The Hippo pathway plays essential roles in organ size control and cancer prevention via restricting its downstream effector, Yes-associated protein (YAP). Previous studies have revealed an oncogenic function of YAP in reprogramming glucose metabolism, while the underlying mechanism remains to be fully clarified. Accumulating evidence suggests long noncoding RNAs (lncRNAs) as attractive therapeutic targets, given their roles in modulating various cancer-related signaling pathways. In this study, we report that lncRNA breast cancer anti-estrogen resistance 4 (BCAR4) is required for YAP-dependent glycolysis. Mechanistically, YAP promotes the expression of BCAR4, which subsequently coordinates the Hedgehog signaling to enhance the transcription of glycolysis activators HK2 and PFKFB3. Therapeutic delivery of locked nucleic acids (LNAs) targeting BCAR4 attenuated YAP-dependent glycolysis and tumor growth. The expression levels of BCAR4 and YAP are positively correlated in tissue samples from breast cancer patients, where high expression of both BCAR4 and YAP is associated with poor patient survival outcome. Taken together, our study not only reveals the mechanism by which YAP reprograms glucose metabolism, but also highlights the therapeutic potential of targeting YAP-BCAR4-glycolysis axis for breast cancer treatment.

Cancer cells entail metabolic adaptation and microenvironmental remodeling to survive and progress. Both calcium (Ca) flux and Ca-dependent signaling play a crucial role in this process, although the underlying mechanism has yet to be elucidated. Through RNA screening, we identified one long noncoding RNA (lncRNA) named CamK-A (lncRNA for calcium-dependent kinase activation) in tumorigenesis. CamK-A is highly expressed in multiple human cancers and involved in cancer microenvironment remodeling via activation of Ca-triggered signaling. Mechanistically, CamK-A activates Ca/calmodulin-dependent kinase PNCK, which in turn phosphorylates IκBα and triggers calcium-dependent nuclear factor κB (NF-κB) activation. This regulation results in the tumor microenvironment remodeling, including macrophage recruitment, angiogenesis, and tumor progression. Notably, our human-patient-derived xenograft (PDX) model studies demonstrate that targeting CamK-A robustly impaired cancer development. Clinically, CamK-A expression coordinates with the activation of CaMK-NF-κB axis, and its high expression indicates poor patient survival rate, suggesting its role as a potential biomarker and therapeutic target.

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Voltage-gated Na and Ca2+ channels represent two major ion channel families that enable myriad biological functions including the generation of action potentials and the coupling of electrical and chemical signaling in cells. Calmodulin regulation (calmodulation) of these ion channels comprises a vital feedback mechanism with distinct physiological implications. Though long-sought, a shared understanding of the channel families remained elusive for two decades as the functional manifestations and the structural underpinnings of this modulation often appeared to diverge. Here, we review recent advancements in the understanding of calmodulation of Ca2+ and Na channels that suggest a remarkable similarity in their regulatory scheme. This interrelation between the two channel families now paves the way towards a unified mechanistic framework to understand vital calmodulin-dependent feedback and offers shared principles to approach related channelopathic diseases. An exciting era of synergistic study now looms.

Long noncoding RNAs (lncRNAs) are emerging as a new class of important regulators of signal transduction in tissue homeostasis and cancer development. Liquid-liquid phase separation (LLPS) occurs in a wide range of biological processes, while its role in signal transduction remains largely undeciphered. In this study, we uncovered a lipid-associated lncRNA, small nucleolar RNA host gene 9 (SNHG9) as a tumor-promoting lncRNA driving liquid droplet formation of Large Tumor Suppressor Kinase 1 (LATS1) and inhibiting the Hippo pathway. Mechanistically, SNHG9 and its associated phosphatidic acids (PA) interact with the C-terminal domain of LATS1, promoting LATS1 phase separation and inhibiting LATS1-mediated YAP phosphorylation. Loss of SNHG9 suppresses xenograft breast tumor growth. Clinically, expression of SNHG9 positively correlates with YAP activity and breast cancer progression. Taken together, our results uncover a novel regulatory role of a tumor-promoting lncRNA (i.e., SNHG9) in signal transduction and cancer development by facilitating the LLPS of a signaling kinase (i.e., LATS1).

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Förster resonance energy transfer (FRET) is a versatile method for analyzing protein-protein interactions within living cells. This protocol describes a nondestructive live-cell FRET assay for robust quantification of relative binding affinities for protein-protein interactions. Unlike other approaches, our method correlates the measured FRET efficiencies to relative concentration of interacting proteins to determine binding isotherms while including collisional FRET corrections. We detail how to assemble and calibrate the equipment using experimental and theoretical procedures. A step-by-step protocol is given for sample preparation, data acquisition and analysis. The method uses relatively inexpensive and widely available equipment and can be performed with minimal training. Implementation of the imaging setup requires up to 1 week, and sample preparation takes ∼1-3 d. An individual FRET experiment, including control measurements, can be completed within 4-6 h, with data analysis requiring an additional 1-3 h.

SUMMARY Biology has been revolutionized by tools that allow the detection and characterization of protein-protein interactions (PPI). Förster resonance energy transfer (FRET)-based methods have become particularly attractive as they allow quantitative studies of PPIs within the convenient and relevant context of living cells. We describe here an approach that allows the rapid construction of live-cell, FRET-based binding curves using a commercially-available flow cytometer. We illustrate a simple method for absolutely calibrating the cytometer, validate our binding assay against the gold-standard isothermal calorimetry (ITC), and use flow cytometric FRET to uncover the structural and functional effects of the Cushing syndrome-causing mutation (L206R) on PKA’s catalytic subunit. We discover that this mutation not only differentially affects PKAcat’s binding to its multiple partners, but also impacts its rate of catalysis. These findings improve our mechanistic understanding of this disease-causing mutation, while illustrating the simplicity, general applicability and power of flow cytometric FRET.