Switching from entecavir to tenofovir alafenamide for chronic hepatitis B patients with low-level viraemia.

BACKGROUND AND AIMS: About 20% of patients receiving nucleos(t)ide analogues treatment experienced low-level viraemia (LLV), which is associated with progression of liver fibrosis and high risk of hepatocellular carcinoma. We aimed to evaluate the effectiveness and safety of switching from entecavir (ETV) to tenofovir alafenamide fumarate (TAF) in ETV-treated patients with LLV. METHODS: In this prospective study, ETV-treated patients with LLV, presented to our hospital from December 2018 to October 2019, were enrolled. Switching to TAF or continuing ETV was given. The primary effectiveness endpoint was complete virological response (CVR) at 24 weeks, and the safety endpoint was the first occurrence of any clinical adverse event during the treatment. RESULTS: Totally, 211 patients were recruited and propensity score matching (PSM) generated 75 patients in either TAF or ETV group. After PSM, baseline characteristics were balanced in two groups. After 24-week treatment, the CVR and ALT normalization in TAF group were 62.7% and 47.6%, which were higher than 9.3% and 10.5% in ETV group (OR 16.4, 95% CI 6.6-40.0, P < .001) respectively. Subgroup analysis showed that switching to TAF achieved favours CVR regardless of the status of sex, age, CHB family history, HBV DNA, HBeAg and cirrhosis, whereas alcohol consumption and diabetes mellitus might compromise the CVR of switching to TAF. Both therapies were well tolerated and had satisfying renal safety. CONCLUSIONS: For ETV-treated patients with LLV, switching to TAF is safe enough and superior compared with continuing ETV monotherapy regarding both virological and biochemical benefits.

Gß1 is required for neutrophil migration in zebrafish.

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 Gß1 is critical for neutrophil migration in response to wounding. In embryos treated with small-molecule inhibitors of Gßγ signaling, neutrophils failed to migrate to wound sites. Although both the Gß1 and Gß4 isoforms are expressed in migrating neutrophils, only deficiency for the former (morpholino-based knockdown) interfered with the directed migration of neutrophils towards wounds. The Gß1 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 Gß1 in neutrophil migration is cell autonomous. Finally, live imaging revealed that Gß1 is required for polarized activation of PI3K, and for the actin dynamics that enable neutrophil migration. Collectively, our data indicate that Gß1 signaling controls proper neutrophil migration by activating PI3K and modulating actin dynamics. Moreover, they illustrate a role for a specific Gß isoform in chemotaxis in vivo.

Microcephaly-dystonia due to mutated PLEKHG2 with impaired actin polymerization.

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 Gßγ- 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.

Gß1 controls collective cell migration by regulating the protrusive activity of leader cells in the posterior lateral line primordium.

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 Gß1 is essential for proper pLLP migration. Although both Gß1 and Gß4 are expressed in the pLLP and NMs, depletion of Gß1 but not Gß4 resulted in an arrest of pLLP migration. In embryos deficient for Gß1, 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, Gß1 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 Gß1, Cxcr4b or Cxcl12a do not. Finally, synergy experiments revealed that Gß1 and Cxcr4b interact genetically in regulating pLLP migration. Collectively, our data indicate that Gß1 controls migration of the pLLP, likely by acting downstream of the Cxcl12a/Cxcr4b signaling. This study also provides compelling evidence for functional specificity among Gß isoforms in vivo.

Gßγ signaling controls the polarization of zebrafish primordial germ cells by regulating Rac activity.

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 Gßγ is required to regulate cell polarization. PGCs that are defective for Gßγ 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 Gßγ 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 Gßγ in PGCs increases Rac activity. Our results indicate that during PGC migration in vivo, Gßγ signaling regulates Rac activity to control cell polarity, which is required for the responsiveness to chemokine signaling.

WDR26 functions as a scaffolding protein to promote Gßγ-mediated phospholipase C ß2 (PLCß2) activation in leukocytes.

We have recently identified WDR26 as a novel WD40 repeat protein that binds Gßγ and promotes Gßγ signaling during leukocyte migration. Here, we have determined the mechanism by which WDR26 enhances Gßγ-mediated phospholipase C ß2 (PLCß2) activation in leukocytes. We show that WDR26 not only directly bound Gßγ but also PLCß2. The binding sites of WDR26 and PLCß2 on Gß1γ2 were overlapping but not identical. WDR26 used the same domains for binding Gßγ and PLCß but still formed a signaling complex with Gßγ and PLCß2 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 PLCß2 interaction with and activation by Gßγ. Moreover, WDR26 was required for PLCß2 translocation from the cytosol to the membrane in polarized leukocytes, and the translocation of PLCß2 was sufficient to cause partial activation of PLCß2. Collectively, our data indicate that WDR26 functions as a scaffolding protein to promote PLCß2 membrane translocation and interaction with Gßγ, thereby enhancing PLCß2 activation in leukocytes. These findings have identified a novel mechanism of regulating Gßγ signaling through a scaffolding protein.

[Percutaneous pedicle screw anchored vertebral augmentation for the treatment of Kümmell disease without neurological symptoms].

OBJECTIVE: To explore the clinical effect of percutaneous pedicle screw anchored vertebral augmentation(PPSAVA) in the treatment of asymptomatic Kümmell disease without neurological symptoms. METHODS: The clinical data of 20 patients with Kümmell disease without neurological symptoms treated with PPSAVA in our hospital from January 2019 to December 2021 were analyzed retrospectively, including 5 males and 15 females, aged 56 to 88 (74.95±9.93) years old. and the course of disease was 7 to 60 days with an average of (21.35±14.46) days. All patients were treated with PPSAVA. The time of operation, the amount of bone cement injected and the leakage of bone cement were recorded. The visual analogue scale(VAS), Oswestry disability index(ODI), vertebral body angle(VBA), anterior edge height and midline height of vertebral body were compared among the before operation, 3 days after operation and during the final follow-up. The loosening and displacement of bone cement were observed during the final follow-up. RESULTS: All the 20 patients completed the operation successfully. The operation time was 30 to 56 min with an average of (41.15±7.65) min, and the amount of bone cement injection was 6.0 to 12.0 ml with an average of (9.30±1.49) ml. Bone cement leakage occurred in 6 cases and there were no obvious clinical symptoms. The follow-up time was 6 to 12 months with an average of (8.43±2.82) months. The VBA, anterior edge height and midline height of of injured vertebral body were significantly improved 3 days after operation and the final follow-up(P<0.05), and the VBA, anterior edge height and midline height of of injured vertebral body were lost in different degrees at the final follow-up (P<0.05). The VAS and ODI at 3 days after operation and at the final follow-up were significantly lower than those at preoperatively(P<0.05), but the VAS score and ODI at the final follow-up were not significantly different from those at 3 d after operation(P>0.05). At the last follow-up, no patients showed loosening or displacement of bone cement. CONCLUSION: PPSAVA is highly effective in treating Kümmell disease without neurological symptoms, improving patients' pain and functional impairment, and reducing the risk of cement loosening and displacement postoperatively.

FAM53B promotes pancreatic ductal adenocarcinoma metastasis by regulating macrophage M2 polarization.

BACKGROUND: Our study investigated the role of FAM53B in regulating macrophage M2 polarization and its potential mechanisms in promoting pancreatic ductal adenocarcinoma (PDAC) metastasis. AIM: To further investigate the role of FAM53B in regulating macrophage M2 polarization and its potential mechanism in promoting PDAC metastasis. Our goal is to determine how FAM53B affects macrophage M2 polarization and to define its underlying mechanism in PDAC metastasis. METHODS: Cell culture and various experiments, including protein analysis, immunohistochemistry, and animal model experiments, were conducted. We compared FAM53B expression between PDAC tissues and healthy tissues and assessed the correlation of FAM53B expression with clinical features. Our study analyzed the role of FAM53B in macrophage M2 polarization in vitro by examining the expression of relevant markers. Finally, we used a murine model to study the role of FAM53B in PDAC metastasis and analyzed the potential underlying mechanisms. RESULTS: Our research showed that there was a significant increase in FAM53B levels in PDAC tissues, which was linked to adverse tumor features. Experimental findings indicated that FAM53B can enhance macrophage M2 polarization, leading to increased anti-inflammatory factor release. The results from the mouse model further supported the role of FAM53B in PDAC metastasis, as blocking FAM53B prevented tumor cell invasion and metastasis. CONCLUSION: FAM53B promotes PDAC metastasis by regulating macrophage M2 polarization. This discovery could lead to the development of new strategies for treating PDAC. For example, interfering with the FAM53B signaling pathway may prevent cancer spread. Our research findings also provide important information for expanding our understanding of PDAC pathogenesis.

Clinical characteristics and prognosis of non-APAP drug-induced acute liver failure: a large multicenter cohort study.

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 < 0.05), numerically higher than 0.905 of ALFSG-PI but without statistical difference (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.

Active Gαi/o mutants accelerate breast tumor metastasis via the c-Src pathway.

Constitutively active mutations in the Gαi2 and GαoA subunits 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 migration in vitro as well as dissemination into multiple organs in vivo by activating c-Src signaling. Thus, our study uncovers a critical function of Gαi/o signaling 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/o mutations or elevated Gαi/o signaling by targeting c-Src to inhibit breast cancer metastasis.

Active Gαi/o Mutants Accelerate Breast Tumor Metastasis via the c-Src Pathway.

Constitutively active mutations in the Gαi2 and GαoA subunits of heterotrimeric G proteins have been found in various human cancers, including breast cancer, but their precise roles in tumor formation, progression, and metastasis remain poorly understood. This study focused on GαoAR243H and Gαi2R179C mutants in breast cancer. These mutants alone were insufficient to initiate mammary tumor formation in mice. However, when introduced into transgenic mouse models of breast cancer induced by Neu expression or PTEN loss, the Gαi2R179C mutant notably enhanced spontaneous lung metastasis, without affecting primary tumor initiation and growth. Ectopic expression of the GαoAR243H and Gαi2R179C mutants in tumor cells promoted cell migration in vitro and dissemination into multiple organs in vivo by activating the c-Src signaling pathway. These mutants activate c-Src through direct interaction, involving specific residues in the switch domains II of Gαi subunits, which only partially overlap with those involved in inhibiting adenylyl cyclases. This study uncovers a critical role of Gαi/o signaling in accelerating breast cancer metastasis through the c-Src pathway. These findings hold clinical significance as they may pave the way for personalized therapies targeting c-Src to inhibit breast cancer metastasis in patients with active Gαi/o mutations or elevated Gαi/o signaling.

The WD40 repeat protein WDR26 binds Gßγ and promotes Gßγ-dependent signal transduction and leukocyte migration.

The Gßγ subunits of heterotrimeric G proteins transmit signals to control many cellular processes, including leukocyte migration. Gßγ signaling may regulate and be regulated by numerous signaling partners. Here, we reveal that WDR26, a member of the WD40 repeat protein family, directly bound free Gßγ in vitro, and formed a complex with endogenous Gßγ in Jurkat T cells stimulated by the chemokine SDF1α. Suppression of WDR26 by siRNAs selectively inhibited Gßγ-dependent phospholipase Cß and PI3K activation, and attenuated chemotaxis in Jurkat T cells and differentiated HL60 cells in vitro and Jurkat T cell homing to lymphoid tissues in scid mice. Similarly, disruption of the WDR26/Gßγ interaction via expression of a WDR26 deletion mutant impaired Gßγ signaling and Jurkat T cell migration, indicating that the function of WDR26 depends on its binding to Gßγ. Additional data show that WDR26 also controlled RACK1, a negative regulator, in binding Gßγ and inhibiting leukocyte migration. Collectively, these experiments identify WDR26 as a novel Gßγ-binding protein that is required for the efficacy of Gßγ signaling and leukocyte migration.

A critical role of Gbetagamma in tumorigenesis and metastasis of breast cancer.

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 Gßγ subunits. Here we evaluated the role of Gßγ in breast tumor growth and metastasis both in vitro and in vivo. Our data show that blocking Gßγ 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 Gßγ 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 Gßγ signaling may also play a role in the generation of a tumor microenvironment permissive for tumor progression, because the inhibition of Gßγ signaling attenuated leukocyte infiltration and angiogenesis in primary breast tumors. Taken together, our data demonstrate a critical role of Gßγ signaling in promoting breast tumor growth and metastasis and suggest that targeting Gßγ may represent a novel therapeutic approach for breast cancer.

Identification and expression patterns of members of the protease-activated receptor (PAR) gene family during zebrafish development.

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.

Blocking Gi/o-Coupled Signaling Eradicates Cancer Stem Cells and Sensitizes Breast Tumors to HER2-Targeted Therapies to Inhibit Tumor Relapse.

Cancer 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 the 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.

PRR7-AS1 Correlates with Immune Cell Infiltration and Is a Diagnostic and Prognostic Marker for Hepatocellular Carcinoma.

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.

Primary hepatic neuroendocrine carcinoma with colon adenoma: A case report with literature review.

INTRODUCTION AND IMPORTANCE: 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. CASE PRESENTATION: 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. CLINICAL DISCUSSION: 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. CONCLUSION: 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.

DEP domains: More than just membrane anchors.

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.

Targeting Gi/o protein-coupled receptor signaling blocks HER2-induced breast cancer development and enhances HER2-targeted therapy.

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.

Glypican 4 mediates Wnt transport between germ layers via signaling filopodia.

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.