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.

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.

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.

Unraveling the Complex Interplay Between Transcription Factors and Signaling Molecules in Thyroid Differentiation and Function, From Embryos to Adults.

Thyroid differentiation of progenitor cells occurs during embryonic development and in the adult thyroid gland, and the molecular bases of these complex and finely regulated processes are becoming ever more clear. In this Review, we describe the most recent advances in the study of transcription factors, signaling molecules and regulatory pathways controlling thyroid differentiation and development in the mammalian embryo. We also discuss the maintenance of the adult differentiated phenotype to ensure the biosynthesis of thyroid hormones. We will focus on endoderm-derived thyroid epithelial cells, which are responsible for the formation of the thyroid follicle, the functional unit of the thyroid gland. The use of animal models and pluripotent stem cells has greatly aided in providing clues to the complicated puzzle of thyroid development and function in adults. The so-called thyroid transcription factors - Nkx2-1, Foxe1, Pax8 and Hhex - were the first pieces of the puzzle identified in mice. Other transcription factors, either acting upstream of or directly with the thyroid transcription factors, were subsequently identified to, almost, complete the puzzle. Among them, the transcription factors Glis3, Sox9 and the cofactor of the Hippo pathway Taz, have emerged as important players in thyroid differentiation and development. The involvement of signaling molecules increases the complexity of the puzzle. In this context, the importance of Bmps, Fgfs and Shh signaling at the onset of development, and of TSH, IGF1 and TGFß both at the end of terminal differentiation in embryos and in the adult thyroid, are well recognized. All of these aspects are covered herein. Thus, readers will be able to visualize the puzzle of thyroid differentiation with most - if not all - of the pieces in place.

Regulation of Foxe1 by Thyrotropin and Transforming Growth Factor Beta Depends on the Interplay Between Thyroid-Specific, CREB and SMAD Transcription Factors.

Background: Thyroid follicular cells are characterized by the expression of a specific set of genes necessary for the synthesis and secretion of thyroid hormones, which are in turn regulated by the transcription factors Nkx2-1, Pax8, and Foxe1. Thyroid differentiation is finely tuned by the balance between positive regulatory signals, including thyrotropin (TSH), and by negative regulatory signals, such as transforming growth factor beta (TGF-ß), which counteracts the action of TSH. A role for Foxe1 as a mediator of hormonal and growth-factor control of thyroid differentiation has been previously suggested. Therefore, the aim of this work was to study the mechanisms governing Foxe1 expression to define the ligands and signals that regulate one of the important factors in thyroid differentiation. Methods: Expression of Foxe1 was evaluated in rat PCCl3 thyroid follicular cells under different treatments. The mouse Foxe1 promoter was cloned, and site-directed mutagenesis was undertaken to study its transcriptional regulation and to identify response elements. Protein/DNA binding assays were performed to evaluate the binding of different transcription factors, and gene-silencing approaches were used to elucidate their functional roles. Results:In silico analysis of the Foxe1 promoter identified binding sites for Nkx2-1, Pax8, Foxe1, and Smad proteins, as well as cAMP-response element (CRE) sites. It was found that both CRE-binding protein and CRE modulator were necessary for the TSH-mediated induction of Foxe1 expression via the cAMP/PKA signaling pathway. Moreover, transcription of Foxe1 was regulated by Nkx2-1 and Pax8 and by itself, suggesting an autoregulatory mechanism of activation and an important role for thyroid transcription factors. Finally, TGF-ß, through Smad proteins, inhibited the TSH-induced Foxe1 expression. Conclusions: This study shows that Foxe1 is the final target of TSH/cAMP and TGF-ß regulation that mediates expression of thyroid differentiation genes, and provides evidence of an interplay between CRE-binding proteins, thyroid transcription factors, and Smad proteins in its regulation. Thus, Foxe1 plays an important role in the complex transcriptional network that regulates thyroid follicular cell differentiation.