Accepted Manuscript

Title: Breast cancer cells: focus on the consequences ofepithelial-to-mesenchymal transition

Authors: Alice H.L. Bong, Gregory R. Monteith

PII: S1357-2725(17)30066-3DOI: http://dx.doi.org/doi:10.1016/j.biocel.2017.03.014Reference: BC 5100

To appear in: The International Journal of Biochemistry & Cell Biology

Received date: 12-12-2016Revised date: 16-3-2017Accepted date: 18-3-2017

Please cite this article as: Bong, Alice HL., & Monteith, GregoryR., Breast cancer cells: focus on the consequences of epithelial-to-mesenchymal transition.International Journal of Biochemistry and Cell Biologyhttp://dx.doi.org/10.1016/j.biocel.2017.03.014

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Breast cancer cells: focus on the consequences of epithelial-to-mesenchymal transition

Alice H.L. Bonga, Gregory R. Monteitha,b

a, School of Pharmacy, University of Queensland, Brisbane, Queensland, Australia

b, Mater Research, University of Queensland, Brisbane , Queensland, Australia.

Cell Facts

Breast tumors are heterogeneous, and cancer cells within a tumor may exhibit

epithelial and mesenchymal-like characteristics

Epithelial breast cancer cells can be induced to form mesenchymal-like cells during

EMT

The mesenchymal phenotype as a consequence of EMT can be associated with

increased resistance to current breast cancer therapies

ABSTRACT

Breast cancers are highly heterogeneous and successful treatment of those subtypes with a

high frequency of metastases and resistance to clinically available therapies remains a

challenge. An understanding of mechanisms which may contribute to this heterogeneity and

generation of more resilient cancer cells is therefore essential. Epithelial-to-mesenchymal

transition (EMT) is a dynamic two-way process that occurs during embryonic development

and wound healing whereby epithelial cells can gain plasticity and switch to a mesenchymal-

like phenotype. EMT has received interest from cancer researchers due to its potential role in

2

processes important in cancer progression and metastasis. Recent evidence has revealed a

clear association between EMT and resistance to therapeutics. Targeting of EMT and/or the

mesenchymal-like phenotype may be a promising avenue for future therapeutic intervention.

This review provides a brief summary of the functional consequences of EMT in breast

cancer, with a focus on the mesenchymal-like phenotype.

Abbreviations:

ABC, ATP-binding Cassette; EGF, Epidermal Growth Factor; EMT, Epithelial-to-

mesenchymal Transition; IGF, Insulin Growth; MET, Mesenchymal-to-epithelial Transition;

MMP, Matrix Metalloprotease; PD-L1, Programmed Death Ligand 1; PDGFR-A, Platelet-

derived Growth Factor Receptor Alpha; Rac1, Ras-related C3 botulinum toxin substrate 1;

RhoA, Ras homolog gene family member A; TGF-β, Transforming Growth Factor Beta;

TRPM7, Transient Receptor Potential Melastatin-like 7

KEYWORDS:

Breast carcinoma, mesenchymal, plasticity, heterogeneity

3

1. Introduction

Breast cancer is the most commonly diagnosed cancer in women worldwide (Chao et al.,

2010). The female breast is a complex organ comprised of a branched network of ducts and

lobules with extensive structural remodeling after adulthood. Distinct cell types comprise the

ductal and lobular network, with polarized luminal epithelial cells and basal myoepithelial

cells residing on the supporting basal lamina. Other cell types such as adipocytes, fibroblasts

and cells of the immune and vascular system form the surrounding stroma (Polyak and

Kalluri, 2010). Within a tumor, breast cancer cells can present histologically as either

epithelial or poorly-differentiated and mesenchymal-like (Pattabiraman et al., 2016; Polyak

and Kalluri, 2010). Breast cancers are diverse and often heterogeneous in terms of

morphology, molecular expression of common treatment targets, clinical disease progression

and outcome. Based on the intrinsic molecular classification, breast cancers can be

categorized into at least five distinct subtypes: luminal A and B, HER2-enriched, basal-like

and claudin-low intrinsic subtypes (Prat et al., 2010; Sorlie et al., 2001). The luminal

subtypes are generally epithelial-like whereas the basal-like and claudin-low subtypes are

often enriched in mesenchymal markers and associated with poor prognosis (Prat et al.,

2010). Adding to this heterogeneity is the fact that breast cancer cells are dynamic, with

microenvironmental factors capable of promoting the development of a more mesenchymal

phenotype (Polyak and Kalluri, 2010), which is the focus of this review.

2. Cell origin and plasticity

4

Mesenchymal breast cancer cells can be transformed from epithelial cells during induction of

a developmental program termed epithelial-to-mesenchymal transition (EMT) (Nieto et al.,

2016; Thiery et al., 2009). EMT is important during normal embryonic development and

wound healing, but is also implicated in cancer progression. EMT in breast cancer cells can

be activated by a complex array of signals; these include cues from the tumor

microenvironment such as hypoxia, and chemotherapeutic stress and autocrine and/or

paracrine signals from stromal and immune cells such as transforming growth factor beta

(TGF-β), insulin growth factor (IGF) and epidermal growth factor (EGF) ((Nieto et al., 2016;

Thiery et al., 2009); refer to Fig. 1). These signals activate canonical EMT transcription

factors including Twist, Snail and Zeb, which then produce a variety of key downstream

molecular changes. EMT-activating transcription factors generally repress the expression of

markers associated with the epithelial phenotype and facilitate acquisition of a mesenchymal

phenotype. Indeed, the EMT gene expression signature enriched in expression of EMT-

inducing genes is closely associated with the mesenchymal-like claudin-low molecular

subtype (Prat et al., 2010). Many studies assessing the downstream signaling and functional

consequences of EMT in breast cancer have utilized experimentally inducible models of

EMT, in particular TGF-β or EGF-mediated EMT models. The remodeling of specific

signaling networks occurs in response to these inducers. An example is the remodeling of

calcium signaling as evidenced by several in vitro studies (Davis et al., 2011; Davis et al.,

2014). This includes altered expression of the calcium-permeable channel P2X5 (Davis et al.,

2011) in an EGF-induced EMT model in the basal-like MDA-MB-468 breast cancer cell line,

and the ability of inhibiting the calcium permeable ion channel TRPM7 (Davis et al., 2014) to

suppress EGF-mediated EMT induction of vimentin expression in the same model.

5

EMT is a dynamic two-way process with the reverse process termed mesenchymal-to-

epithelial transition (MET) (Chao et al., 2010; Pattabiraman et al., 2016) Cells in the EMT-

MET spectrum can exist in intermediate states possessing both epithelial and mesenchymal

traits to varying degrees (Nieto et al., 2016; Tan et al., 2014). These intermediate “hybrid”

states are proposed to be highly plastic and can more readily revert to fully epithelial or

mesenchymal phenotypes in response to local stimuli (Tan et al., 2014). Indeed, there is

evidence for the presence of intermediate or “partial EMT” circulating breast tumor cells, and

an EMT scoring tool predictive of clinical response to chemotherapy has recently been

developed to quantify the extent of EMT in cells (Tan et al., 2014).

3. Functions

Epithelial cells undergo extensive molecular and cytoskeletal remodeling as a consequence of

EMT. A key hallmark of EMT is loss of the cell adhesion molecule E-cadherin (Thiery et al.,

2009). During EMT there is a general downregulation of many epithelial markers with a

concomitant gain in expression of molecules typically associated with mesenchymal cells

such as N-cadherin and vimentin (Fig. 2) (Nieto et al., 2016; Thiery et al., 2009).

Morphologically this results in a switch from polarized epithelial-like cells to spindle-shaped

mesenchymal cells with front-rear polarity (Nieto et al., 2016; Thiery et al., 2009). The loss

of E-cadherin also results in the activation of downstream EMT signaling pathways

associated with malignant progression (Onder et al., 2008). The most commonly reported

functional consequences of EMT in breast cancer cells are summarized below.

4. Associated pathologies

4.1 EMT and breast cancer cell invasion and migration

6

The acquisition of pro-invasive and migratory capabilities requires extensive cytoskeletal

remodeling, involving actin polymerization and intermediate filaments. The formation of

membranous protrusions called invadopodia, which are capable of releasing matrix

metalloproteases (MMP) is important for cancer cell invasion. EMT-inducing transcription

factors such as Twist and canonical proteins associated with the mesenchymal phenotype

such as vimentin have been linked to some of these pro-invasive traits, as shown in various

breast cancer cell lines including MCF7, MDA-MB-231 as well as mouse-derived mammary

carcinomas (Asiedu et al., 2011; Schoumacher et al., 2010; Zhao et al., 2015). Vimentin in

particular was shown to be essential for the elongation of mature invadopodia, which

facilitates the breaching of MDA-MB-231 cells through the basement membrane to invade

the surrounding stroma (Schoumacher et al., 2010).

During EMT, the downregulation of cell-cell adhesion and increased detachment from

underlying extracellular matrices allows increased cell motility and migratory capacity. EMT

induction also activates signaling pathways involved in actin-myosin stimulation and cellular

morphological changes essential for generating the traction force enabling cell migration. In

particular, the upregulation of tyrosine kinase receptor AXL in mesenchymal cells induced by

EMT and its association with enhanced cell motility and migration has been shown by several

studies (Dang et al., 2015; Mak et al., 2016; Vuoriluoto et al., 2011). During EGF-induced

EMT in various breast cancer cell lines, EMT activation was shown to be associated with

increased RhoA GTPase and Rac1 activity and enhanced breast cancer migration and

invasion (Zhang et al., 2014). This migratory capability was confirmed in a recent in vivo

study of EMT using fluorescent-labelled MMTV-PyMT transgenic mice (Beerling et al.,

2016). These studies provided evidence that spontaneous EMT without the use of

experimental inducers can generate mesenchymal cells with enhanced migratory capability,

7

although still not metastatic (Beerling et al., 2016). Although increased migratory potential is

often attributed to the acquisition of mesenchymal characteristics, this was shown to not

always be at the expense of complete loss of epithelial features, and cells with an

intermediate phenotype are also highly migratory (Dang et al., 2015; Yu et al., 2013).

Mammary tumor cells with a mesenchymal phenotype as a result of EMT can also remain

non-migratory or temporarily “dormant”, as demonstrated in a recent study using an EMT-

driven fluorescent color-switching transgenic mouse model (Zhao et al., 2016). It is important

to note that EMT may also facilitate the acquisition of other non-mesenchymal phenotypes

capable of migration, such as an amoeboid phenotype (Zhao et al., 2016).

4.2 EMT confers resistance to anoikis

Detachment of breast epithelial cells from supporting extracellular matrix can activate an

apoptotic program termed anoikis. As such, to detach and escape from the primary tumor and

traverse through the circulation, breast cancer cells must overcome anoikis (Kumar et al.,

2011). EMT activation and downregulation of E-cadherin has been shown to confer anoikis

resistance to breast cancer cells, whereas inhibition of EMT by silencing Twist reverses this

effect (Onder et al., 2008; Kumar et al., 2011). This phenomenon can be partly explained by

the reduced E-cadherin-mediated repression of survival signaling pathways such as the

Wnt/β-catenin signaling pathway (Onder et al., 2008). Further evidence that EMT is activated

and may be important for matrix attachment-independent cell survival comes from the

observation that circulating breast tumor cells exhibit EMT-like plasticity, capable of

switching between epithelial and mesenchymal phenotypes (Yu et al., 2013). The EMT-

8

mediated protection against anoikis is also widely suggested to be one of the traits favorable

for cancer cell metastasis (Kumar et al., 2011).

4.3 EMT and breast cancer metastasis

EMT has long been proposed to initiate the metastatic cascade, a series of events

encompassing escape from the primary tumor to the seeding of tumors at distant secondary

organs (Micalizzi et al., 2009; Thiery et al., 2009). Studies in support of this theory include

various in vivo studies demonstrating the increased incidence of secondary metastases as a

result of induced EMT (Micalizzi et al., 2009; Zhao et al., 2015). MET has also been

suggested to be required for the seeding of tumor cells at the secondary metastatic site, as

secondary tumor cells have been shown to be phenotypically epithelial and must exhibit a

proliferative phenotype to establish a metastatic colony (Bonnomet et al., 2012). Clinical

observations of EMT-like and mesenchymal gene expression signatures in circulating tumor

cells and in cancers with poor prognosis and response to chemotherapy further supports the

link between EMT and cancer progression (Bonnomet et al., 2012; Bulfoni et al., 2016).

Despite these findings, other studies have suggested that EMT is not essential for breast

cancer metastasis but is instead important in cancer cell adaptability to deleterious

environmental stimuli such as hypoxia (Trimboli et al., 2008; Fischer et al., 2015). Using

Cre-recombinant triple-transgenic mice with EMT-lineage tracing to detect cells that have

undergone EMT, it was recently shown that breast cancer cells can metastasize independently

of EMT, as established secondary tumors did not exhibit a fluorescent color-switch indicative

of EMT (Fischer et al., 2015). These conflicting findings highlight the complexity of

9

assessing the significance of EMT in cancer metastases and the need for diverse

physiologically-relevant methodologies and studies in other model systems (Nieto et al.,

2016). The contribution of the microenvironment in regulating EMT/MET and enhancing

pro-invasive and metastatic signaling should be the focus of future studies assessing the

potential link between EMT and metastasis (Nieto et al., 2016).

4.4 EMT is associated with therapeutic resistance

There is mounting evidence for the link between EMT and therapeutic resistance in various

cancers. Acquisition of a mesenchymal phenotype was shown to protect breast cancer cells

against many conventional anti-proliferative chemotherapy agents as well as radiation

therapy (Asiedu et al., 2011). However, this effect could be limited to certain cancers and

appears dependent on the degree of differentiation along the EMT spectrum, as cells that are

"fully" mesenchymal may actually be more chemo-sensitive compared to "intermediate"

mesenchymal phenotypes (Tan et al., 2014). EMT activation has also been shown to

upregulate the CD44+/CD24- ratio, a marker of "stemness" in breast cancer cells (Asiedu et

al., 2011; Mani et al., 2008). The link between EMT and "stemness" has been reviewed

elsewhere (Nieto et al., 2016). Increased "stemness" may promote resistance to

chemotherapy-induced cell death (Asiedu et al., 2011) as demonstrated in TGF-β induced

EMT in a mouse mammary carcinoma cell line. Consistent with this, breast cancer

populations that survive chemotherapy treatment have been shown clinically to have a more

mesenchymal gene profile with enrichment in stem cell-like markers (Creighton et al., 2009).

Using microarray analysis, patient tumors with EMT gene expression signatures were also

shown to be less responsive to various clinically used chemotherapy agents (Taube et al.,

2010). These findings have been consolidated recently in an EMT-lineage tracing in vivo

study by Fischer et al., showing that cells undergoing EMT are more resistant against

10

cyclophosphamide treatment (Fischer et al., 2015). The contribution of EMT to therapeutic

resistance may also be partly explained by EMT-induced upregulation of multi-drug

resistance genes such as the ABC drug transporters and drug metabolizing enzymes,

enhancing removal of the drug from breast tumors (Asiedu et al., 2011; Fischer et al., 2015).

5. Therapeutic opportunities and future directions

The role of EMT in various cellular functions associated with cancer progression and

therapeutic response suggests that pharmacological intervention of this process may represent

a therapeutic target for breast cancer (Thiery et al., 2009). Given the association between an

EMT-like signature and clinical prognosis, EMT-targeting agents could be used in

combination with other chemotherapeutic agents. Agents that are selectively cytotoxic to the

mesenchymal phenotype could be administered after chemotherapy, and these agents could

be identified through the use of phenotypic-based drug-drug combination screens. A recent

study which derived a generic EMT signature across different cancer types has identified

potential targets upregulated in mesenchymal-like cells: AXL, immune checkpoint protein

PD-L1 and PDGFR-A (Mak et al., 2016). Importantly, therapeutic sensitivity is increased

with inhibition of these proteins (Mak et al., 2016). Alternatively, inhibiting EMT induction

or inducing MET in breast cancer cells that may have acquired the mesenchymal-like

phenotype may also reduce resistance towards chemotherapeutic agents. Reversing the stem

cell-like, mesenchymal phenotype is another particularly attractive approach since this

phenotype is closely associated with resistance to many conventional anti-proliferative

chemotherapeutic agents (Pattabiraman et al., 2016). The recent identification of protein

kinase A activators that can induce and maintain epithelial characteristics in mesenchymal-

like breast cancer cells and increase sensitivity to doxorubicin and paclitaxel treatments also

11

demonstrates the promising potential of targeting EMT (Pattabiraman et al., 2016).

Nevertheless, each of the EMT-targeting strategies described has potential drawbacks. The

induction of MET may facilitate secondary metastatic outgrowths due to the reversion to a

potentially more proliferative, epithelial phenotype (Bonnomet et al., 2012, Nieto et al.,

2016). In addition, partial MET reversion may generate more therapeutically-resistant hybrid

phenotypes, further complicating treatment (Tan et al., 2014). In summary, breast cancers are

diverse and dynamic. A better understanding and assessment of the mesenchymal phenotype

generated as a consequence of EMT is required and may open up a new avenue for the

development of a novel set of therapeutic agents to combat breast cancer progression.

Acknowledgements

This work was supported by the Australian National Health and Medical Research Council

(Project grants 569645 and 1022263). AB was funded by an Australian Postgraduate Award.

GRM is supported by the Mater Foundation. The Translational Research Institute is

supported by a grant from the Australian Government.

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Figure Legends

Fig. 1: EMT inducers and the EMT-MET spectrum. Epithelial cells can be induced to form

mesenchymal-like cells as a result of EMT-activating stimuli such as chemotherapy, hypoxia,

inflammation, metabolic stress, immune factors and growth factors such as IGF and EGF.

The reverse can also occur via MET. Cells can undergo incomplete or partial EMT along an

EMT spectrum of varying extents of epithelial or mesenchymal phenotypes; i.e. they are in

highly plastic, intermediate EMT cell states and can become either epithelial or mesenchymal

in response to local stimuli. Figure adapted from Thiery et al., (2009) and Nieto et al., (2016).

18

Fig. 2: Characteristics of epithelial and mesenchymal cells and commonly used experimental

molecular markers. Epithelial-like cells (left) are characterized by cell-cell adhesions and

presence of tight junctions, with apico-basal polarity and are typically proliferative and non-

motile. Mesenchymal-like cells (right) are characterized by a lack of cell-cell adhesions and

have a fibroblast-like morphology with front-rear polarity. They have increased motility and

are generally not as proliferative. Figure adapted and/or compiled from references: Asiedu et

al., 2011; Beerling et al., 2016; Nieto et al., 2016