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Background There is growing recognition of natural history, complications, and outcomes of patients who develop non-acetaminophen (APAP) drug-induced acute liver failure (ALF). To clarify high-risk factors and develop a nomogram model to predict transplant-free survival (TFS) in patients with non-APAP drug-induced ALF. Methods Patients with non-APAP drug-induced ALF from 5 participating centers were retrospectively analyzed. The primary endpoint was 21-day TFS. Total sample size was 482 patients. Results Regarding causative agents, the most common implicated drugs were herbal and dietary supplements (HDS) (57.0%). The hepatocellular type (R ≥ 5) was the main liver injury pattern (69.0%). International normalized ratio, hepatic encephalopathy grades, the use of vasopressor, N-acetylcysteine, or artificial liver support system were associated with TFS and incorporated to construct a nomogram model (drug-induced acute liver failure-5, DIALF-5). The AUROC of DIALF-5 for 7-day, 21-day, 60-day, and 90-day TFS in the internal cohort were 0.886, 0.915, 0.920, and 0.912, respectively. Moreover, the AUROC of DIALF-5 for 21-day TFS had the highest AUROC, which was significantly higher than 0.725 of MELD and 0.519 of KCC (p p > 0.05). These results were successfully validated in the external cohort (147 patients). Conclusions Based on easily identifiable clinical data, the novel DIALF-5 model was developed to predict transplant-free survival in non-APAP drug-induced ALF, which was superior to KCC, MELD and had a similar prediction performance to ALFSG-PI but is more convenient, which can directly calculate TFS at multiple time points.

Pancreatic ductal adenocarcinoma (PDAC) is an incurable, highly metastatic disease that is largely resistant to existing treatments. A better understanding of the genetic basis of PDAC metastasis should facilitate development of improved therapies. To that end, we developed a novel mouse xenograft model of PDAC metastasis to expedite testing of candidate genes associated with the disease. Human PDAC cell lines BxPC-3, MiaPaCa-2, and Panc-1 stably expressing luciferase were generated and introduced by intracardiac injections into immunodeficient mice to model hematogenous dissemination of cancer cells. Tumor development was monitored by bioluminescence imaging. Bioluminescent MiaPaCa-2 cells most effectively recapitulated PDAC tumor development and metastatic distribution in vivo. Tumors formed in nearly 90% of mice and in multiple tissues, including normal sites of PDAC metastasis. Effects of p14ARF, a known suppressor of PDAC, were tested to validate the model. In vitro, p14ARF acted through a CtBP2-dependent, p53-independent pathway to inhibit MiaPaCa-2–invasive phenotypes, which correlated with reduced tumor cell colonization in vivo. These findings establish a new bioluminescent mouse tumor model for rapidly assessing the biological significance of suspected PDAC metastasis genes. This system may also provide a valuable platform for testing innovative therapies. Mol Cancer Res; 9(7); 867–77. ©2011 AACR.

Constitutively active mutations in the Gαi2and GαoAsubunits of heterotrimeric G proteins have been identified in several human cancers including breast cancer, but their functional significance in tumorigenesis and metastasis has not been well characterized. In this study, we show that expression of the constitutively active GαoAR243H and Gαi2R179C mutants alone was insufficient to induce mammary tumor formation in mice. However, in transgenic mouse models of breast cancer induced by Neu expression or PTEN loss, we found that the Gαi2R179C mutant enhanced spontaneous lung metastasis while having no effect on primary tumor initiation and growth. Additionally, we observed that ectopic expression of the GαoAR243H and Gαi2R179C mutants in tumor cells promote cell migrationin vitroas well as dissemination into multiple organsin vivoby activating c-Src signaling. Thus, our study uncovers a critical function of Gαi/osignaling in accelerating breast cancer metastasis via the c-Src pathway. This work is clinically significant, as it can potentially pave the way to personalized therapies for patients who present with active Gαi/omutations or elevated Gαi/osignaling by targeting c-Src to inhibit breast cancer metastasis.

SummaryCancer stem cells (CSCs) are a small subpopulation of cells within tumors that are resistant to anti-tumor therapies, making them a likely origin of tumor relapse after treatment. In many cancers including breast cancer, CSC function is regulated by G protein-coupled receptors (GPCRs), making GPCR signaling an attractive target for new therapies designed to eradicate CSCs. Yet, CSCs overexpress multiple GPCRs that are redundant in maintaining CSC function, so it is unclear how to target all the various GPCRs to prevent relapse. Here, in a model of HER2+ breast cancer (i.e., transgenic MMTV-Neu mice), we were able to block tumorsphere- and tumor-forming capability of CSCs by targeting GPCRs coupled to Gi/o proteins (Gi/o-GPCRs). Similarly, in HER2+ breast cancer cells, blocking signaling downstream of Gi/o-GPCRs in the PI3K/AKT and Src pathways also enhanced HER2-targeted elimination of CSCs. In a proof-of-concept study, when CSCs were selectively ablated (via a suicide gene construct), loss of CSCs from HER2+ breast cancer cell populations mimicked the effect of targeting Gi/o-GPCR signaling, suppressing their capacity for tumor initiation and progression and enhancing HER2-targeted therapy. Thus, targeting Gi/o-GPCR signaling in HER2+ breast cancer is a promising approach for eradicating CSCs, enhancing HER2+ targeted therapy and blocking tumor reemergence.

Hepatocellular carcinoma (HCC) is a highly aggressive malignant disease, and numerous studies have shown that certain functional long noncoding RNAs (lncRNAs) are implicated in the progression of several cancers. The purpose of the research was to determine, using a database, bioinformatics, and statistical analysis, whether or not lncRNA PRR7-AS1 (PRR7-AS1) was related to HCC. TCGA datasets were used to conduct research on the PRR7-AS1 expression pattern in HCC. In order to evaluate the efficacy of GIHCG as a prognostic tool, both survival and Cox regression analyses were carried out. Furthermore, an investigation of the connection between the expression of PRR7-AS1 and immune infiltration in HCC was carried out. In this study, we identified 125 lncRNAs that were significantly dysregulated in HCC and were associated with long-term survival. Among the above 125 lncRNAs, our attention focused on PRR7-AS1. We found that PRR7-AS1 expressions were distinctly overexpressed in HCC samples compared with nontumor samples. ROC assays revealed that PRR7-AS1 effectively differentiated HCC specimens from normal tissues with an AUC of 0.875 (95% CI: 0.840 to 0.911). Moreover, the high PRR7-AS1 expression was associated with advanced clinical stage and poor prognosis of HCC patients. Importantly, the multivariate Cox proportional hazards model suggested that up-expression of PRR7-AS1 was an independent prognostic marker indicating shorter overall survival and disease-specific survival for HCC patients. Finally, we found that PRR7-AS1 expression was associated with the expression of NK CD56bright cells, Th2 cells, TFH, macrophages, Th1 cells, aDC, T helper cells, cytotoxic cells, DC, Tgd, neutrophils, and Th17 cells. Overall, the results of our study show that PRR7-AS1 was a biomarker that could be utilized to predict the prognosis of HCC patients and was linked to the infiltration of immune cells in HCC.

Glypicans influence signaling pathways by regulating morphogen trafficking and reception. However, the underlying mechanisms in vertebrates are poorly understood. In zebrafish, Glypican 4 (Gpc4) is required for convergence and extension (C&E) of both the mesoderm and endoderm. Here, we show that transgenic expression of GFP-Gpc4 in the endoderm of gpc4 mutants rescued C&E defects in all germ layers. The rescue of mesoderm was likely mediated by Wnt5b and Wnt11f2 and depended on signaling filopodia rather than on cleavage of the Gpc4 GPI anchor. Gpc4 bound both Wnt5b and Wnt11f2 and regulated formation of the filopodia that transport Wnt5b and Wnt11f2 to neighboring cells. Moreover, this rescue was suppressed by blocking signaling filopodia that extend from endodermal cells. Thus, GFP-Gpc4–labeled protrusions that emanated from endodermal cells transported Wnt5b and Wnt11f2 to other germ layers, rescuing the C&E defects caused by a gpc4 deficiency. Our study reveals a new mechanism that could explain in vivo morphogen distribution involving Gpc4.

GPCRs are highly desirable drug targets for human disease. Although GPCR dysfunction drives development and progression of many tumors, including breast cancer (BC), targeting individual GPCRs has limited efficacy as a cancer therapy because numerous GPCRs are activated. Here, we sought a new way of blocking GPCR activation in HER2+ BC by targeting a subgroup of GPCRs that couple to Gi/o proteins (Gi/o-GPCRs). In mammary epithelial cells of transgenic mouse models, and BC cell lines, HER2 hyperactivation altered GPCR expression, particularly, Gi/o-GPCR expression. Gi/o-GPCR stimulation transactivated EGFR and HER2 and activated the PI3K/AKT and Src pathways. If we uncoupled Gi/o-GPCRs from their cognate Gi/o proteins by pertussis toxin (PTx), then BC cell proliferation and migration was inhibited in vitro and HER2-driven tumor formation and metastasis were suppressed in vivo. Moreover, targeting Gi/o-GPCR signaling via PTx, PI3K, or Src inhibitors enhanced HER2-targeted therapy. These results indicate that, in BC cells, HER2 hyperactivation drives aberrant Gi/o-GPCR signaling and Gi/o-GPCR signals converge on the PI3K/AKT and Src signaling pathways to promote cancer progression and resistance to HER2-targeted therapy. Our findings point to a way to pharmacologically deactivate GPCR signaling to block tumor growth and enhance therapeutic efficacy.

G protein coupled receptors (GPCRs) are among the most desirable drug targets for human disease. Although GPCR dysfunction drives the development and progression of many tumors including breast cancer (BC), targeting individual GPCRs has limited efficacy as a cancer therapy because numerous GPCRs are activated. In this study, we sought a new way of blocking GPCR activation in HER2+-BC by targeting a subgroup of GPCRs that couple to Gi/o proteins (Gi/o-GPCRs). Using cell lines and transgenic mouse models, we showed in mammary epithelial cells, HER2 hyperactivation altered GPCR expression, particularly, Gi/o-GPCRs. Gi/o-GPCR stimulation transactivated EGFR and HER2, which in turn activated the PI3K/AKT and Src pathways. Uncoupling Gi/o-GPCRs from cognate Gi/o proteins by pertussis toxin (PTx) inhibited BC cell proliferation and migration in vitro and suppressed HER2-driven tumor formation and metastasis in vivo. Moreover, targeting Gi/o-GPCR signaling via PTx, PI3K, or Src inhibitors enhanced HER2-targeted therapy. These results indicate that HER2 hyperactivation in BC cells drives aberrant Gi/o-GPCR signaling, and Gi/o-GPCR signals converge on PI3K/AKT and Src signaling pathways to promote cancer progression and the development of resistance to HER2-targeted therapy. Our findings suggest a new way to pharmacologically deactivate GPCR signaling to block tumor growth and enhance therapeutic efficacy.

Primary hepatic neuroendocrine tumors (PHNETs) are extremely rare, and the clinical symptoms, test results, and imaging characteristics are nonspecific in most patients; thus, it is difficult to differentiate from other liver masses before surgery. Histopathology and immunohistochemistry are the main basis for the diagnosis. PHNETs and colon tumors co-occur in a patient and are non-homologous, as reported in the English-language literature for the first time.We present a case of a 60-year-old woman with right hepatic lobe mass accidentally discovered on abdominal ultrasonography during a routine examination. Preoperative liver contrast-enhanced computed tomography suggested hepatocellular carcinoma; then, surgery were performed. Pathological results revealed a Grade 2 neuroendocrine tumor of the liver. In search of the primary tumor, upper and lower endoscopy of the GI tract was performed and revealed a mass in the ascending colon. Ascending colon cancer was considered; then, laparoscopic right hemicolectomy was performed. Pathological results suggested tubular villous adenoma of the ascending colon. The final diagnosis was not colon cancer with liver metastases but was PHNETs with colon adenoma.PHNETs are rare cancers that are difficult to diagnose, requiring not only differentiation from other liver masses but also exclusion of metastases from extrahepatic sources. The pathological results play an important in making an accurate diagnosis.Pathology, postoperative follow-up, and comprehensive imaging examinations are powerful tools in the diagnosis of PHNETs. Currently, surgery is the best treatment to achieve a potential cure and prolong the patient's survival.

Glypicans influence signaling pathways by regulating morphogen trafficking and reception. However, the underlying mechanisms in vertebrates are poorly understood. In zebrafish, Glypican 4 (Gpc4) is required for convergence and extension (C&E) of both the mesoderm and endoderm. Here we show that transgenic expression of GFP-Gpc4 in the endoderm ofgpc4mutants rescues C&E defects in all germ layers. The rescue of mesoderm was likely mediated by Wnt5b and Wnt11f2, and depended on signaling filopodia rather than on cleavage of the Gpc4 GPI anchor. Gpc4 bound Wnt5b and regulated formation of the filopodia that transport Wnt5b to neighboring cells. Blocking signaling filopodia that extend from endodermal cells suppressed this rescue. Thus, endodermal signaling filopodia that expressed GFP-Gpc4 transported Wnt5b, and likely Wnt11f2, to other germ layers, rescuing the C&E defects caused by agpc4deficiency. Our study reveals a new mechanism that could explainin vivomorphogen distribution involving Gpc4.

Obesity is associated with elevated intracellular nitric oxide (NO) production, which promotes nitrosative stress in metabolic tissues such as liver and skeletal muscle, contributing to insulin resistance. The onset of obesity-associated insulin resistance is due, in part, to the compromise of hepatic autophagy, a process that leads to lysosomal degradation of cellular components. However, it is not known how NO bioactivity might impact autophagy in obesity. Here, we establish that S-nitrosoglutathione reductase (GSNOR), a major protein denitrosylase, provides a key regulatory link between inflammation and autophagy, which is disrupted in obesity and diabetes. We demonstrate that obesity promotes S-nitrosylation of lysosomal proteins in the liver, thereby impairing lysosomal enzyme activities. Moreover, in mice and humans, obesity and diabetes are accompanied by decreases in GSNOR activity, engendering nitrosative stress. In mice with a GSNOR deletion, diet-induced obesity increases lysosomal nitrosative stress and impairs autophagy in the liver, leading to hepatic insulin resistance. Conversely, liver-specific overexpression of GSNOR in obese mice markedly enhances lysosomal function and autophagy and, remarkably, improves insulin action and glucose homeostasis. Furthermore, overexpression of S-nitrosylation–resistant variants of lysosomal enzymes enhances autophagy, and pharmacologically and genetically enhancing autophagy improves hepatic insulin sensitivity in GSNOR-deficient hepatocytes. Taken together, our data indicate that obesity-induced protein S-nitrosylation is a key mechanism compromising the hepatic autophagy, contributing to hepatic insulin resistance.

Signaling mediated by G protein-coupled receptors (GPCRs) is essential for the migration of cells toward chemoattractants. The recruitment of neutrophils to injured tissues in zebrafish larvae is a useful model for studying neutrophil migration and trafficking in vivo. Indeed, the study of this process led to the discovery that PI3Kγ is required for the polarity and motility of neutrophils, features that are necessary for the directed migration of these cells to wounds. However, the mechanism by which PI3Kγ is activated remains to be determined. Here we show that signaling by specifically the heterotrimeric G protein subunit Gβ1 is critical for neutrophil migration in response to wounding. In embryos treated with small-molecule inhibitors of Gβγ signaling, neutrophils failed to migrate to wound sites. Although both the Gβ1 and Gβ4 isoforms are expressed in migrating neutrophils, only deficiency for the former (morpholino-based knockdown) interfered with the directed migration of neutrophils towards wounds. The Gβ1 deficiency also impaired the ability of cells to change cell shape and reduced their general motility, defects that are similar to those in neutrophils deficient for PI3Kγ. Transplantation assays showed that the requirement for Gβ1 in neutrophil migration is cell autonomous. Finally, live imaging revealed that Gβ1 is required for polarized activation of PI3K, and for the actin dynamics that enable neutrophil migration. Collectively, our data indicate that Gβ1 signaling controls proper neutrophil migration by activating PI3K and modulating actin dynamics. Moreover, they illustrate a role for a specific Gβ isoform in chemotaxis in vivo.

// Prakash Paudyal 1 , Qing Xie 1 , Prasanna Kuma Vaddi 1 , Michael D. Henry 2, 3, 4, 6 , Songhai Chen 1, 5, 6 1 The Department of Pharmacology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA 2 The Department of Molecular Physiology and Biophysics, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA 3 The Department of Pathology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA 4 The Department of Urology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA 5 The Holden Comprehensive Cancer Center, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA 6 The Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA Correspondence to: Songhai Chen, email: songhai-chen@uiowa.edu Keywords: G protein-coupled receptors, Gβγ, prostate cancer growth and metastasis, cancer stem cells Received: August 17, 2016 Accepted: March 11, 2017 Published: March 21, 2017 ABSTRACT Aberrant activation of G protein-coupled receptors (GPCRs) is implicated in prostate cancer progression, but targeting them has been challenging because multiple GPCRs are involved in cancer progression. In this study, we tested the effect of blocking signaling via a hub through which multiple GPCRs converge — the G-protein Gβγ subunits. Inhibiting Gβγ signaling in several castration-resistant prostate cancer cell lines (i.e. PC3, DU145 and 22Rv1), impaired cell growth and migration in vitro , and halted tumor growth and metastasis in nude mice. The blockade of Gβγ signaling also diminished prostate cancer stem cell-like activities, by reducing tumorsphere formation in vitro and tumor formation in a limiting dilution assay in nude mice. Furthermore, Gβγ blockade enhanced the sensitivity of prostate cancer cells to paclitaxel treatment, both in vitro and in vivo . Together, our results identify a novel function of Gβγ in regulating prostate cancer stem-cell-like activities, and demonstrate that targeting Gβγ signaling is an effective approach in blocking prostate cancer progression and augmenting response to chemotherapy.

SOX2 and OCT4 are key regulators of embryonic stem cell pluripotency. They are overexpressed in prostate cancers and have been associated with cancer stem cell (CSC) properties. However, reliable tools for detecting and targeting SOX2/OCT4-overexpressing cells are lacking, limiting our understanding of their roles in prostate cancer initiation, progression, and therapeutic resistance. Here, we show that a fluorescent reporter called SORE6 can identify SOX2/OCT4-overexpressing prostate cancer cells. Among tumor cells, the SORE6 reporter identified a small fraction with CSC hallmarks: rapid self-renewal, the capability to form tumors and metastasize, and resistance to chemotherapies. Transcriptome and biochemical analyses identified PI3K/AKT signaling as critical for maintaining the SORE6+ population. Moreover, a SORE6-driven herpes simplex virus thymidine kinase (TK) expression construct could selectively ablate SORE6+ cells in tumors, blocking tumor initiation and progression, and sensitizing tumors to chemotherapy. This study demonstrates a key role of SOX2/OCT4-associated prostate cancer stem cells in tumor development and therapeutic resistance, and identifies the SORE6 reporter system as a useful tool for characterizing CSCs functions in a native tumor microenvironment.

Rearrangement of the actin cytoskeleton is controlled by RhoGTPases which are activated by RhoGEFs. We identified homozygosity for Arg204Trp mutation in the Rho guanidine exchange factor (RhoGEF) PLEKHG2 gene in five patients with profound mental retardation, dystonia, postnatal microcephaly, and distinct neuroimaging pattern. The activity of the mutant PLEKHG2 was significantly decreased, both in basal state and when Gβγ- or lysophosphatidic acid (LPA)-stimulated. SDF1a-stimulated actin polymerization was significantly impaired in patient cells, and this abnormality was duplicated in control cells when PLEKHG2 expression was downregulated. These results underscore the role of PLEKHG2 in actin polymerization and delineate the clinical and radiological findings in PLEKHG2 deficiency.

Collective cell migration is critical for normal development, tissue repair and cancer metastasis. Migration of the posterior lateral line primordium (pLLP) generates the zebrafish sensory organs (neuromasts, NMs). This migration is promoted by the leader cells at the leading edge of the pLLP, which express the G protein-coupled chemokine receptor Cxcr4b and respond to the chemokine Cxcl12a. However, the mechanism by which Cxc112a/Cxcr4b signaling regulates pLLP migration remains unclear. Here we report that signal transduction by the heterotrimeric G protein subunit Gβ1 is essential for proper pLLP migration. Although both Gβ1 and Gβ4 are expressed in the pLLP and NMs, depletion of Gβ1 but not Gβ4 resulted in an arrest of pLLP migration. In embryos deficient for Gβ1, the pLLP cells migrated in an uncoordinated fashion and were unable to extend protrusions at the leading front, phenocopying those in embryos deficient for Cxcl12a or Cxcr4b. A transplantation assay showed that, like Cxcr4b, Gβ1 is required only in the leader cells of the pLLP. Analysis of F-actin dynamics in the pLLP revealed that whereas wild-type leader cells display extensive actin polymerization in the direction of pLLP migration, counterparts defective for Gβ1, Cxcr4b or Cxcl12a do not. Finally, synergy experiments revealed that Gβ1 and Cxcr4b interact genetically in regulating pLLP migration. Collectively, our data indicate that Gβ1 controls migration of the pLLP, likely by acting downstream of the Cxcl12a/Cxcr4b signaling. This study also provides compelling evidence for functional specificity among Gβ isoforms in vivo.

G protein coupled receptors (GPCRs) are the largest family of cell surface receptors with over 800 members. Aberrant activation of GPCRs has been implicated in cancer initiation and development. However, many of GPCRs are overexpressed in cancer cells, making it difficult to target individual GPCRs for cancer treatment. In this study, we have tested a new approach of targeting GPCRs in HER2+ breast cancer by blocking their common pathways downstream of G proteins. By mammary gland-specific expression of the pertussis toxin (PTx) catalytic subunit, a selective inhibitor that uncouples a subgroup of GPCRs from activating Gi/o proteins, we demonstrated that blocking GPCR downstream signaling delayed the onset of HER2-driven spontaneous mammary tumor formation and suppressed lung metastasis. Moreover, we showed that aberrant HER2 signaling upregulated Gi/o-GPCR expression in breast cancer cells, which in turn activated the ErbB/HER family of protein-tyrosine kinases via Src and PI3K pathways to maintain cancer stem cell tumorigenicity. Targeting Gi/o-GPCR signaling by PTx, Src or PI3K inhibitors sensitizes HER2+ breast cancer to HER2-targeted therapies. Together, our data demonstrate that targeting GPCR downstream signal pathways may represent a new approach to ablate CSCs to block tumor progression and augment HER2-targeted therapeutics. Citation Format: Wei Wang, Dharmendra Bhargava, Yuan Chao Ye, Songhai Chen. Targeting GPCR signaling in HER2+ breast cancer suppresses cancer stem cell tumorigenicity and sensitizes HER2-targeted therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4683.

Cancer metastasis is the major cause of tumor mortality and has been attributed in part to the presence of a minority subpopulation of cancer stem cells (CSCs) in the bulk of tumor cells. We showed previously that WDR26, a scaffolding/adaptor protein that is highly upregulated in breast cancer, promotes breast cancer growth and metastasis. Here we show WDR26 was required for maintaining the CSC populations in breast cancer cells and the formation of lung metastases. Downregulation of WDR26 in breast cancer cells impaired the CSC-like activities and reduced the CSC population. Mammary gland-specific deletion of WDR26 in the MMTV-PyMT mouse model of breast cancer had a little effect on primary tumor formation but largely abolished spontaneous lung metastasis. WDR26 promoted β-catenin activation via AKT and GSK3, and the activity of AKT, GSK3 and β-catenin was required for maintaining the CSC population in breast cancer cells. Our results have identified a novel, WDR26-dependent pathway that links breast CSC activities to tumor metastatic potential. Citation Format: Dharmendra K. Bhargava, Wei Wang, Maddison Lensing, Songhai Chen. WDR26 regulates an AKT-Gsk3-Wnt/b-catenin signaling cascade to maintain the breast cancer stem cell population and controls cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4672.

PLEKHG2 is a Dbl family Rho guanine nucleotide exchange factor (RhoGEF) whose gene was originally identified as being upregulated in a leukemia mouse model and was later shown to be activated by heterotrimeric G protein βγ (Gβγ) subunits. However, its function and activation mechanisms remain elusive. Here we show that, compared to its expression in primary human T cells, its expression is upregulated in several leukemia cell lines, including Jurkat T cells. Downregulation of PLEKHG2 in Jurkat T cells by small interfering RNAs (siRNAs) specifically inhibited Gβγ-stimulated Rac and Cdc42, but not RhoA, activation. Consequently, suppressing PLEKHG2 expression blocked actin polymerization and SDF1α-stimulated lymphocyte migration. Additional studies indicate that Gβγ likely activates PLEKHG2, in part by binding the N terminus of PLEKHG2 to release an autoinhibition imposed by its C terminus, which interacts with a region encompassing the catalytic Dbl homology (DH) domain. As a result, overexpressing either the N terminus or the C terminus of PLEKHG2 blocked Gβγ-stimulated Rac and Cdc42 activation and prevented Jurkat T cells from forming membrane protrusions and migrating. Together, our studies have provided the first evidence for the endogenous function of PLEKHG2, which may serve as a key Gβγ-stimulated RhoGEF that regulates lymphocyte chemotaxis via Rac and Cdc42 activation and actin polymerization.

A splice variant of Gβ3, termed Gβ3s, has been associated with the C825T polymorphism in the Gβ3 gene and linked with many human disorders. However, the biochemical properties and functionality of Gβ3s remain controversial. Here, using multidisciplinary approaches including co-immunoprecipitation analysis and bioluminescence resonance energy transfer (BRET) measurements, we showed that unlike Gβ3, Gβ3s failed to form complexes with either Gγ or Gα subunits. Moreover, using a mutant Gγ2 deficient in lipid modification to purify Gβ3s from Sf9 cells without the use of detergents, we further showed that the failure of Gβ3s to form dimers with Gγ was not due to the instability of the dimers in detergents, but rather, reflected the intrinsic properties of Gβ3s. Additional studies indicated that Gβ3s is unstable, and unable to localize properly to the plasma membrane and to activate diverse Gβγ effectors including PLCβ2/3, PI3Kγ, ERKs and the Rho guanine exchange factor (RhoGEF) PLEKHG2. Thus, these data suggest that the pathological effects of Gβ3 C825T polymorphism may result from the downregulation of Gβ3 function. However, we found that the chemokine SDF1α transmits signals primarily through Gβ1 and Gβ2, but not Gβ3, to regulate chemotaxis of several human lymphocytic cell lines, indicating the effects of Gβ3 C825T polymorphism are likely to be tissue and/or stimuli specific and its association with various disorders in different tissues should be interpreted with great caution.

The phosphatidylinositol 3-kinase (PI3K)/AKT pathway transmits signals downstream of receptor tyrosine kinases and G protein-coupled receptors (GPCRs), and is one of the most dysregulated pathways in breast cancer. PI3Ks and AKTs consist of multiple isoforms that play distinct and even opposite roles in breast cancer cell growth and metastasis. However, it remains unknown how the activities of various PI3K and AKT isoforms are coordinated during breast cancer progression. Previously, we showed WDR26 is a novel WD40 protein that binds Gβγ and promotes Gβγ signaling. Here, we demonstrate that WDR26 is overexpressed in highly malignant breast tumor cell lines and human breast cancer samples, and that WDR26 overexpression correlates with shortened survival of breast cancer patients. In highly malignant cell lines (MDA-MB231, DU4475 and BT549), downregulation of WDR26 expression selectively alleviated GPCR- but not EGF receptor-stimulated PI3K/AKT signaling and tumor cell growth, migration and invasion. In contrast, in a less malignant cell line (MCF7), WDR26 overexpression had the opposite effect. Additional studies indicate that downstream of GPCR stimulation, WDR26 serves as a scaffold that fosters assembly of a specific signaling complex consisting of Gβγ, PI3Kβ and AKT2. In an orthotopic xenograft mouse model of breast cancer, disrupting formation of this complex, by overexpressing WDR26 mutants in MDA-MB231 cells, abrogated PI3K/AKT activation and tumor cell growth and metastasis. Together, our results identify a novel mechanism regulating GPCR-dependent activation of the PI3K/AKT signaling axis in breast tumor cells, and pinpoint WDR26 as a potential therapeutic target for breast cancer.

During development, primordial germ cells (PGCs) migrate from the sites of their specification towards the region in which the future gonad develops. This cell migration requires polarization of PGCs and their responsiveness to external guidance cues. In zebrafish, the directed migration and polarization of PGCs are regulated independently, by the chemokine Cxcl12a and the Rho GTPase Rac1, respectively. However, the upstream signals controlling Rac activity in this context have not yet been identified. By investigating the role of G proteins in PGC migration, we found that signaling mediated by G protein subunits Gβγ is required to regulate cell polarization. PGCs that are defective for Gβγ signaling failed to polarize, and developed multiple protrusions in random locations, resembling the defects observed in PGCs with decreased Rac activity. These defects render PGCs incapable of migrating actively and responding to directional cues. FRET-based assays showed that PGCs require Gβγ signaling for polarized Rac activation and actin organization at the leading front, as well as for maintaining overall Rac levels in these cells. Conversely, overexpression of Gβγ in PGCs increases Rac activity. Our results indicate that during PGC migration in vivo, Gβγ signaling regulates Rac activity to control cell polarity, which is required for the responsiveness to chemokine signaling.

The ability of cells to generate a highly polarized intracellular signal through G protein-coupled receptors (GPCRs) is essential for their migration toward chemoattractants. The Gβγ subunits of heterotrimeric G proteins play a critical role in transmitting chemotactic signals from GPCRs via the activation of diverse effectors, including PLCβ and PI3K, primarily at the leading edge of cells. Although Gβγ can directly activate many of these effectors through protein-protein interactions in vitro, it remains unclear how Gβγ spatially and temporally orchestrates the activation of these effectors in vivo. A yeast two-hybrid screen for Gβ interacting proteins identified two WD40-repeat domain containing proteins, RACK1 and WDR26, which are predicted to serve as scaffolding/adaptor proteins. Previous data indicates that RACK1 negatively regulates Gβγ-mediated leukocyte migration by inhibiting Gβγ-stimulated PLCβ and PI3K activities. In contrast, recently published work by Sun et al. indicates that WDR26 promotes leukocyte migration by enhancing Gβγ-mediated signal transduction. These findings reveal a novel mechanism regulating Gβγ signaling during chemotaxis, namely through the positive and negative regulation of WDR26 and RACK1 on Gβγ to promote and fine tune Gβγ-mediated effector activation, ultimately governing the ability of cells to polarize and migrate toward a chemoattractant gradient.

A growing body of evidence indicates that G protein-coupled receptors (GPCRs) are involved in breast tumor progression and that targeting GPCRs may be a novel adjuvant strategy in cancer treatment. However, due to the redundant role of multiple GPCRs in tumor development, it may be necessary to target a common signaling component downstream of these receptors to achieve maximum efficacy. GPCRs transmit signals through heterotrimeric G proteins composed of Gα and Gβγ subunits. Here we evaluated the role of Gβγ in breast tumor growth and metastasis both in vitro and in vivo. Our data show that blocking Gβγ signaling with Gα(t) or small molecule inhibitors blocked serum-induced breast tumor cell proliferation as well as tumor cell migration induced by various GPCRs in vitro. Moreover, induced expression of Gα(t) in MDA-MB-231 cells inhibited primary tumor formation and retarded growth of existing breast tumors in nude mice. Blocking Gβγ signaling also dramatically reduced the incidence of spontaneous lung metastasis from primary tumors and decreased tumor formation in the experimental lung metastasis model. Additional studies indicate that Gβγ signaling may also play a role in the generation of a tumor microenvironment permissive for tumor progression, because the inhibition of Gβγ signaling attenuated leukocyte infiltration and angiogenesis in primary breast tumors. Taken together, our data demonstrate a critical role of Gβγ signaling in promoting breast tumor growth and metastasis and suggest that targeting Gβγ may represent a novel therapeutic approach for breast cancer.

Protease-activated receptors (PARs) play critical roles in hemostasis in vertebrates including zebrafish. However, the zebrafish gene classification appears to be complex, and the expression patterns of par genes are not established. Based on analyses of genomic organization, phylogenetics, protein primary structure, and protein internalization, we report the identification of four zebrafish PARs: par1, par2a, par2b, and par3. This classification differs from one reported previously. We also show that these genes have distinct spatiotemporal expression profiles in embryos and larvae, with par1, par2a, and par2b expressed maternally and ubiquitously during gastrula stages and their expression patterns refined at later stages, and par3 expressed only in 3-day-old larvae. Notably, the expression patterns of zebrafish par1 and par2b resemble those of their mammalian counterparts, suggesting that receptor function is conserved among vertebrates. This conservation is supported by our findings that Par1 and Par2b are internalized following exposure to thrombin and trypsin, respectively.

Epiboly spreads and thins the blastoderm over the yolk cell during zebrafish gastrulation, and involves coordinated movements of several cell layers. Although recent studies have begun to elucidate the processes that underlie these epibolic movements, the cellular and molecular mechanisms involved remain to be fully defined. Here, we show that gastrulae with altered Galpha(12/13) signaling display delayed epibolic movement of the deep cells, abnormal movement of dorsal forerunner cells, and dissociation of cells from the blastoderm, phenocopying e-cadherin mutants. Biochemical and genetic studies indicate that Galpha(12/13) regulate epiboly, in part by associating with the cytoplasmic terminus of E-cadherin, and thereby inhibiting E-cadherin activity and cell adhesion. Furthermore, we demonstrate that Galpha(12/13) modulate epibolic movements of the enveloping layer by regulating actin cytoskeleton organization through a RhoGEF/Rho-dependent pathway. These results provide the first in vivo evidence that Galpha(12/13) regulate epiboly through two distinct mechanisms: limiting E-cadherin activity and modulating the organization of the actin cytoskeleton.

Receptor for Activated C Kinase 1 (RACK1), a novel Gβγ-interacting protein, selectively inhibits the activation of a subclass of Gβγ effectors such as phospholipase C β2 (PLCβ2) and adenylyl cyclase II by direct binding to Gβγ (Chen, S., Dell, E. J., Lin, F., Sai, J., and Hamm, H. E. (2004) J. Biol. Chem. 279, 17861-17868). Here we have mapped the RACK1 binding sites on Gβγ. We found that RACK1 interacts with several different Gβγ isoforms, including Gβ1γ1, Gβ1γ2, and Gβ5γ2, with similar affinities, suggesting that the conserved residues between Gβ1 and Gβ5 may be involved in their binding to RACK1. We have confirmed this hypothesis and shown that several synthetic peptides corresponding to the conserved residues can inhibit the RACK1/Gβγ interaction as monitored by fluorescence spectroscopy. Interestingly, these peptides are located at one side of Gβ1 and have little overlap with the Gα subunit binding interface. Additional experiments indicate that the Gβγ contact residues for RACK1, in particular the positively charged amino acids within residues 44-54 of Gβ1, are also involved in the interaction with PLCβ2 and play a critical role in Gβγ-mediated PLCβ2 activation. These data thus demonstrate that RACK1 can regulate the activity of a Gβγ effector by competing for its binding to the signal transfer region of Gβγ.

Heterotrimeric G-proteins, composed of Galpha and Gbetagamma subunits, transmit numerous and diverse extracellular stimuli via a large family of heptahelical cell-surface receptors to various intracellular effector molecules. The Gbetagamma subunit plays a central role in G-protein signaling. The Gbeta subunit belongs to a large family of WD40 repeat proteins, which adopt a circular beta-bladed propeller structure. This unique structural feature confers interactions of Gbetagamma with a variety of proteins to play diverse functions. Intriguingly, we recently found that Gbetagamma can interact with three other WD40 repeat proteins, receptor for activated C kinase 1 (RACK1), dynein intermediate chain-1A and a protein of unknown function. This raises the following questions: are interactions among WD40 proteins a common theme and does the formation of a WD40-WD40 repeat protein complex constitute a protein scaffold for integrating signals from different cellular processes. We are beginning to address these issues by studying the interaction between Gbetagamma and RACK1. Here we will describe the molecular mechanism underlying this interaction and the implications of the interaction on the signal transduction of G-protein and RACK1.

We showed previously that Gbetagamma interacts with Receptor for Activated C Kinase 1 (RACK1), a protein that not only binds activated protein kinase C (PKC) but also serves as an adaptor/scaffold for many signaling pathways. Here we report that RACK1 does not interact with Galpha subunits or heterotrimeric G proteins but binds free Gbetagamma subunits released from activated heterotrimeric G proteins following the activation of their cognate receptors in vivo. The association with Gbetagamma promotes the translocation of RACK1 from the cytosol to the membrane. Moreover, binding of RACK1 to Gbetagamma results in inhibition of Gbetagamma-mediated activation of phospholipase C beta2 and adenylyl cyclase II. However, RACK1 has no effect on other functions of Gbetagamma, such as activation of the mitogen-activated protein kinase signaling pathway or chemotaxis of HEK293 cells via the chemokine receptor CXCR2. Similarly, RACK1 does not affect signal transduction through the Galpha subunits of G(i), G(s), or G(q). Collectively, these findings suggest a role of RACK1 in regulating specific functions of Gbetagamma.

A yeast two-hybrid approach was used to discern possible new effectors for the betagamma subunit of heterotrimeric G proteins. Three of the clones isolated are structurally similar to Gbeta, each exhibiting the WD40 repeat motif. Two of these proteins, the receptor for activated C kinase 1 (RACK1) and the dynein intermediate chain, co-immunoprecipitate with Gbetagamma using an anti-Gbeta antibody. The third protein, AAH20044, has no known function; however, sequence analysis indicates that it is a WD40 repeat protein. Further investigation with RACK1 shows that it not only interacts with Gbeta(1)gamma(1) but also unexpectedly with the transducin heterotrimer Galpha(t)beta(1)gamma(1). Galpha(t) alone does not interact, but it must contribute to the interaction because the apparent EC(50) value of RACK1 for Galpha(t)beta(1)gamma(1) is 3-fold greater than that for Gbeta(1)gamma(1) (0.1 versus 0.3 microm). RACK1 is a scaffold that interacts with several proteins, among which are activated betaIIPKC and dynamin-1 (1). betaIIPKC and dynamin-1 compete with Gbeta(1)gamma(1) and Galpha(t)beta(1)gamma(1) for interaction with RACK1. These findings have several implications: 1) that WD40 repeat proteins may interact with each other; 2) that Gbetagamma interacts differently with RACK1 than with its other known effectors; and/or 3) that the G protein-RACK1 complex may constitute a signaling scaffold important for intracellular responses.

Neutrophils play an essential role in the innate immune response to infection. Neutrophils migrate from the vasculature into the tissue in response to infection. Recently, a neutrophil cell surface receptor, CD177, was shown to help mediate neutrophil migration across the endothelium through interactions with PECAM1. We examined a publicly available gene array dataset of CD177 expression from human neutrophils following pulmonary endotoxin instillation. Among all 22,214 genes examined, CD177 mRNA was the most upregulated following endotoxin exposure. The high level of CD177 expression is also maintained in airspace neutrophils, suggesting a potential involvement of CD177 in neutrophil infiltration under infectious diseases. To determine the role of CD177 in neutrophils in vivo, we constructed a CD177-genetic knockout mouse model. The mice with homozygous deletion of CD177 have no discernible phenotype and no significant change in immune cells, other than decreased neutrophil counts in peripheral blood. We examined the role of CD177 in neutrophil accumulation using a skin infection model with Staphylococcus aureus. CD177 deletion reduced neutrophil counts in inflammatory skin caused by S. aureus. Mechanistically we found that CD177 deletion in mouse neutrophils has no significant impact in CXCL1/KC- or fMLP-induced migration, but led to significant cell death. Herein we established a novel genetic mouse model to study the role of CD177 and found that CD177 plays an important role in neutrophils. Electronic supplementary material The online version of this article (doi:10.1007/s13238-014-0109-1) contains supplementary material, which is available to authorized users.

G protein β3 (Gβ3) is an isoform of heterotrimeric G protein β subunits involved in transducing G protein coupled receptor (GPCR) signaling. Polymorphisms in Gβ3 (GNB3) are associated with many human disorders (e.g. hypertension, diabetes and obesity) but the role of GNB3 in these pathogeneses remains unclear. Here, Gβ3-null mice (GNB3(-/-)) were characterized to determine how Gβ3 functions to regulate blood pressure, body weight and metabolism. We found Gβ3 expression restricted to limited types of tissues, including the retina, several regions of the brain and heart ventricles. Gβ3-deficient mice were normal as judged by body weight gain by age or by feeding with high-fat diet (HFD); glucose tolerance and insulin sensitivity; baseline blood pressure and angiotensin II infusion-induced hypertension. During tail-cuff blood pressure measurements, however, Gβ3-null mice had slower heart rates (~450 vs ~500 beats/min). This bradycardia was not observed in isolated and perfused Gβ3-null mouse hearts. Moreover, mouse hearts isolated from GNB3(-/-) and controls responded equivalently to muscarinic receptor- and β-adrenergic receptor-stimulated bradycardia and tachycardia, respectively. Since no difference was seen in isolated hearts, Gβ3 is unlikely to be involved directly in the GPCR signaling activity that controls heart pacemaker activity. These results demonstrate that although Gβ3 appears dispensable in mice for the regulation of blood pressure, body weight and metabolic features associated with obesity and diabetes, Gβ3 may regulate heart rate.

Pharmacophore mapping of adrenergic receptors indicates that the phenyl ring of catecholamine agonists is involved in receptor binding and activation. Here we evaluated Phe310, Phe311, and Phe303 in transmembrane VI (TMVI), as well as Tyr348 in TMVII of the alpha1B-adrenergic receptor (alpha1B-AR), which have been implicated in a catechol-ring interaction. Neither catecholamine docking studies nor mutagenesis studies of Phe311, Phe303, or Tyr348 supported a role for these residues in catechol-ring binding. By contrast, docking studies indicated that the Phe310 side chain is well positioned to interact with the catechol-ring, and substituted cysteine accessibility method studies revealed that the side chain of the 310, but not 311 residue, is both solvent accessible and directed into the agonist-binding pocket. Also, saturation mutagenesis of both Phe310 and Phe311 revealed for the former, but not for the latter, a direct relationship between side chain volume and agonist affinity, and that aromaticity is essential for wild-type agonist binding, and for both wild-type agonist potency and efficacy. Moreover, studies of Phe310 mutants combined with a previously described constitutively active alpha1B-AR mutant, A293E, indicated that although not required for spontaneous receptor isomerization from the basal state, R, to a partially activated conformation R', interaction of Phe310 with catecholamine agonists is essential for isomerization from R' to the fully activated state, R.

Bone metastasis is one of the most common and severe complications in advanced prostate cancer . It is the major cause of mortality and morbidities, due to the development of bone pain, hypercalcemia, fractures, spinal cord compression and consequent paralysis. The current regimen for these patients is largely palliative and non-curative, because metastatic tumors are resistant to most of the current anti-cancer treatments. Thus, it is imperative to develop novel therapeutic approaches for the treatment of advance prostate cancer. This proposal aims to define the key role of the G subunits of heterotrimeric G proteins in the development of prostate tumor bone metastasis and the associated bone pain, as well as determine the potential therapeutic efficacy of targeting G with small molecule inhibitors in preclinical models of bone-metastasized prostate cancer. G proteins mediate the function of a large group of cell surface receptor proteins called G protein-coupled receptors (GPCRs). Comparative experimental and clinical evidence has indicated that excessive activation of the GPCR systems due to overexpression of the receptors and their ligands in prostate tumor cells or their surrounding cells contributes to the metastatic spread of tumor cells to bones, their subsequent growth there and the consequent bone destruction. Moreover, continue activation of GPCRs in the sensory nerve fibers adjacent to bones results in increased activity/expression of key pain-sensing receptor channels, such as TRPV1, such that the channels are constitutively activated, leading to the sensation of chronic pain without any overt stimulation. Thus, the GPCR system represents an attractive target for the therapeutics of bone tumor metastasis and the associated bone pain.

We have recently identified WDR26 as a novel WD40 repeat protein that binds Gβγ and promotes Gβγ signaling during leukocyte migration. Here, we have determined the mechanism by which WDR26 enhances Gβγ-mediated phospholipase C β2 (PLCβ2) activation in leukocytes. We show that WDR26 not only directly bound Gβγ but also PLCβ2. The binding sites of WDR26 and PLCβ2 on Gβ1γ2 were overlapping but not identical. WDR26 used the same domains for binding Gβγ and PLCβ but still formed a signaling complex with Gβγ and PLCβ2 probably due to the fact that WDR26 formed a higher order oligomer through its Lis homology and C-terminal to LisH (LisH-CTLH) and WD40 domains. Additional studies indicated that the formation of higher order oligomers was required for WDR26 to promote PLCβ2 interaction with and activation by Gβγ. Moreover, WDR26 was required for PLCβ2 translocation from the cytosol to the membrane in polarized leukocytes, and the translocation of PLCβ2 was sufficient to cause partial activation of PLCβ2. Collectively, our data indicate that WDR26 functions as a scaffolding protein to promote PLCβ2 membrane translocation and interaction with Gβγ, thereby enhancing PLCβ2 activation in leukocytes. These findings have identified a novel mechanism of regulating Gβγ signaling through a scaffolding protein.

The ability of cells to generate a highly polarized intracellular signal through G protein-coupled receptors (GPCRs) is essential for their migration toward chemoattractants. The Gβγ subunits of heterotrimeric G proteins play a critical role in transmitting chemotactic signals from GPCRs via the activation of diverse effectors, including PLCβ and PI3K, primarily at the leading edge of cells. Although Gβγ can directly activate many of these effectors through protein-protein interactions in vitro, it remains unclear how Gβγ spatially and temporally orchestrates the activation of these effectors in vivo. A yeast two-hybrid screen for Gβ interacting proteins identified two WD40-repeat domain containing proteins, RACK1 and WDR26, which are predicted to serve as scaffolding/adaptor proteins. Previous data indicates that RACK1 negatively regulates Gβγ-mediated leukocyte migration by inhibiting Gβγ-stimulated PLCβ and PI3K activities. In contrast, recently published work by Sun et al. indicates that WDR26 promotes leukocyte migration by enhancing Gβγ-mediated signal transduction. These findings reveal a novel mechanism regulating Gβγ signaling during chemotaxis, namely through the positive and negative regulation of WDR26 and RACK1 on Gβγ to promote and fine tune Gβγ-mediated effector activation, ultimately governing the ability of cells to polarize and migrate toward a chemoattractant gradient.

Tissue transglutaminase (TGase II) is a Ca2+- and thiol-dependent enzyme that catalyzes the post-translational modification of proteins via the formation of epsilon(gamma-glutamyl) lysine bonds. We have shown previously that the GTP-binding protein, Gh, is a TGase II that mediates intracellular signaling by the alpha1B-adrenergic receptor (AR) (Nakaoka, H., Perez, D. M., Baek, K. J., Das, T., Husain, A., Mison, K., Im, M.-J., and Graham, R. M. (1994) Science 264, 1593-1596). Here, we evaluated the ability of Gh as compared with Gq to mediate receptor-stimulated inositol phosphate turnover by the three alpha1-subtypes (alpha1A, alpha1B, and alpha1D). In addition, we questioned if the transglutaminase function of Gh is involved in its receptor signaling activity. A mutant form of a human TGase II cDNA in which the codon for the active site cysteine (Cys277) was replaced by serine was cloned into the mammalian expression vector pMT2'. Compared with wild-type TGase II, no transglutaminase activity was observed with transient transfection of this Cys--Ser mutant in COS-1 cells. However, like wild-type TGase, the Cys--Ser mutant mediated receptor-stimulated inositol phosphate turnover when cotransfected with an alpha1B-AR cDNA. Galphaq supported alpha1-AR-mediated inositol phosphate turnover by all three receptor subtypes. By contrast, although both the wild-type and Cys--Ser construct mediated receptor signaling by the alpha1B AR and alpha1D AR, the alpha1A-AR was unable to interact with Gh. However, a Gh-dependent signaling phenotype could be rescued by a chimeric alpha1A construct in which the third intracellular loop of the alpha1A-AR was replaced by that of the alpha1B-AR. Thus, the signaling function of Gh is independent of its transglutaminase activity and is alpha1-AR subtype specific. This subtype specificity of the interaction between alpha1 ARs and Gh involves important determinants in their third intracellular loops.

Pancreatic ductal adenocarcinoma (PDAC) is an incurable, highly metastatic disease that is largely resistant to existing treatments. A better understanding of the genetic basis of PDAC metastasis should facilitate development of improved therapies. To that end, we developed a novel mouse xenograft model of PDAC metastasis to expedite testing of candidate genes associated with the disease. Human PDAC cell lines BxPC-3, MiaPaCa-2, and Panc-1 stably expressing luciferase were generated and introduced by intracardiac injections into immunodeficient mice to model hematogenous dissemination of cancer cells. Tumor development was monitored by bioluminescence imaging. Bioluminescent MiaPaCa-2 cells most effectively recapitulated PDAC tumor development and metastatic distribution in vivo. Tumors formed in nearly 90% of mice and in multiple tissues, including normal sites of PDAC metastasis. Effects of p14ARF, a known suppressor of PDAC, were tested to validate the model. In vitro, p14ARF acted through a CtBP2-dependent, p53-independent pathway to inhibit MiaPaCa-2–invasive phenotypes, which correlated with reduced tumor cell colonization in vivo. These findings establish a new bioluminescent mouse tumor model for rapidly assessing the biological significance of suspected PDAC metastasis genes. This system may also provide a valuable platform for testing innovative therapies. Mol Cancer Res; 9(7); 867–77. ©2011 AACR.

PLEKHG2 is a novel Gβγ-activated Rho guanine nucleotide exchange factor (GEF) which catalyzes the activation of the RhoGTPase family of proteins. Overexpression of PLEKHG2 has been associated with ...

Migration of cells up the chemoattractant gradients is mediated by the binding of chemoattractants to G protein-coupled receptors and activation of a network of coordinated excitatory and inhibitory signals. Although the excitatory process has been well studied, the molecular nature of the inhibitory signals remains largely elusive. Here we report that the receptor for activated C kinase 1 (RACK1), a novel binding protein of heterotrimeric G protein betagamma (G betagamma) subunits, acts as a negative regulator of directed cell migration. After chemoattractant-induced polarization of Jurkat and neutrophil-like differentiated HL60 (dHL60) cells, RACK1 interacts with G betagamma and is recruited to the leading edge. Down-regulation of RACK1 dramatically enhances chemotaxis of cells, whereas overexpression of RACK1 or a fragment of RACK1 that retains G betagamma-binding capacity inhibits cell migration. Further studies reveal that RACK1 does not modulate cell migration through binding to other known interacting proteins such as PKC beta and Src. Rather, RACK1 selectively inhibits G betagamma-stimulated phosphatidylinositol 3-kinase gamma (PI3K gamma) and phospholipase C (PLC) beta activity, due to the competitive binding of RACK1, PI3K gamma, and PLC beta to G betagamma. Taken together, these findings provide a novel mechanism of regulating cell migration, i.e., RACK1-mediated interference with G betagamma-dependent activation of key effectors critical for chemotaxis.

The DEP domain is present in a number of signaling molecules, including Regulator of G protein Signaling (RGS) proteins, and has been implicated in membrane targeting. New findings in yeast, however, demonstrate a major role for a DEP domain in mediating the interaction of an RGS protein to the C-terminal tail of a GPCR, thus placing RGS in close proximity with its substrate G protein alpha subunit.