Poster S3 - Impact of translation regulation during the metastasis cascade

Introduction: In humans, more than 80% of tumors originate from epithelial cells and their progression leads to metastasis, which is responsible for close to 90% of cancer-related death. Cancer progression and dissemination are complex processes that requires several interrelated biological mechanisms that force cancer cells to rapidly adapt to survive and progress through the metastatic cascade. First, to leave the primary tumor, cancer cells undergo an epithelial to mesenchymal transition (EMT) and invade the surrounding stroma. Tumor cells that underwent EMT must then intravasate the circulatory network to become circulating tumor cells (CTCs), survive the shear stress of the blood flow, and then extravasate when they reach a distal metastatic niche. For that, cells must adapt to different stresses (i.e. shear stress) to progress through the metastatic cascade. Our initial work on the metastatic cell adhesion allowed us to unveil the importance of mRNA translation during the early phases of the adhesion. Moreover, we identified adhesion-related proteins that are highly translated during the initial phase of adhesion of mesenchymal-like cells. In addition, we identified HSP70 and HSP90, which are known for their essential role in the unfolded protein response. We thus hypothesize that these HSPs could be involved in the proper folding of newly translated mRNAs involved in this mesenchymal-like adhesion mechanism. To assess this possibility, we investigated this adhesion specific translational regulation and the role of the HSPs in this critical step of the metastatic cascade.

Materials and

Methods: Our experiments were performed on MDA-MB-231, a highly metastatic cancer cell line that mainly expresses mesenchymal markers. As a counterpart, we used a less invasive cell line (MDA-MB-468), that predominantly expresses epithelial markers. To visualize how the de novo translated proteins affect CTCs adhesion to endothelium and how this impact their extravasation under shear stress conditions, we designed and three-dimensional micro-fluidic chambers. HUVECs (Human Umbilical Vein Endothelial Cells) were seeded throughout the microfluidic chamber channels to create an endothelial monolayer on which we can assess how translation inhibition/activation affect CTCs adhesion and invasion. This allowed us to evaluate the impact of adhesion-regulated translation in controlled conditions with well-characterized drugs (i.e. cycloheximide (inhibition) and ISRIB (activation)).

Results, Conclusions, and Discussions: Our results showed that this translation regulation during adhesion was essential, suggesting a mesenchymal specific adhesion-induced translation reprogramming. We also showed an orchestrated increase in the expression of adhesion specific proteins, as well as key HSPs (Fig. 1). Consistently, repressing their translation, via well-characterized translation inhibitors, greatly impaired mesenchymal-like cells capacity to adhere to different substrates which is not observed in epithelial cells (Fig.2). Moreover, we found that blocking adhesion through the inhibition of de novo translation impacts the transendothelial invasion capacity (Fig. 3). Finally, using our microfluidic chamber, we observed adhesion of MDA-MB-231 GFP+ on the HUVECs monolayer with the flow (Fig. 4) and we are now testing the impact of ISRIB and cycloheximide during the adhesion. Our findings indicate that mesenchymal-like CTCs can exploit stress-induced response mechanisms to reprogram their translation, enabling their adaptation and survival during bloodstream invasion and their subsequent extravasation. Indeed, our results suggest that resuspension-induced stress restricts adhesion by significantly reducing mRNA translation in mesenchymal cells. Similar mechanisms appear to operate in metastatic cells progressed through EMT, suggesting a maintained translational regulation in response to bloodstream shear stress. This implies that spatio-temporal translation of adhesion proteins can influence the decision to initiate adhesion to facilitate CTCs extravasation and colonization.

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