The clinical success of immunotherapy that inhibits the negative immune regulatory pathway programmed cell death protein 1/PD-1 ligand (PD-1/PD-L1) has initiated a new era in the treatment of metastatic cancer. PD-L1 expression is upregulated in many solid tumors including lung cancer and functions predominantly in lactate-enriched tumor microenvironments. Here, we provided evidence for PD-L1 induction in response to lactate stimulation in lung cancer cells. Lactate-induced PD-L1 induction was mediated by its receptor GPR81. The silencing of GPR81 signaling in lung cancer cells resulted in a decrease in PD-L1 protein levels and functional inactivation of PD-L1 promoter activity. In addition, GPR81-mediated upregulation of PD-L1 in glucose-stimulated lung cancer cells that recapitulates the enhanced glycolysis in vivo was dependent on lactate dehydrogenase A (LDHA). We also demonstrated that activation of GPR81 decreases intracellular cAMP levels and inhibits protein kinase A (PKA) activity, leading to activation of the transcriptional coactivator TAZ. Interaction of TAZ with the transcription factor TEAD was essential for TAZ activation of PD-L1 and induction of its expression. Furthermore, we found that lactate-induced activation of PD-L1 in tumor cells led to reduced production of interferon-γ and induction of apoptosis of cocultured Jurkat T-cell leukemia cells. Our findings reveal an unexpected role of lactate in contributing to tumor cell protection from cytotoxic T-cell targeting and establishes a direct connection between tumor cell metabolic reprograming and tumor evasion from the immune response.

The Hippo-YAP/TAZ signaling pathway is an evolutionarily conserved signaling pathway involved in a broad spectrum of biological processes, including tumorigenesis. Whilst aberrant Hippo-YAP/TAZ signaling is frequently reported in various cancers, the genetic alterations of this pathway are relatively rare, suggesting regulation at the post-transcriptional level. MicroRNAs play key role in tumorigenesis by regulating gene expression post-transcriptionally. Amongst the cancer-relevant microRNAs, miR-582-5p suppresses cell growth and tumorigenesis by inhibiting the expression of oncogenes, including AKT3, MAP3K2 and NOTCH1. Given the oncogenic role of YAP/TAZ in solid tumors, we scrutinized the possible interplay between miR-582-5p and Hippo-YAP/TAZ signaling. Correlation analysis in NSCLC cells revealed a positive relationship between the expression of mature miR-582-5p and the proportion of phosphorylated YAP/TAZ. Intriguingly, YAP/TAZ knockdown reduced the expression of mature miR-582-5p but increased that of primary miR-582. Overexpression of miR-582-5p resulted in increased phosphorylation of YAP/TAZ with a concomitant reduction in cell proliferation and enhanced apoptosis. Mechanistically, we find that miR-582-5p targets actin regulators NCKAP1 and PIP5K1C, which may be responsible for the observed alteration in F-actin, known to modulate YAP/TAZ. We postulate that regulation of the actin cytoskeleton by miR-582-5p may attenuate YAP/TAZ activity. Altogether, this study reveals a novel mechanism of YAP/TAZ regulation by miR-582-5p in a cytoskeleton-dependent manner and suggests a negative feedback loop, highlighting the therapeutic potential of restoring miR-582-5p expression in treating NSCLC.

Long non-coding RNAs (lncRNAs) regulate numerous biological processes involved in both development and carcinogenesis. Hippo-YAP/TAZ signaling, a critical pathway responsible for organ size control, is often dysregulated in a variety of cancers. However, the nature and function of YAP/TAZ-regulated lncRNAs during tumorigenesis remain largely unexplored. By profiling YAP/TAZ-regulated lncRNAs, we identified SFTA1P as a novel transcriptional target and a positive feedback regulator of YAP/TAZ signaling. Using non-small cell lung cancer (NSCLC) cell lines, we show that SFTA1P is transcriptionally activated by YAP/TAZ in a TEAD-dependent manner. Functionally, knockdown of SFTA1P in NSCLC cell lines inhibited proliferation, induced programmed cell death, and compromised their tumorigenic potential. Mechanistically, SFTA1P knockdown decreased TAZ protein abundance and consequently, the expression of YAP/TAZ transcriptional targets. We provide evidence that this phenomenon could potentially be mediated via its interaction with TAZ mRNA to regulate TAZ translation. Our results reveal SFTA1P as a positive feedback regulator of Hippo-YAP/TAZ signaling, which may serve as the molecular basis for lncRNA-based therapies against YAP/TAZ-driven cancers.

YAP and its paralog TAZ are the nuclear effectors of the Hippo tumour-suppressor pathway, and function as transcriptional co-activators to control gene expression in response to mechanical cues. To identify both common and unique transcriptional targets of YAP and TAZ in gastric cancer cells, we carried out RNA-sequencing analysis of overexpressed YAP or TAZ in the corresponding paralogous gene-knockouts (KOs), TAZ KO or YAP KO, respectively. Gene Ontology (GO) analysis of the YAP/TAZ-transcriptional targets revealed activation of genes involved in platelet biology and lipoprotein particle formation as targets that are common for both YAP and TAZ. However, the GO terms for cell-substrate junction were a unique function of YAP. Further, we found that YAP was indispensable for the gastric cancer cells to re-establish cell-substrate junctions on a rigid surface following prolonged culture on a soft substrate. Collectively, our study not only identifies common and unique transcriptional signatures of YAP and TAZ in gastric cancer cells but also reveals a dominant role for YAP over TAZ in the control of cell-substrate adhesion.

The 2i-media, composed of two small molecule inhibitors (PD0325901 and CHIR99021) against MEK and GSK3-kinases, respectively, is known to establish naïve ground state pluripotency in mouse embryonic stem cells (mESCs). These inhibitors block MEK-mediated differentiation, while driving β-catenin dependent de-repression of pluripotency promoting targets. However, accumulating evidence suggest that β-catenin's association with activating TCFs (TCF7 and TCF7L2) can induce expression of several lineage-specific prodifferentiation genes. We posited that CHIR-induced upregulation of β-catenin levels could therefore compromise the stability of the naïve state in long-term cultures. Here, we investigated whether replacing CHIR with iCRT3, a small molecule that abrogates β-catenin-TCF interaction, can still retain ground state pluripotency in mESCs. Our data suggests that iCRT3 + PD mediated coinhibition of MEK and β-catenin/TCF-dependent transcriptional activity over multiple passages significantly reduces expression of differentiation markers, as compared to 2i. Furthermore, the ability to efficiently contribute toward chimera generation and germline transmission suggests that the inhibition of β-catenin's TCF-dependent transcriptional activity, independent of its protein expression level, retains the naïve ground state pluripotency in mESCs. Additionally, growth medium containing iCRT3 + PD can provide an alternative to 2i as a stable culture method. Stem Cells 2017;35:1924-1933.