Epithelial-mesenchymal plasticity determines estrogen receptor positive (ER+) breast cancer dormancy and reacquisition of an epithelial state drives awakening

Patrick Aouad, Yueyun Zhang, Céline Stibolt, Sendurai A. Mani, George Sflomos, Cathrin Brisken

Posted on: 6 September 2021

Preprint posted on 26 July 2021

Article now published in Nature Communications at http://dx.doi.org/10.1038/s41467-022-32523-6

Sleeping Beauties and the case of Epithelial-Mesenchymal Plasticity. Aouad et al., use a mouse intraductal xenografting approach (MIND) to study tumour cell dormancy in ER+ breast cancer.

Selected by Sagar Varankar

Categories: cancer biology, cell biology

Background:

Breast cancer is a lethal gynaecological malignancy accounting for ~30% of overall cases and ~15% of cancer-associated deaths¹. Decades of research have provided effective screening methods and identified clinically relevant subtypes for this disease^2,3. Based on the expression of hormone receptors for estrogen (ER), progesterone (PR) and the receptor tyrosine kinase Her2, four broad subtypes viz., ER⁺, PR⁺, Her2⁺ and ER^–PR^–Her2^– (or triple negative breast cancers/TNBC), have been defined. While additional clinical subtypes exist, over 70% of breast cancer cases are identified as ER⁺ and often associate with better 5-year survival rates than TNBC/Her2+ patients. However, despite good initial prognosis, several patients with ER⁺breast cancer present with disease relapse which can be challenging to treat⁴.

Disease recurrence is often attributed to the presence of disseminated tumour cells (DTCs) which are dormant and can be activated years after the primary diagnosis⁵. Dormant DTCs have been identified across multiple cancers and can influence disease outcome by reducing the efficacy of therapeutic interventions. Hence, it is crucial to understand DTC biology and dynamics during disease progression. While DTCs have been implicated in ER⁺breast cancer relapse, existing experimental models do not recapitulate several physiological events that generate this population. These limitations may result from the absence of an appropriate microenvironment or may be inherent to the experimental system under consideration.

In a previous study, the authors developed an intraductal model of cell transplantation for breast cancer cell lines and cells from patient derived xenografts (PDXs)^6,7. Inoculation of cells in the mammary duct captured several clinical aspects of the disease and identified differential growth in the primary and metastatic tumours. In this preprint, the authors expand on their previous findings to demonstrate the role of epithelial-mesenchymal plasticity induced dormancy in ER⁺ breast cancer progression.

Key Findings:

Recapitulating the clinical progression and dormancy of ER⁺ breast cancer:

Figure 1. Schematic depiction of the experimental approach. (Adapted from Figure 1 of Aouad et al.,)

The authors injected red/green fluorescent protein-expressing ER⁺ breast cancer cells into mouse mammary ducts. To comprehensively examine the phenomenon of disease relapse, the authors used ER⁺ cancer cell lines and patient derived xenografts; ER^– samples were used as controls (Figure 1). They observed greater proliferation, distant dissemination, and eventual metastatic growth of ER^– compared to ER⁺cells. While ER⁺cells did disseminate to distant organs, they only formed small tumour lesions compared to their ER^– counterparts, due to a lower rate of proliferation. The organ-specific homing of tumour cells and their differential expansion was reminiscent of known clinical features. Thus, the authors developed an experimental system to recapitulate several events associated with ER+ breast cancer progression including the dormancy of disseminated cells.

Cellular plasticity during ER⁺ breast cancer progression:

Characterization of primary and distant tumour lesions, formed by the injected cells, identified an upregulation of mesenchymal markers in ER⁺ compared to ER^– distant lesions. The authors observed that epithelial to mesenchymal transition (EMT) programs were activated in dormant DTCs of ER+ intraductal xenografts. Artificial induction of EMT in ER⁺cells dramatically reduced their capacity to form primary and secondary tumours. The authors validated these observations across multiple systems to eliminate cell line-, mouse model- and expression construct- specific biases and obtained consistent results emphasizing that activation of EMT in ER⁺ cancers is a late stage event.

Artificial expression of E-cadherin breaks the dormancy of disseminated tumour cells

As the authors observed that EMT was associated with the acquisition of a dormant cellular state, they examined whether the forced induction of epithelial properties could overcome this dormancy. Constitutive and inducible over-expression of E-cadherin resulted in outgrowth of distant metastatic lesions by virtue of enhanced proliferation, thus affirming the exit from dormancy.

Why I Chose This Preprint:

Disease relapse is a major hurdle in cancer treatment. The frequency at which different cancers recur is quite varied and the associated cellular processes continue to be poorly understood. In this preprint, the authors attempt to understand late stage recurrence of ER⁺ breast cancer and its association with dormancy. Their findings are clinically relevant and emphasize the use of appropriate animal models in cancer biology. On a personal note, I was interested in this study as similar mouse models for ovarian cancer would have greatly benefitted my graduate research and may be useful for other researchers in the field of cancer research.

Questions For the Authors:

Recent studies have defined distinct states along the epithelial-mesenchymal continuum^8,9. What are your thoughts regarding the enrichment of specific epithelial/mesenchymal cell states in ER⁺ versus ER^– negative cancers and its contribution to differential dormancy? Have you examined the differences in these states in primary versus secondary tumours in your mouse model?
Do you intend to examine the expression of transcription factors that are responsible for mesenchymal to epithelial transition and their role in tumour dormancy?
From a future perspective, do you think that differences in dormancy may arise from changes in tumour-stroma and tumour-ECM crosstalk?

References:

Siegel, R. L., Miller, K. D., Fuchs, H. E. & Jemal, A. Cancer Statistics, 2021. CA. Cancer J. Clin. 71, 7–33 (2021).
Tan, P. H. et al. The 2019 World Health Organization classification of tumours of the breast. Histopathology 77, 181–185 (2020).
Bhushan, A., Gonsalves, A. & Menon, J. U. Current state of breast cancer diagnosis, treatment, and theranostics. Pharmaceutics 13, 1–24 (2021).
Pan, H. et al. 20-Year Risks of Breast-Cancer Recurrence after Stopping Endocrine Therapy at 5 Years. N. Engl. J. Med. 377, 1836–1846 (2017).
Santos-de-Frutos, K. & Djouder, N. When dormancy fuels tumour relapse. Commun. Biol. 4, (2021).
Fiche, M. et al. Intraductal patient-derived xenografts of estrogen receptor α-positive breast cancer recapitulate the histopathological spectrum and metastatic potential of human lesions. J. Pathol. 247, 287–292 (2019).
Sflomos, G. et al. A Preclinical Model for ERα-Positive Breast Cancer Points to the Epithelial Microenvironment as Determinant of Luminal Phenotype and Hormone Response. Cancer Cell 29, 407–422 (2016).
Jolly, M. K. et al. Hybrid epithelial/mesenchymal phenotypes promote metastasis and therapy resistance across carcinomas. Pharmacol. Ther. (2018) doi:10.1016/j.pharmthera.2018.09.007.
Pastushenko, I. et al. Identification of the tumour transition states occurring during EMT. Nature 556, 463–468 (2018).

doi: https://doi.org/10.1242/prelights.30509

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Author's response

Cathrin Brisken, Patrick Aouad shared

Recent studies have defined distinct states along the epithelial-mesenchymal continuum^7,8. What are your thoughts regarding the enrichment of specific epithelial/mesenchymal cell states in ER⁺ versus ER^– negative cancers and its contribution to differential dormancy? Have you examined the differences in these states in primary versus secondary tumours in your mouse model?

We concur with the idea that there is a continuum of different epithelial-mesenchymal states and used the term epithelial-mesenchymal plasticity to reflect this. Indeed, we wonder whether cells are ever completely locked in a mesenchymal state or always somewhat plastic. To us, the hypothesis that different depth of mesenchymal states may affect the ability to proliferate is attractive. It is very challenging to isolate the DTCs from different organs; multiple technical hurdles still need to be overcome in order to isolate DTCs in their entirety from different organs so we can study them in a comprehensive way.

Do you intend to examine the expression of transcription factors that are responsible for mesenchymal to epithelial transition and their role in tumour dormancy?

Yes, that is an important point. It seems that ZEB1/2 and VIM might have a preponderant role in the models that we analyzed, but clearly other transcription factors may well be important. It might also be that dependent on the metastatic site and/or the specific awakening stimulus as different transcription factors may have different roles to play.

From a future perspective, do you think that differences in dormancy may arise from changes in tumour-stroma and tumour-ECM crosstalk?

I think that all options are open. There is some evidence in the literature that strong emotional events, like the loss of a partner, may trigger recurrence. Stress hormones might act directly on the dormant cells, affect their interactions with the ECM and/or modify tumor stromal interactions. It will be exciting to explore the relationship between the endocrine, nervous and immune systems, and the dormant cells and their microenvironment.

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Epithelial-mesenchymal plasticity determines estrogen receptor positive (ER+) breast cancer dormancy and reacquisition of an epithelial state drives awakening

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