Focal adhesion kinase-YAP signaling axis drives drug-tolerant persister cells and residual disease in lung cancer.

Targeted therapy is effective in many tumor types including lung cancer, the leading cause of cancer mortality. Paradigm defining examples are targeted therapies directed against non-small cell lung cancer (NSCLC) subtypes with oncogenic alterations in EGFR, ALK and KRAS. The success of targeted therapy is limited by drug-tolerant persister cells (DTPs) which withstand and adapt to treatment and comprise the residual disease state that is typical during treatment with clinical targeted therapies. Here, we integrate studies in patient-derived and immunocompetent lung cancer models and clinical specimens obtained from patients on targeted therapy to uncover a focal adhesion kinase (FAK)-YAP signaling axis that promotes residual disease during oncogenic EGFR-, ALK-, and KRAS-targeted therapies. FAK-YAP signaling inhibition combined with the primary targeted therapy suppressed residual drug-tolerant cells and enhanced tumor responses. This study unveils a FAK-YAP signaling module that promotes residual disease in lung cancer and mechanism-based therapeutic strategies to improve tumor response.

Basolateral Sorting of the Sodium/Iodide Symporter Is Mediated by Adaptor Protein 1 Clathrin Adaptor Complexes.

Background: The sodium/iodide symporter (NIS) is a transmembrane protein located on the basolateral membrane of thyrocytes. Despite its physiological and clinical relevance, little is known about the mechanisms that mediate NIS subcellular sorting. In the present study, we examined NIS basolateral trafficking in vitro using non-thyroid and thyroid epithelial cells. Methods: Immunofluorescence and Western blotting were performed to analyze NIS subcellular location and function in cells grown in monolayers under unpolarized and/or polarized conditions. Strategic NIS residues were mutated, and binding of NIS to clathrin adaptor complexes was determined by immunoprecipitation. Results: We show that NIS reaches the plasma membrane (PM) through a thyrotropin-dependent mechanism 24 hours after treatment with the hormone. We demonstrate that NIS basolateral trafficking is a clathrin-mediated mechanism, in which the clathrin adaptor complexes AP-1 (A and B) sort NIS from the trans-Golgi network (TGN) and recycling endosomes (REs). Specifically, we show that the AP-1B µ1 subunit controls NIS basolateral sorting through common REs. In its absence, NIS is apically missorted but remains functional. Additionally, direct NIS basolateral transport from the TGN to the basolateral membrane is mediated by AP-1A through clathrin-coated vesicles that also carry the transferrin receptor. Loss of the µ1 subunit of AP-1A is functionally compensated by AP-1B. Furthermore, loss of both subunits diminishes NIS trafficking to the PM. Finally, we demonstrate that AP-1A binds to the L121 and LL562/563 residues on NIS, whereas AP-1B binds to L583. Conclusions: Our findings highlight the novel involvement of the clathrin-coated machinery in basolateral NIS trafficking. Given that AP-1A expression is reduced in tumors, and its expression correlates with that of NIS, these findings will help uncover new targets in thyroid cancer treatment.

A Critical Balance Between PAX8 and the Hippo Mediator TAZ Determines Sodium/Iodide Symporter Expression and Function.

Background: The Hippo pathway has a fundamental role in tissue homeostasis, but little is known about how this signaling cascade is controlled in the thyroid. PAX8 is an essential driver of thyroid differentiation and is involved in the control of genes crucial for thyroid hormone biosynthesis, including the sodium/iodide symporter (NIS; SLC5A5). A role for the Hippo mediator transcriptional coactivator with PDZ-binding motif (TAZ) as a coactivator of PAX8 to promote thyroglobulin expression has been previously described. Here, we studied the role of TAZ on thyroid differentiation focusing on PAX8-mediated Slc5a5 transcription. Methods: Gene silencing and overexpression assays were performed in rat PCCl3 thyroid follicular cells (TFCs) to determine the role of TAZ in the regulation of Slc5a5. Transcriptional activity of the Hippo mediators was investigated by chromatin immunoprecipitation and promoter-reporter gene activity. Hippo component levels and location were analyzed in PCCl3 cells and in mouse thyroid under different treatment conditions. Results: By suppressing the expression of PAX8 and its binding to the Slc5a5 upstream enhancer, TAZ inhibits Slc5a5 expression, impairing NIS membrane location and activity. Other Hippo effectors such as YAP1 and TEAD1 were not required for the repressor effect of TAZ. We also found an interplay between the Hippo, thyrotropin (TSH), and transforming growth factor ß1 (TGFß) pathways in TFCs. TSH via cyclic adenosine monophosphate activated Hippo signaling pathway and, consequently, TAZ was excluded from the nucleus. We confirmed this in hypothyroid mice, characterized by elevated TSH serum levels, which showed downregulated activation of Hippo signaling in thyroid. Conversely, TAZ nuclear retention was promoted by TGFß, a potent NIS repressor, and TAZ silencing markedly relieved the TGFß-induced inhibition of the symporter. Conclusions: We demonstrate that the effects of TAZ are promoter specific, as it functions as a corepressor of PAX8 to modulate Slc5a5 expression in TFCs. Overall, our data place TAZ as an integrator of the different signaling pathways that control NIS expression, pointing to a role for TAZ in thyroid differentiation and identifying the Hippo pathway as a relevant target to recover NIS levels in thyroid cancer cells.