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Regulation of mammary epithelial morphogenesis by microtubule plus-end tracking proteins

Final Report Summary - 3D HMEC-MT-TIP (Regulation of mammary epithelial morphogenesis by microtubule plus-end tracking proteins)

Introduction

Cell migration is a complex process that requires cell interaction with the extracellular matrix and the coordination of the two major cytoskeletal networks, actin filaments and microtubules. While actin dynamics was extensively studied in the context of cell migration in both two-dimensional (2D) and three-dimensional (3D) culture systems, relatively little is known about the role of microtubules in this process. Moreover, it is admitted that extrapolation of the results obtained in 2D cell culture models to malignant tissue process has a limited predictive power. The invasion of the basement membrane and the trans-stromal cell migration that are thought to contribute to the metastatic dissemination in carcinoma obviously occur in the 3D tissue context, and therefore should be studied using 3D models. Besides, molecular mechanisms underlying early metastatic steps in breast tumor cells is complicated by the morphological plasticity displayed by malignant cells. The process called epithelial-mesenchymal transition (EMT), which physiologically drives crucial morphogenetic events in both vertebrate and invertebrate embryos, is currently thought to contribute to both collective and single-cell invasion in breast cancer. Strikingly, the role of microtubules is this process has been poorly investigated. Finally, microtubules are one of the key targets of cancer chemotherapy and it is becoming increasingly accepted that mitosis-unrelated functions of microtubules are highly relevant for cancer therapy and that interphase microtubule functions can be an important target for therapy improvement. Now, a model allowing to study interphase microtubule role in 3D matrix invasion is needed to better understand how breast cancer cells achieve the initial step of the metastatic cascade and how those functions can be altered by microtubule-targeted therapeutic strategy.

Objectives

The goal of this project was to identify microtubule related functions that regulate the behavior of human mammary epithelial cells grown in 3D and control the invasive process driven by their mesenchymal transformed counterpart. This project fits very well within European priorities for scientific research in biomedical sciences due to the relevance of metastasis in human breast cancer, which leads to considerable costs for the society and individuals in the European Union.

Results

In order to study the role of microtubules in breast cancer cell invasion, we established several original breast cancer cell lines showing mesenchymal invasion when grown in a 3D matrix and expressing fluorescent makers for microtubule and actin. Notably, the 3D growth of MDA-MB-231 cells expressing EB3-GFP, a marker for growing microtubule plus end, and Lifeact-mCherry, a marker for polymerized actin, combined with a new microscopy setup permitted to capture dynamics of these cytoskeletal structures in 3D with a comparable resolution to what is generally achieved in 2D cultures. This original setup allowed us to establish that microtubule plus ends target the cell edge at the tip of 3D invasive protrusions in a much different fashion than they do in the lamella of 2D growing cells. The most striking result is the persistence of quasi-stationary growing plus ends at the tip of 3D protrusions for 20 seconds in average and up to almost 2 minutes whereas similar events typically last for 1-2 seconds in average in 2D. Moreover, the inactivation of SLAIN2, a recently identified plus end tracking protein (+TIP) in the Akhmanova lab that act as a “molecular glue” by binding to multiple microtubule regulators and recruiting the microtubule polymerase ch-TOG to microtubule plus ends to ensure persistent microtubule growth. We found that the inactivation of SLAIN2 and the subsequent attenuation of persistent microtubule growth without major impairment of the overall microtubule network has a dramatic effect on 3D migrating cells by suppressing the formation of long protrusions required for mesenchymal invasion while this has no effect on 2D growing cell morphology and migration. We completed this demonstration by using microtubule specific targeting agents like vinblastine and paclitaxel currently employed in first line for the treatment of metastatic breast cancer. Moderate doses of those agents that have different mechanism of action commonly resulted in the attenuation of attenuation of persistent microtubule growth without major disruption of the overall microtubule network and 2D migration. Those treatments, similarly to SLAIN2 inactivation, suppressed mesenchymal invasion. Finally, by using a new EMT-inducible model, we demonstrated that persistent microtubule growth is specifically required for EMT-driven 3D matrix invasion in human mammary cells.

Conclusions and socio-economic impact

This project unravels a new function of microtubule that specifically applies to 3D invasive cells. In this context, invasion displays a much higher sensitivity to microtubule targeting than expected from 2D studies. This project has enhanced the overall competitiveness of European science in the key fields of cancer research and cytoskeleton biology. Based on this project, the rationale behind the use of current microtubule-targeting drugs and the design of new molecules with similar mechanism of action for the treatment of breast cancer should be significantly refined. The consequences of such an improvement are a better management of metastatic breast cancer patients with a decreased mortality in EU citizens and, in parallel, a potentially huge benefit in term of drug discovery-related profit for the European Union.