Su An

G protein-coupled receptors as promising cancer targets

G protein-coupled receptors (GPCRs) regulate an array of fundamental biological processes, such as growth, metabolism and homeostasis. Specifically, GPCRs are involved in cancer initiation and progression. However, compared with the involvement of the epidermal growth factor receptor in cancer, that of GPCRs have been largely ignored. Recent findings have implicated many GPCRs in tumorigenesis, tumor progression, invasion and metastasis. Moreover, GPCRs contribute to the establishment and maintenance of a microenvironment which is permissive for tumor formation and growth, including effects upon surrounding blood vessels, signaling molecules and the extracellular matrix. Thus, GPCRs are considered to be among the most useful drug targets against many solid cancers. Development of selective ligands targeting GPCRs may provide novel and effective treatment strategies against cancer and some anticancer compounds are now in clinical trials. Here, we focus on tumor related GPCRs, such as G protein-coupled receptor 30, the lysophosphatidic acid receptor, angiotensin receptors 1 and 2, the sphingosine 1-phosphate receptors and gastrin releasing peptide receptor. We also summarize their tissue distributions, activation and roles in tumorigenesis and discuss the potential use of GPCR agonists and antagonists in cancer therapy.

Methods used to study the oligomeric structure of G protein-coupled receptors.

G-protein-coupled receptors (GPCRs), which constitute the largest family of cell surface receptors, were originally thought to function as monomers, but are now recognized as being able to act in a wide range of oligomeric states and indeed, it is known that the oligomerization state of a GPCR can modulate its pharmacology and function. A number of experimental techniques have been devised to study GPCR oligomerization including those based upon traditional biochemistry such as blue-native PAGE (BN-PAGE), co-immunoprecipitation (Co-IP) and protein-fragment complementation assays (PCAs), those based upon resonance energy transfer, FRET, time-resolved FRET (TR-FRET), FRET spectrometry and bioluminescence resonance energy transfer (BRET). Those based upon microscopy such as FRAP, total internal reflection fluorescence microscopy (TIRFM), spatial intensity distribution analysis (SpIDA) and various single molecule imaging techniques. Finally with the solution of a growing number of crystal structures, X-ray crystallography must be acknowledged as an important source of discovery in this field. A different, but in many ways complementary approach to the use of more traditional experimental techniques, are those involving computational methods that possess obvious merit in the study of the dynamics of oligomer formation and function. Here, we summarize the latest developments that have been made in the methods used to study GPCR oligomerization and give an overview of their application.

A-Raf: A new star of the family of raf kinases

Abstract The Ras-Raf-MEK-MAPK (mitogen-activated protein kinase)-signaling pathway plays a key role in the regulation of many cellular functions, including cell proliferation, differentiation and transformation, by transmitting signals from membrane receptors to various cytoplasmic and nuclear targets. One of the key components of this pathway is the serine/threonine protein kinase, Raf. The Raf family kinases (A-Raf, B-Raf and C-Raf) have been intensively studied since being identified in the early 1980s as retroviral oncogenes, especially with respect to the discovery of activating mutations of B-Raf in a large number of tumors which led to intensified efforts to develop drugs targeting Raf kinases. This also resulted in a rapid increase in our knowledge of the biological functions of the B-Raf and C-Raf isoforms, which may in turn be contrasted with the little that is known about A-Raf. The biological functions of A-Raf remain mysterious, although it appears to share some of the basic properties of the other two isoforms. Recently, emerging evidence has begun to reveal the functions of A-Raf, of which some are kinase-independent. These include the inhibition of apoptosis by binding to MST2, acting as safeguard against oncogenic transformation by suppressing extracellular signal-regulated kinases (ERK) activation and playing a role in resistance to Raf inhibitors. In this review, we discuss the regulation of A-Raf protein expression, and the roles of A-Raf in apoptosis and cancer, with a special focus on its role in resistance to Raf inhibitors. We also describe the scaffold functions of A-Raf and summarize the unexpected complexity of Raf signaling.

Raf‐interactome in tuning the complexity and diversity of Raf function

Raf kinases have been intensely studied subsequent to their discovery 30 years ago. The Ras‐Raf‐mitogen‐activated protein kinase/extracellular signal‐regulated kinase kinase‐extracellular signal‐regulated kinase/mitogen‐activated protein kinase (Ras‐Raf‐MEK‐ERK/MAPK) signaling pathway is at the heart of the signaling networks that control many fundamental cellular processes and Raf kinases takes centre stage in the MAPK pathway, which is now appreciated to be one of the most common sources of the oncogenic mutations in cancer. The dependency of tumors on this pathway has been clearly demonstrated by targeting its key nodes; however, blockade of the central components of the MAPK pathway may have some unexpected side effects. Over recent years, the Raf‐interactome or Raf‐interacting proteins have emerged as promising targets for protein‐directed cancer therapy. This review focuses on the diversity of Raf‐interacting proteins and discusses the mechanisms by which these proteins regulate Raf function, as well as the implications of targeting Raf‐interacting proteins in the treatment of human cancer.

Rap-Interacting Proteins are Key Players in the Rap Symphony Orchestra

Rap, a member of the Ras-like small G-protein family, is a key node among G-protein coupled receptors (GPCR), receptor tyrosine kinases (RTKs), ion channels and many other downstream pathways. Rap plays a unique role in cell morphogenesis, adhesion, migration, exocytosis, proliferation, apoptosis and carcinogenesis. The complexity and diversity of Rap functions are tightly regulated by Rap-interacting proteins such as GEFs, GAPs, Rap effectors and scaffold proteins. These interacting proteins decide the subcellular localization of Rap, the interaction modes with downstream Rap effectors and tune Rap as an atypical molecular conductor, coupling extra- and intracellular signals to various pathways. In this review, we summarize four groups of Rap-interacting proteins, highlight their distinctions in Rap-binding properties and interactive modes and discuss their contribution to the spatiotemporal regulation of Rap as well as the implications of targeting Rap-interacting proteins in human cancer therapy.

Emerging role of the Jun N-terminal kinase interactome in human health: The JNK interactome

The c‐Jun N‐terminal kinases (JNKs) are located downstream of Ras‐mitogen activated protein kinase signaling cascades. More than 20 years of study has shown that JNKs control cell fate and many cellular functions. JNKs and their interacting proteins form a complicated network with diverse biological functions and physiological effects. Members of the JNK interactome include Jun, amyloid precursor protein, and insulin receptor substrate. Recent studies have shown that the JNK interactome is involved in tumorigenesis, neuron development, and insulin resistance. In this review, we summarize the features of the JNK interactome and classify its members into three groups: upstream regulators, downstream effectors, and scaffold partners. We also highlight the unique cellular signaling mechanisms of JNKs and provide more insights into the roles of the JNK interactome in human diseases.

Video-assisted thoracoscopic surgery lobectomy for lung cancer versus thoracotomy: a less decrease in sVEGFR2 level after surgery.

BACKGROUND Angiogenic and anti-angiogenic factors play an important role in tumor biology and tumor recurrence after surgical resection. Antiangiogenic factors such as vascular endothelial growth factor (VEGF)-receptor 1 (sVEGFR1) and sVEGFR2, two soluble form receptor proteins of VEGF, are critical for angiogenesis. VEGF can be sequestered by soluble forms of these receptors, which result in decreasing VEGF amount available to bind to its receptor on vascular endothelial cell surface. This study aimed to investigate the influences of video-assisted thoracoscopic surgery (VATS) lobectomy and open by thoracotomy for early stage non-small cell lung cancer (NSCLC) on postoperative circulating sVEGFR1 and sVEGFR2 levels. METHODS Forty-eight lung cancer patients underwent lobectomy through either VATS (n=26) or thoracotomy (n=22). Blood samples were collected from all patients preoperatively and postoperatively on days 1, 3 and 7. ELISA analysis was used to determine the plasma levels of sVEGFR1 and sVEGFR2. Data are reported as means and standard deviations, and were assessed with the Wilcoxon signed-Rank test (P<0.05). RESULTS For all patients undergoing lobectomy, postoperative sVEGFR1 levels on days 1 and 3 were markedly increased, while postoperative sVEGFR2 levels on days 1 and 3 were significantly decreased. Moreover, VATS group had significantly higher plasma level of sVEGFR2 postoperative in comparison with open thoracotomy (OT) on day 1 (VATS 6,953±1,535 pg/mL; OT 5,874±1,328 pg/mL, P<0.05). CONCLUSIONS Major pulmonary resection for early stage NSCLC resulted in the increased sVEGFR1 and decreased sVEGFR2 productions. VATS is associated with enhanced anti-angiogenic response with higher circulating sVEGFR2 levels compared with that with OT. Such differences in anti-angiogenic response may have an important effect on cancer biology and recurrence after surgery.

C-RAF function at the genome-wide transcriptome level: A systematic view

C-RAF was the first member of the RAF kinase family to be discovered. Since its discovery, C-RAF has been found to regulate many fundamental cell processes, such as cell proliferation, cell death, and metabolism. However, the majority of these functions are achieved through interactions with different proteins; the genes regulated by C-RAF in its active or inactive state remain unclear. In the work, we used RNA-seq analysis to study the global transcriptomes of C-RAF bearing or C-RAF knockout cells in quiescent or EGF activated states. We identified 3353 genes that are promoted or suppressed by C-RAF. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed that these genes are involved in drug addiction, cardiomyopathy, autoimmunity, and regulation of cell metabolism. Our results provide a panoramic view of C-RAF function, including known and novel functions, and have revealed potential targets for elucidating the role of C-RAF.

Decoding the full picture of Raf1 function based on its interacting proteins

Raf1 is a member of the Raf kinase family and regulates many fundamental cell processes, including proliferation, differentiation, apoptosis, motility, and metabolism. However, the functions of Raf1 have not been completely elucidated. To better understand Raf1 function, we investigated the proteins that interacted with Raf1. We identified 198 Raf1 interacting proteins and our data suggested that Raf1 may regulate cell processes through these interactions. These interaction partners were involved in all ten hallmarks of cancer, suggesting that Raf1 is involved in different aspects of carcinogenesis. In addition, we showed that Raf1 interacting proteins were enriched in six signaling pathways and many human diseases. The interaction partners identified in this study may represent oncological candidates for future investigations into Raf1 function. Our findings have provided an overview of Raf1 function from a systems biology perspective.Raf1 is a member of the Raf kinase family and regulates many fundamental cell processes, including proliferation, differentiation, apoptosis, motility, and metabolism. However, the functions of Raf1 have not been completely elucidated. To better understand Raf1 function, we investigated the proteins that interacted with Raf1. We identified 198 Raf1 interacting proteins and our data suggested that Raf1 may regulate cell processes through these interactions. These interaction partners were involved in all ten hallmarks of cancer, suggesting that Raf1 is involved in different aspects of carcinogenesis. In addition, we showed that Raf1 interacting proteins were enriched in six signaling pathways and many human diseases. The interaction partners identified in this study may represent oncological candidates for future investigations into Raf1 function. Our findings have provided an overview of Raf1 function from a systems biology perspective.

Assessing the real-time activation of the cannabinoid CB1 receptor and the associated structural changes using a FRET biosensor

The cannabinoid receptor 1 (CB1) is mainly expressed in the nervous system and regulates learning, memory processes, pain and energy metabolism. However, there is no way to directly measure its activation. In this study, we constructed a CB1 intramolecular fluorescence resonance energy transfer (FRET) sensor, which could measure CB1 activation by monitoring structural changes between the third intracellular loop and the C-terminal tail. CB1 agonists induced a time- and concentration-dependent increase in the FRET signal, corresponding to a reduction in the distance between the third intracellular loop and the C-terminal tail. This, in turn, mobilized intracellular Ca2+, inhibited cAMP accumulation, and increased phosphorylation of the ERK1/2 MAP kinases. The activation kinetics detected using this method were consistent with those from previous reports. Moreover, the increased FRET signal was markedly inhibited by the CB1 antagonist rimonabant, which also reduced phosphorylation of the ERK1/2 MAP kinases. We mutated a single cysteine residue in the sensor (at position 257 or 264) to alanine. Both mutation reduced the agonist-induced increase in FRET signal and structural changes in the CB1 receptor, which attenuated phosphorylation of the ERK1/2 MAP kinases. In summary, our sensor directly assesses the kinetics of CB1 activation in real-time and can be used to monitor CB1 structure and function.