Nattapon Panupinthu

Regulation of the Hippo-YAP pathway by G-protein coupled receptor signaling

The Hippo pathway is crucial in organ size control, and its dysregulation contributes to tumorigenesis. However, upstream signals that regulate the mammalian Hippo pathway have remained elusive. Here, we report that the Hippo pathway is regulated by G-protein-coupled receptor (GPCR) signaling. Serum-borne lysophosphatidic acid (LPA) and sphingosine 1-phosphophate (S1P) act through G12/13-coupled receptors to inhibit the Hippo pathway kinases Lats1/2, thereby activating YAP and TAZ transcription coactivators, which are oncoproteins repressed by Lats1/2. YAP and TAZ are involved in LPA-induced gene expression, cell migration, and proliferation. In contrast, stimulation of Gs-coupled receptors by glucagon or epinephrine activates Lats1/2 kinase activity, thereby inhibiting YAP function. Thus, GPCR signaling can either activate or inhibit the Hippo-YAP pathway depending on the coupled G protein. Our study identifies extracellular diffusible signals that modulate the Hippo pathway and also establishes the Hippo-YAP pathway as a critical signaling branch downstream of GPCR.

Growth of Triple-Negative Breast Cancer Cells Relies upon Coordinate Autocrine Expression of the Proinflammatory Cytokines IL-6 and IL-8

Triple-negative breast cancers (TNBC) are aggressive with no effective targeted therapies. A combined database analysis identified 32 inflammation-related genes differentially expressed in TNBCs and 10 proved critical for anchorage-independent growth. In TNBC cells, an LPA-LPAR2-EZH2 NF-κB signaling cascade was essential for expression of interleukin (IL)-6, IL-8, and CXCL1. Concurrent inhibition of IL-6 and IL-8 expression dramatically inhibited colony formation and cell survival in vitro and stanched tumor engraftment and growth in vivo. A Cox multivariable analysis of patient specimens revealed that IL-6 and IL-8 expression predicted patient survival times. Together these findings offer a rationale for dual inhibition of IL-6/IL-8 signaling as a therapeutic strategy to improve outcomes for patients with TNBCs.

Lysophosphatidic acid augments human hepatocellular carcinoma cell invasion through LPA1 receptor and MMP-9 expression

Lysophosphatidic acid (LPA), produced extracellularly by autotaxin (ATX), has diverse biological activities implicated in tumor initiation and progression, including increasing cell survival, angiogenesis, invasion and metastasis. ATX, LPA and the matrix metalloproteinase (MMP)-9 have all been implicated in hepatocellular carcinoma (HCC) invasion and metastasis. We, thus sought to determine whether ATX with subsequent LPA production and action, including induction of MMP-9 could provide a unifying mechanism. ATX transcripts and LPA receptor type 1 (LPA1) protein are elevated in HCC compared with normal tissues. Silencing or pharmacological inhibition of LPA1 significantly attenuated LPA-induced MMP-9 expression and HCC cell invasion. Further, reducing MMP-9 activity or expression significantly inhibits LPA-induced HCC cell invasion, demonstrating that MMP-9 is downstream of LPA1. Inhibition of phosphoinositide-3 kinase (PI3K) signaling or dominant-negative mutants of protein kinase Cδ and p38 mitogen-activated protein kinase (MAPK) abrogated LPA-induced MMP-9 expression and subsequent invasion. We thus demonstrate a mechanistic cascade of ATX-producing LPA with LPA activating LPA1 and inducing MMP-9 through coordinate activation of the PI3K and the p38 MPAK signaling cascades, providing novel biomarkers and potential therapeutic targets for HCC.

Lysophosphatidic acid production and action: critical new players in breast cancer initiation and progression

Lysophosphatidic acid (LPA) is a potent lipid mediator that acts on a series of specific G protein-coupled receptors, leading to diverse biological actions. Lysophosphatidic acid induces cell proliferation, survival and migration, which are critically required for tumour formation and metastasis. This bioactive lipid is produced by the ectoenzyme lysophospholipase D or autotaxin (ATX), earlier known as an autocrine motility factor. The ATX–LPA signalling axis has emerged as an important player in many types of cancer. Indeed, aberrant expression of ATX and LPA receptors occurs during the development and progression of breast cancer. Importantly, expression of either ATX or LPA receptors in the mammary gland of transgenic mice is sufficient to induce the development of a high frequency of invasive and metastatic mammary cancers. The focus of research now turns to understanding the mechanisms by which ATX and LPA promote mammary tumourigenesis and metastasis. Targeting the ATX–LPA signalling axis for drug development may further improve outcomes in patients with breast cancer.

ATX-LPA receptor axis in inflammation and cancer

Lysophosphatidic acid (LPA, 1- or 2-acyl-sn-glycerol 3-phosphate) mediates a plethora of physiological and pathological activities via interactions with a series of high affinity G protein-coupled receptors (GPCR). Both LPA receptor family members and autotaxin (ATX/LysoPLD), the primary LPA-producing enzyme, are aberrantly expressed in many human breast cancers and several other cancer lineages. Using transgenic mice expressing either an LPA receptor or ATX, we recently demonstrated that the ATX-LPA receptor axis plays a causal role in breast tumorigenesis and cancerrelated inflammation, further validating the ATX-LPA receptor axis as a rich therapeutic target in cancer.

Benzyl and naphthalene methylphosphonic acid inhibitors of autotaxin with anti-invasive and anti-metastatic activity

Autotaxin (ATX, NPP2) is a member of the nucleotide pyrophosphate phosphodiesterase enzyme family. ATX catalyzes the hydrolytic cleavage of lysophosphatidylcholine (LPC) by lysophospholipase D activity, which leads to generation of the growth‐factor‐like lipid mediator lysophosphatidic acid (LPA). ATX is highly upregulated in metastatic and chemotherapy‐resistant carcinomas and represents a potential target to mediate cancer invasion and metastasis. Herein we report the synthesis and pharmacological characterization of ATX inhibitors based on the 4‐tetradecanoylaminobenzylphosphonic acid scaffold, which was previously found to lack sufficient stability in cellular systems. The new 4‐substituted benzylphosphonic acid and 6‐substituted naphthalen‐2‐ylmethylphosphonic acid analogues block ATX activity with Ki values in the low micromolar to nanomolar range against FS3, LPC, and nucleotide substrates through a mixed‐mode inhibition mechanism. None of the compounds tested inhibit the activity of related enzymes (NPP6 and NPP7). In addition, the compounds were evaluated as agonists or antagonists of seven LPA receptor (LPAR) subtypes. Analogues 22 and 30 b, the two most potent ATX inhibitors, inhibit the invasion of MM1 hepatoma cells across murine mesothelial and human vascular endothelial monolayers in vitro in a dose‐dependent manner. The average terminal half‐life for compound 22 is 10±5.4 h and it causes a long‐lasting decrease in plasma LPA levels. Compounds 22 and 30 b significantly decrease lung metastasis of B16‐F10 syngeneic mouse melanoma in a post‐inoculation treatment paradigm. The 4‐substituted benzylphosphonic acids and 6‐substituted naphthalen‐2‐ylmethylphosphonic acids described herein represent new lead compounds that effectively inhibit the ATX–LPA–LPAR axis both in vitro and in vivo.

EGFR mediates LPA-induced proteolytic enzyme expression and ovarian cancer invasion: Inhibition by resveratrol

Lysophosphatidic acid (LPA) augments proliferation and metastasis of various cancer cells. We recently identified a critical role of the Rho/ROCK pathway for LPA‐induced proteolytic enzyme expression and cancer cell progression. In the present study, we elucidate the underlying mechanisms by which LPA induces Rho activation and subsequent cellular invasion, and the reversal of these effects by resveratrol. We observed that both Gi and G13 contribute to LPA‐induced EGFR activation. The activated EGFR in turn initiates a Ras/Rho/ROCK signaling cascade, leading to proteolytic enzyme secretion. Further we provide evidence that resveratrol inhibits EGFR phosphorylation and subsequent activation of a Ras/Rho/ROCK signaling. Therefore, we demonstrate a mechanistic cascade of LPA activating EGFR through Gi and G13 thus inducing a Ras/Rho/ROCK signaling for proteolytic enzyme expression and ovarian cancer cell invasion, as well as interference of the cascade by resveratrol through blocking EGFR phosphorylation.

Self-reinforcing loop of amphiregulin and Y-box binding protein-1 contributes to poor outcomes in ovarian cancer

The Y-box binding protein-1 (YB-1) transcription factor is associated with unfavorable clinical outcomes. However, the mechanisms underlying this association remain to be fully elucidated. We demonstrate that YB-1 phosphorylation, indicative of YB-1 activation, is a powerful marker of outcomes for ovarian cancer patients. In ovarian cancer, YB-1 phosphorylation is induced by activation of the lysophosphatidic acid (LPA) receptor (LPAR) via SRC-dependent transactivation of the epidermal growth factor receptor (EGFR) that is coupled to MAPK/p90 ribosomal S6 kinase (p90RSK), but not phosphatidylinositol 3-kinase (PI3K)/AKT signaling. Activation of the LPAR/SRC/EGFR/MAPK/p90RSK/YB-1 axis leads to production of the EGFR ligand amphiregulin (AREG). AREG induces ongoing YB-1 phosphorylation as well as YB-1-dependent AREG expression, thus constituting an AREG/YB-1 self-reinforcing loop. Disruption of transactivation of the EGFR and the downstream self-reinforcing loop decreases invasiveness of ovarian cancer cells in vitro and limits ovarian cancer growth in xenograft models. These findings established the regulation and significance of YB-1 phosphorylation, therefore further exploration of this signaling axis as a therapeutic avenue in ovarian cancer is warranted.

Synthesis and pharmacological evaluation of the stereoisomers of 3-carba cyclic-phosphatidic acid.

Cyclic phosphatidic acid (CPA) is a naturally occurring analog of lysophosphatidic acid (LPA) in which the sn-2 hydroxy group forms a five-membered ring with the sn-3 phosphate. Here, we describe the synthesis of R-3-CCPA and S-3-CCPA along with their pharmacological properties as inhibitors of lysophospholipase D/autotaxin, agonists of the LPA(5) GPCR, and blockers of lung metastasis of B16-F10 melanoma cells in a C57BL/6 mouse model. S-3CCPA was significantly more efficacious in the activation of LPA(5) compared to the R-stereoisomer. In contrast, no stereoselective differences were found between the two isomers toward the inhibition of autotaxin or lung metastasis of B16-F10 melanoma cells in vivo. These results extend the potential utility of these compounds as potential lead compounds warranting evaluation as cancer therapeutics.

YAP/TAZ-Mediated Upregulation of GAB2 Leads to Increased Sensitivity to Growth Factor–Induced Activation of the PI3K Pathway

The transcription regulators YAP and TAZ function as effectors of the HIPPO signaling cascade, critical for organismal development, cell growth, and cellular reprogramming, and YAP/TAZ is commonly misregulated in human cancers. The precise mechanism by which aberrant YAP/TAZ promotes tumor growth remains unclear. The HIPPO tumor suppressor pathway phosphorylates YAP and TAZ, resulting in cytosolic sequestration with subsequent degradation. Here, we report that the PI3K/AKT pathway, which is critically involved in the pathophysiology of endometrial cancer, interacts with the HIPPO pathway at multiple levels. Strikingly, coordinate knockdown of YAP and TAZ, mimicking activation of the HIPPO pathway, markedly decreased both constitutive and growth factor-induced PI3K pathway activation by decreasing levels of the GAB2 linker molecule in endometrial cancer lines. Furthermore, targeting YAP/TAZ decreased endometrial cancer tumor growth in vivo In addition, YAP and TAZ total and phosphoprotein levels correlated with clinical characteristics and outcomes in endometrial cancer. Thus, YAP and TAZ, which are inhibited by the HIPPO tumor suppressor pathway, modify PI3K/AKT pathway signaling in endometrial cancer. The cross-talk between these key pathways identifies potential new biomarkers and therapeutic targets in endometrial cancer. Cancer Res; 77(7); 1637-48. ©2017 AACR.