure 3). In addition, Yap/Taz is often viewed as a tumor-driving villain, whereas Yap/Taz apparently also can act as tumor suppressors in some contexts.^{26, 53, 54} It is likely that Yap/Taz have differential tumor suppressor or oncogenic activities depending on the cell in which they are activated. For example, Yap/Taz were shown to act as tumor suppressors in normal liver cells and as oncogenes in liver cancer cells.⁵⁵ This further exacerbates the need to study the function of Yap/Taz in different breast epithelial cell types during different stages of cancer progression to properly elucidate the role of Yap/Taz in breast cancer.

4 YAP AND TAZ IN DIVERSE BREAST EPITHELIAL CELL TYPES DURING THE EARLIEST STAGES OF PROGRESSION

Our analysis of the Yap/Taz literature in different breast epithelial cell types prior to immortalization has led us to the following three hypotheses:

4.1 Hypothesis 1: Yap/Taz dysfunction acts in the earliest stages of breast cancer progression

The earliest stages of breast cancer progression have been relatively understudied in the past, particularly because a majority of breast epithelial cells studied in culture have already undergone many steps of malignant progression. However, most of the oncogenes present in fulminant cancers also are detected earlier in progression, in pre-malignant ductal carcinoma in situ (DCIS) cancer precursor cells. An approach to studying Yap/Taz in earlier stages of breast cancer progression could take advantage of accurate cell culture models of breast cancer progression that start with normal HMEC from women that have undergone breast reduction surgery (Figure 5). The cell culture model that was developed by Martha Stampfer accurately reflects the earliest phases of breast cancer progression that occur in vivo, and supports the growth of different subtypes of normal breast epithelial cells, including: myoepithelial, luminal, and progenitor cells.^56-62 This model has been used to good effect to study early events in breast cancer progression and the roles of putative oncogenes that promote breast cancer development.

Aging is arguably the most significant risk factor for the development of breast cancer.^{63, 64} We previously proposed that specific molecular features associated with an aging phenotype act in breast cancer precursor cells and contribute to early breast cancer progression.⁶⁵ In normal HMEC the subcellular localization and function of Yap changes as a function of age (Figure 6).^{31, 42} Therefore, Yap is a good candidate protein whose regulation and function changes during the aging process and may contribute early on to the development of breast cancer, perhaps before there is anything recognizable as a pathological lesion. To further test this hypothesis, it will be important to perturb Yap signaling in HMEC from different aged specimens and assay for changes in oncogenic and early progression phenotypes such as proliferation, malignant properties, or barrier bypass.

5 SUMMARY/FUTURE OUTLOOKS

Here, we take a focused look at Yap/Taz function in breast cancer. Our analysis of the literature agrees with the interpretation that Yap/Taz are involved in breast cancer and that Taz in particular is associated with the basal/triple-negative subtype in fulminant cancers. Furthermore, the literature in total supports the concept that Yap/Taz allow access to cell functions needed for stereotypical malignant behavior and cancer stem cell states, such as EMT genes. However, we also notice that much more information is needed to understand and further elucidate the precise timing, location, and function of Yap/Taz in diverse mammary epithelial cell contexts and its relationship to the development of breast cancer.

To this end, the function of Yap has been studied in a cell culture model of early breast cancer progression, starting with normal finite cells. These studies have led us to hypothesize that Yap dysregulation may act at the earliest stages of breast cancer progression. In addition, Yap may play different roles in distinct mammary epithelial cell types that altogether leads to the early progression of breast cancer. If Yap/Taz contributes to breast cancer phenotypes at the earliest stages of progression, this may lead to the development of novel therapeutics that are better aimed to prevent breast cancer. One can develop therapeutics that can stop either Yap/Taz themselves or some of their downstream transcriptional targets that contribute to Yap/Taz-induced early cancer phenotypes.

AUTHOR CONTRIBUTIONS

TF and ML contributed to conceptualization, investigation, writing and editing, and funding acquisition. ML provided supervision.

CONFLICTS OF INTEREST

TF and ML have no conflicts of interest to declare