Summary The Hippo pathway plays a crucial role in organ size control and tumor suppression, but its precise regulation has not been fully understood. In this study, we discovered phosphatidic acid (PA)-related lipid signaling as a key regulator of the Hippo pathway. Supplementing PA in various Hippo-activating conditions activates YAP. This PA-related lipid signaling is involved in the Rho-mediated YAP activation. Mechanistically, PA directly interacts with Hippo components LATS and NF2 to respectively disrupt the LATS-MOB1 complex formation and NF2-mediated LATS membrane translocation and activation. Inhibition of phospholipase D (PLD)-dependent PA production suppresses YAP oncogenic activities. PLD1 is highly expressed in breast cancer and positively correlates with YAP activation, suggesting their pathological relevance in breast cancer development. Taken together, our study not only reveals a role of PLD-PA lipid signaling in regulation of the Hippo pathway, but also indicates the PLD-PA-YAP axis as a potential therapeutic target for cancer treatment.

YAP1, the main Hippo pathway effector, is a potent oncogene and is overexpressed in non‐small‐cell lung cancer (NSCLC); however, the YAP1 expression pattern in small‐cell lung cancer (SCLC) has not yet been elucidated in detail. We report that the loss of YAP1 is a special feature of high‐grade neuroendocrine lung tumors. A hierarchical cluster analysis of 15 high‐grade neuroendocrine tumor cell lines containing 14 SCLC cell lines that depended on the genes of Hippo pathway molecules and neuroendocrine markers clearly classified these lines into two groups: the YAP1‐negative and neuroendocrine marker‐positive group (n = 11), and the YAP1‐positive and neuroendocrine marker‐negative group (n = 4). Among the 41 NSCLC cell lines examined, the loss of YAP1 was only observed in one cell line showing the strong expression of neuroendocrine markers. Immunostaining for YAP1, using the sections of 189 NSCLC, 41 SCLC, and 30 large cell neuroendocrine carcinoma (LCNEC) cases, revealed that the loss of YAP1 was common in SCLC (40/41, 98%) and LCNEC (18/30, 60%), but was rare in NSCLC (6/189, 3%). Among the SCLC and LCNEC cases tested, the loss of YAP1 correlated with the expression of neuroendocrine markers, and a survival analysis revealed that YAP1‐negative cases were more chemosensitive than YAP1‐positive cases. Chemosensitivity test for cisplatin using YAP1‐positive/YAP1‐negative SCLC cell lines also showed compatible results. YAP1‐sh‐mediated knockdown induced the neuroendocrine marker RAB3a, which suggested the possible involvement of YAP1 in the regulation of neuroendocrine differentiation. Thus, we showed that the loss of YAP1 has potential as a clinical marker for predicting neuroendocrine features and chemosensitivity.

Disrupted energy metabolism drives cell dysfunction and disease, but approaches to increase or preserve ATP are lacking. To generate a comprehensive metabolic map of genes and pathways that regulate cellular ATP-the ATPome-we conducted a genome-wide CRISPR interference/activation screen integrated with an ATP biosensor. We show that ATP level is modulated by distinct mechanisms that promote energy production or inhibit consumption. In our system HK2 is the greatest ATP consumer, indicating energy failure may not be a general deficiency in producing ATP, but rather failure to recoup the ATP cost of glycolysis and diversion of glucose metabolites to the pentose phosphate pathway. We identify systemslevel reciprocal inhibition between the HIF1 pathway and mitochondria; glycolysis-promoting enzymes inhibit respiration even when there is no glycolytic ATP production, and vice versa. Consequently, suppressing alternative metabolism modes paradoxically increases energy levels under substrate restriction. This work reveals mechanisms of metabolic control, and identifies therapeutic targets to correct energy failure.

Loss of anchorage to the extracellular matrix leads to apoptosis (anoikis) in normal cells, but cancerous cells are usually resistant to such stress. Here we report the pivotal role of an E3 ubiquitin ligase, ring-finger protein 126 (RNF126), in the resistance of cancer cells to the stress associated with non-adherent conditions. Non-adherent cancer cells exhibited increased flux through the tricarboxylic acid cycle via increased conversion of pyruvate to acetyl-CoA. RNF126 was found to act as a ubiquitin ligase for pyruvate dehydrogenase kinases (PDKs), resulting in their proteasomal degradation. This decrease in PDK levels allowed pyruvate dehydrogenases to catalyze the conversion of pyruvate to acetyl-CoA. Moreover, depletion of RNF126 or increased expression of PDK1 in cancer cells suppressed colony formation in soft agar as well as tumorigenicity in mice. RNF126 expression in cancer cells was found to be under the control of the extracellular signal-regulated kinase signaling pathway, which is essential for anoikis resistance. Thus, RNF126 is an attractive molecule for treating cancer by selectively targeting anchorage-independent growth.

Unlike most cells, cancer cells activate hypoxia inducible factor-1 (HIF-1) to use glycolysis even at normal oxygen levels, or normoxia. Therefore, HIF-1 is an attractive target in cancer therapy. However, the regulation of HIF-1 during normoxia is not well characterised, although Mint3 was recently found to activate HIF-1 in cancer cells and macrophages by suppressing the HIF-1 inhibitor, factor inhibiting HIF-1 (FIH-1). In this study, we analysed Mint3-binding proteins to investigate the mechanism by which Mint3 regulates HIF-1. Yeast two-hybrid screening using Mint3 as bait identified N-terminal EF-hand calcium binding protein 3 (NECAB3) as a novel factor regulating HIF-1 activity via Mint3. NECAB3 bound to the phosphotyrosine-binding domain of Mint3, formed a ternary complex with Mint3 and FIH-1, and co-localised with Mint3 at the Golgi apparatus. Depletion of NECAB3 decreased the expression of HIF-1 target genes and reduced glycolysis in normoxic cancer cells. NECAB3 mutants that binds Mint3 but lacks an intact monooxygenase domain also inhibited HIF-1 activation. Inhibition of NECAB3 in cancer cells by either expressing shRNAs or generating a dominant negative mutant reduced tumourigenicity. Taken together, the data indicate that NECAB3 is a promising new target for cancer therapy.