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MENA isoform-driven taxane resistance affects progression through the cell cycle

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The cell cortex is a contractile actin meshwork, which determines cell shape and is essential for cell mechanics, migration and division. Because the cortical thickness is below optical resolution, it has been generally considered as a thin uniform two-dimensional layer. Using two mutually attracted magnetic beads, one inside the cell and the other in the extracellular medium, we pinch the cortex of dendritic cells and provide an accurate and time resolved measure of its thickness. Our observations draw a new picture of the cell cortex as a highly dynamic layer, harboring large fluctuations in its third dimension due to actomyosin contractility. We propose that the cortex dynamics might be responsible for the fast shape changing capacity of highly contractile cells that use amoeboid-like migration.

Cell migration depends on a combination of the cell's intrinsic capacity to move and the proper interpretation of external cues. This multistep process enables leukocytes to travel long distances in organs in just a few hours. This fast migration is partly due to the leukocytes' high level of plasticity, which helps them to adapt to a changing environment. Here, we review recent progress in understanding the mechanisms used by leukocytes to move rapidly and efficiently in intricate anatomical landscapes. We shall focus on specific cytoskeletal rearrangements used by neutrophils and dendritic cells to migrate within confined environments. Lastly, we will describe the properties that facilitate the rapid migration of leukocyte in complex tissue geometries.

Single cells migrate in a myriad of physiological contexts, such as tissue patrolling by immune cells, and during neurogenesis and tissue remodeling, as well as in metastasis, the spread of cancer cells. To understand the basic principles of single-cell migration, a reductionist approach can be taken. This aims to control and deconstruct the complexity of different cellular microenvironments into simpler elementary constrains that can be recombined together. This approach is the cell microenvironment equivalent of in vitro reconstituted systems that combine elementary molecular players to understand cellular functions. In this Cell Science at a Glance article and accompanying poster, we present selected experimental setups that mimic different events that cells undergo during migration in vivo. These include polydimethylsiloxane (PDMS) devices to deform whole cells or organelles, micro patterning, nano-fabricated structures like grooves, and compartmentalized collagen chambers with chemical gradients. We also outline the main contribution of each technique to the understanding of different aspects of single-cell migration.

In multicellular organisms, cell migration is a complex process. Examples of this are observed during cell motility in the interstitial space, full of extracellular matrix fibers, or when cells pass through endothelial layers to colonize or exit specific tissues. A common parameter for both situations is the fast adaptation of the cellular shape to their irregular landscape. In this chapter, we describe two methods to study cell migration in complex environments. The first one consists in a multichamber device for the visualization of cell haptotaxis toward the collagen-binding chemokine CCL21. This method is used to study cell migration as well as deformations during directed motility, as in the interstitial space. The second one consists in microfabricated channels connected to small constrictions. This procedure allows the study of cell deformations when single cells migrate through small holes and it is analogous to passage of cells through endothelial layers, resulting in a simplified system to study the mechanisms operating during transvasation. Both methods combined provide a powerful hub for the study of cell plasticity during migration in complex environments.

In multicellular organisms, cell migration is a complex process. Examples of this are observed during cell motility in the interstitial space, full of extracellular matrix fibers, or when cells pass through endothelial layers to colonize or exit specific tissues. A common parameter for both situations is the fast adaptation of the cellular shape to their irregular landscape. In this chapter, we describe two methods to study cell migration in complex environments. The first one consists in a multichamber device for the visualization of cell haptotaxis toward the collagen-binding chemokine CCL21. This method is used to study cell migration as well as deformations during directed motility, as in the interstitial space. The second one consists in microfabricated channels connected to small constrictions. This procedure allows the study of cell deformations when single cells migrate through small holes and it is analogous to passage of cells through endothelial layers, resulting in a simplified system to study the mechanisms operating during transvasation. Both methods combined provide a powerful hub for the study of cell plasticity during migration in complex environments.

Dendritic cells (DCs) patrol their environment by linking antigen acquisition by macropinocytosis to cell locomotion. DC activation upon bacterial sensing inhibits macropinocytosis and increases DC migration, thus promoting the arrival of DCs to lymph nodes for antigen presentation to T cells. The signaling events that trigger such changes are not fully understood. We show that lysosome signaling plays a critical role in this process. Upon bacterial sensing, lysosomal calcium is released by the ionic channel TRPML1 (transient receptor potential cation channel, mucolipin subfamily, member 1), which activates the actin-based motor protein myosin II at the cell rear, promoting fast and directional migration. Lysosomal calcium further induces the activation of the transcription factor EB (TFEB), which translocates to the nucleus to maintain TRPML1 expression. We found that the TRPML1-TFEB axis results from the down-regulation of macropinocytosis after bacterial sensing by DCs. Lysosomal signaling therefore emerges as a hitherto unexpected link between macropinocytosis, actomyosin cytoskeleton organization, and DC migration.

Taxane therapy remains the standard of care for triple-negative breast cancer. However, high frequencies of recurrence and progression in treated patients indicate that metastatic breast cancer cells can acquire resistance to this drug. The actin regulatory protein MENA and particularly its invasive isoform, MENAINV, are established drivers of metastasis. MENAINV expression is significantly correlated with metastasis and poor outcome in human patients with breast cancer. We investigated whether MENA isoforms might play a role in driving resistance to chemotherapeutics. We find that both MENA and MENAINV confer resistance to the taxane paclitaxel, but not to the widely used DNA-damaging agents doxorubicin or cisplatin. Furthermore, paclitaxel treatment does not attenuate growth of MENAINV-driven metastatic lesions. Mechanistically, MENA isoform expression alters the ratio of dynamic and stable microtubule populations in paclitaxel-treated cells. MENA expression also increases MAPK signaling in response to paclitaxel treatment. Decreasing ERK phosphorylation by co-treatment with MEK inhibitor restored paclitaxel sensitivity by driving microtubule stabilization in MENA isoform–expressing cells. Our results reveal a novel mechanism of taxane resistance in highly metastatic breast cancer cells and identify a combination therapy to overcome such resistance. Mol Cancer Ther; 16(1); 143–55. ©2016 AACR.

For patients with metastatic triple-negative breast cancer (TNBC), where metastasis occurs in 30% of patients, treatment options are limited to chemotherapy, given the lack of any effective targeted therapies approved for this subtype. However, the response rate is low and the time to relapse is short. While most chemotherapeutic drugs target proliferating cells, we set out to investigate the effect of these drugs on metastatic cells and identify metastasis-specific mechanisms of resistance. In preliminary experiments, mice with triple-negative xenograft tumors and known metastatic disease were treated with taxol and doxorubicin, two drugs commonly used to treat metastatic TNBC patients. Both drugs significantly slowed tumor growth; however, taxol, but not doxorubicin, treatment reduced cell motility within the primary tumor as measured by intravital imaging. These data support the idea that chemotherapeutics can have different effects on growth and motility in vivo, and that effects on tumor size might not correlate with changes in cell motility. We first focused on identifying tumor-intrinsic mechanisms of resistance to chemotherapy. We found that cells expressing the proinvasive isoform of the actin regulatory protein Mena are resistant to the taxane paclitaxel, but not to the widely used DNA-damaging agents doxorubicin or cisplatin. Furthermore, paclitaxel treatment does not attenuate growth of MenaINV-driven metastatic lesions or reduce metastatic burden. Mechanistically, Mena isoform expression alters the ratio of dynamic and stable microtubule populations in paclitaxel-treated cells, while also increasing MAPK signaling in response to paclitaxel treatment. Our data indicate that highly metastatic cells may respond differently to chemotherapeutic drugs than nonmetastatic ones. Second, we investigated the role of tumor extrinsic factors on chemoresistance of metastatic cells, focusing on the extracellular matrix, which is known to drive local invasion and metastasis. We found that in vitro, plating TNBC cells on collagen renders cells more resistant to doxorubicin, but not taxol. In vivo, both taxol and doxorubicin treatments led to increases in collagen deposition. Our data suggest that chemotherapeutic treatments can therefore lead to changes in tumor ECM amount and can also change how cells respond to tumor ECM. Further studies are under way to identify the role of the matrix in chemoresistance. Together, these data indicate that chemotherapeutic drugs, commonly used to treat metastatic disease, have different effects on cell growth vs. cell migration, and that chemotherapy can lead to the generation of prometastatic environments. Citation Format: Madeleine J. Oudin, Lucie Barbier, Tatiana Kosciuk, Emanuel Kreidl, Frank Gertler. Novel tumor intrinsic vs. extrinsic mechanisms of resistance to chemotherapy in metastatic disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4031.