Letrozole-Based Near-Infrared Dynamic Imaging Targeting Ductal-Vascular RhoJ From Pancreatic Intraepithelial Neoplasia to Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) relies heavily on neoangiogenesis for its progression, making early detection crucial. Here, LTZi-MHI148 (Letrozole inhibitor bonding with MHI-148 dye), a near-infrared (NIR) fluorescent agent is developed, to target RhoJ (Ras Homolog Family Member J), a protein expressed in neonatal vasculature, for both imaging and therapy of early PDAC. This agent is synthesized by conjugating Letrozole with MHI-148, exhibiting excellent NIR characteristics and photostability. In vitro studies showed that LTZi-MHI148 selectively accumulated within pancreatic cancer cells through Organic Anion Transporting Polypeptide (OATP) transporters and bound to cytoplasmic RhoJ. In vivo, the probe effectively targeted neoangiogenesis and Pancreatic Intraepithelial Neoplasias (PanINs) in various PDAC models, including the orthotopic, ectopic, spontaneous, and tamoxifen-induced tumors. Notably, LTZi-MHI148 detected preneoplastic PanIN lesions with Overexpressed RhoJ and active neoangiogenesis in both spontaneous and tamoxifen-induced PDAC murine models. Longitudinal imaging studies revealed that RhoJ-targeted neoangiogenesis tracks lesion progression, highlighting LTZi-MHI148's utility in monitoring disease progression. Furthermore, multiple LTZi-MHI148 administrations attenuated PanINs to PDAC progression, suggesting its potential as a therapeutic intervention. These findings underscore the translational potential of LTZi-MHI148 for the early detection and targeted therapy of PDAC, utilizing NIR-I/II imaging to monitor RhoJ overexpression in precancerous ductal neoplasia associated with neoangiogenesis.

Towards Understanding the Development of Breast Cancer: The Role of RhoJ in the Obesity Microenvironment.

Obesity is a growing pandemic with an increasing risk of inducing different cancer types, including breast cancer. Adipose tissue is proposed to be a major player in the initiation and progression of breast cancer in obese people. However, the mechanistic link between adipogenicity and tumorigenicity in breast tissues is poorly understood. We used in vitro and in vivo approaches to investigate the mechanistic relationship between obesity and the onset and progression of breast cancer. In obesity, adipose tissue expansion and remodeling are associated with increased inflammatory mediator's release and anti-inflammatory mediators' reduction.. In order to mimic the obesity micro-environment, we cultured cells in an enriched pro-inflammatory cytokine medium to which we added a low concentration of beneficial adipokines. Epithelial cells exposed to the obesity micro-environment were phenotypically transformed into mesenchymal-like cells, characterized by an increase in different mesenchymal markers and the acquisition of the major hallmarks of cancerous cells; these include sustained DNA damage, the activation of the ATR-Chk2 pathway, an increase in proliferation rate, cell invasion, and resistance to conventional chemotherapy. Transcriptomic analysis revealed that several genes, including RhoJ, CCL7, and MMP9, acted as potential major players in the observed phenomenon. The transcriptomics findings were confirmed in vitro using qRT-PCR and in vivo using high-fat-diet-fed mice. Our data suggests RhoJ as a potential novel molecular driver of tumor development in breast tissues and a mediator of cell resistance to conventional chemotherapy through PAK1 activation. These data propose that RhoJ is a potential target for therapeutic interventions in obese breast cancer patients.

Corrigendum: PlexinD1 signaling controls domain-specific dendritic development in newborn neurons in the postnatal olfactory bulb.

[This corrects the article DOI: 10.3389/fnins.2023.1143130.].

Oncogenic Gαq activates RhoJ through PDZ-RhoGEF.

Oncogenic Gαq causes uveal melanoma via non-canonical signaling pathways. This constitutively active mutant GTPase is also found in cutaneous melanoma, lung adenocarcinoma, and seminoma, as well as in benign vascular tumors, such as congenital hemangiomas. We recently described that PDZ-RhoGEF (also known as ARHGEF11), a canonical Gα12/13 effector, is enabled by Gαs Q227L to activate CdcIn addition, and we demonstrated that constitutively active Gαq interacts with the PDZ-RhoGEF DH-PH catalytic module, but does not affect its binding to RhoA or Cdc. This suggests that it guides this RhoGEF to gain affinity for other GTPases. Since RhoJ, a small GTPase of the Cdc42 subfamily, has been involved in tumor-induced angiogenesis and the metastatic dissemination of cancer cells, we hypothesized that it might be a target of oncogenic Gαq signaling via PDZ-RhoGEF. Consistent with this possibility, we found that Gαq Q209L drives full-length PDZ-RhoGEF and a DH-PH construct to interact with nucleotide-free RhoJ-G33A, a mutant with affinity for active RhoJ-GEFs. Gαq Q209L binding to PDZ-RhoGEF was mapped to the PH domain, which, as an isolated construct, attenuated the interaction of this mutant GTPase with PDZ-RhoGEF's catalytic module (DH-PH domains). Expression of these catalytic domains caused contraction of endothelial cells and generated fine cell sprouts that were inhibited by co-expression of dominant negative RhoJ. Using relational data mining of uveal melanoma patient TCGA datasets, we got an insight into the signaling landscape that accompanies the Gαq/PDZ-RhoGEF/RhoJ axis. We identified three transcriptional signatures statistically linked with shorter patient survival, including GPCRs and signaling effectors that are recognized as vulnerabilities in cancer cell synthetic lethality datasets. In conclusion, we demonstrated that an oncogenic Gαq mutant enables the PDZ-RhoGEF DH-PH module to recognize RhoJ, suggesting an allosteric mechanism by which this constitutively active GTPase stimulates RhoJ via PDZ-RhoGEF. These findings highlight PDZ-RhoGEF and RhoJ as potential targets in tumors driven by mutant Gαq.

RHOJ Induces Epithelial-to-Mesenchymal Transition by IL-6/STAT3 to Promote Invasion and Metastasis in Gastric Cancer.

Background: Recently, the molecular classification of gastric cancer (GC) promotes the advances of GC patients' precision therapy and prognosis prediction. According to the Asian Cancer Research Group (ACRG), GC is classified as microsatellite instable (MSI) subtype GC, microsatellite stable/epithelial-to-mesenchymal transition (MSS/EMT) subtype GC, MSS/TP53- subtype GC, and MSS/TP53+ subtype GC. Due to the easy metastasis of EMT-subtype GC, it has the worst prognosis, the highest recurrence rate, and the tendency to occur at a younger age. Therefore, it is curious and crucial for us to understand the molecular basis of EMT-subtype GC. Methods: The expression of RHOJ was detected by quantitative real-time PCR (qPCR) and immunohistochemistry (IHC) in GC cells and tissues. Western blotting and immunofluorescence (IF) were conducted to examine the effects of RHOJ on the EMT markers' expression of GC cells. The GC cells' migration and invasion were investigated by transwell assay. The tumor growth and metastasis were demonstrated correspondingly in different xenograft models. Results: Firstly, it was noticed that RHOJ was significantly upregulated in EMT-subtype GC and RHOJ has close relationships with the EMT process of GC, based on the Gene Expression Omnibus (GEO) and the Cancer Genome Atlas (TCGA) databases. Next, transwell assay and tail vein metastasis models were conducted to verify that RHOJ mediates the EMT to regulate the invasion and metastasis of GC in vitro and in vivo. In addition, weakened tumor angiogenesis was observed after RHOJ knockdown by the angiogenesis assay of HUVEC. RNA-seq and further study unveiled that RHOJ aggravates the malignant progression of GC by inducing EMT through IL-6/STAT3 to promote invasion and metastasis. Finally, blocking the IL-6/STAT3 signaling overcame RHOJ-mediated GC cells' growth and migration. Conclusions: These results indicate that the upregulation of RHOJ contributes to EMT-subtype GC invasion and metastasis via IL-6/STAT3 signaling, and RHOJ is expected to become a promising biomarker and therapeutic target for EMT-subtype GC patients.

Elucidating the Associated Biological Function and Clinical Significance of RHOJ Expression in Urothelial Carcinoma.

Urothelial cancer, a common urinary system malignancy, often presents treatment challenges due to metastasis and chemotherapy side effects. Angiogenesis, crucial for tumor growth, has become a target for drug development. This study explores the expression, prognostic value, and clinical correlation of RHOJ in the TCGA BLCA, GSE31684, and GSE32894 datasets. We identify common differentially expressed genes across these databases and utilize g:Profiler and Cytoscape ClueGO for functional assessment. Further, we perform a gene set enrichment analysis (GSEA) using Hallmark gene sets and use the imsig package for immune cell infiltration analysis. Our analysis indicates that RHOJ expression levels significantly impact survival rates, tumor progression, and immune response in urothelial tumors. High RHOJ expression correlated with poor prognosis, advanced disease stages, and an increase in monocyte population within the tumor microenvironment. This aligns with current literature indicating a key role of immune infiltration in bladder cancer progression and treatment response. Moreover, the GSEA and imsig results further suggest a potential mechanistic link between RHOJ expression and immune-related pathways. Considering the increasing emphasis on immunotherapeutic strategies in bladder cancer management, our findings on RHOJ's potential as a diagnostic biomarker and its association with immune response open new avenues for therapeutic interventions.

PlexinD1 signaling controls domain-specific dendritic development in newborn neurons in the postnatal olfactory bulb.

Newborn neurons show immature bipolar morphology and continue to migrate toward their destinations. After the termination of migration, newborn neurons undergo spatially controlled dendrite formation and change into a complex morphology. The mechanisms of dendritic development of newborn neurons have not been fully understood. Here, we show that in the postnatal olfactory bulb (OB), the Sema3E-PlexinD1 signaling, which maintains bipolar morphology of newborn neurons, also regulates their dendritic development after the termination of migration in a dendritic domain-specific manner. Genetic ablation of Sema3E or PlexinD1 enhanced dendritic branching in the proximal domain of the apical dendrites of OB newborn granule cells, whereas PlexinD1 overexpression suppressed it in a Rho binding domain (RBD)-dependent manner. Furthermore, RhoJ, a small GTPase that directly binds to PlexinD1RBD in vascular endothelial cells, is expressed in migrating and differentiating newborn granule cells in the OB and is also involved in the suppression of proximal branching of their apical dendrites. These results suggest that the Sema3E-PlexinD1-RhoJ axis regulates domain-specific dendrite formation of newborn neurons in the postnatal OB.

Circ-RHOJ.1 regulated myocardial cell proliferation and apoptosis via targeting the miR-124-3p/NRG-1 axis in myocardial ischemia/reperfusion injury.

Introduction: Myocardial ischemia/reperfusion (I/R) injury is a leading cause of cardiac dysfunction. Circular RNAs (circRNAs) are involved in the pathogenesis of myocardial I/R injury. However, the functions and underlying mechanisms are unclear. The present study determined the role of circ-RHOJ.1 in regulating myocardial cell proliferation and apoptosis after I/R injury. Material and methods: Myocardial cells isolated from Sprague-Dawley rats were identified with an immunofluorescence assay using cardiac troponin T antibody. Expression of circ-RHOJ.1, miR-124-3p and neuregulin-1 (NRG1) mRNA was assessed with real-time quantitative polymerase chain reaction. NRG1 protein expression was evaluated with western blot and immunofluorescence assays. Dual-luciferase reporter assay was performed to confirm interaction between miR-124-3p and circ-RHOJ.1, and miR-124-3p and NRG1. Effects of circ-RHOJ.1 overexpression or miR-124-3p inhibition on cell proliferation and apoptosis were evaluated using cell counting kit (CCK)-8 assay and flow cytometry. Cytokines levels were analyzed with an enzyme-linked immunosorbent assay. Results: Myocardial cells were successfully isolated and had down-regulated expression of circ-RHOJ.1 and NRG1, and up-regulated expression of miR-124-3p after I/R injury. circ-RHOJ.1 acted as a sponge for miR-124-3p, and NRG1 served as a target gene of miR-124-3p. circ-RHOJ.1 overexpression or miR-124-3p inhibition increased interleukin (IL)-10 levels and reduced IL-2, IL-6, and tumor necrosis factor-α levels in myocardial cells after I/R injury. Functional assay results illustrated that circ-RHOJ.1 overexpression or miR-124-3p inhibition enhanced proliferation and inhibited apoptosis of myocardial cells after I/R injury. Conclusions: Circ-RHOJ.1 served as a molecular marker of myocardial I/R injury via regulation of miR-124-3p and NRG1 expression.

Structure-based design of CDC42 effector interaction inhibitors for the treatment of cancer.

CDC42 family GTPases (RHOJ, RHOQ, CDC42) are upregulated but rarely mutated in cancer and control both the ability of tumor cells to invade surrounding tissues and the ability of endothelial cells to vascularize tumors. Here, we use computer-aided drug design to discover a chemical entity (ARN22089) that has broad activity against a panel of cancer cell lines, inhibits S6 phosphorylation and MAPK activation, activates pro-inflammatory and apoptotic signaling, and blocks tumor growth and angiogenesis in 3D vascularized microtumor models (VMT) in vitro. Additionally, ARN22089 has a favorable pharmacokinetic profile and can inhibit the growth of BRAF mutant mouse melanomas and patient-derived xenografts in vivo. ARN22089 selectively blocks CDC42 effector interactions without affecting the binding between closely related GTPases and their downstream effectors. Taken together, we identify a class of therapeutic agents that influence tumor growth by modulating CDC42 signaling in both the tumor cell and its microenvironment.

RhoJ facilitates angiogenesis in glioblastoma via JNK/VEGFR2 mediated activation of PAK and ERK signaling pathways.

Glioblastoma (GBM) is a highly vascularized malignant tumor that depends on new blood vessel formation. Small molecules targeting the angiogenic process may be an effective anti-GBM therapeutic strategy. We previously demonstrated that RhoJ promoted the progression and invasion of GBM. RhoJ has also been shown to be expressed in endothelial cells and plays an important role in regulating endothelial cell migration and tumor angiogenesis. Therefore, we aimed to evaluate the role and mechanism of actions of RhoJ in GBM angiogenesis. We analyzed the expression of RhoJ in different grade gliomas and investigated its role in GBM angiogenesis in vivo and in vitro. Furtherly, RNA sequencing (RNA-seq), Western blotting and immunofluorescence were performed to identify the molecular mechanism of RhoJ in regulating endothelial cell behavior and GBM angiogenesis. Here, we found that silencing RhoJ resulted in inhibition of HUVEC cell migration and blood vessel formation. Overexpression of RhoJ promoted the expression of CD31, EpCAM and moesin, suggesting RhoJ facilitated angiogenesis and the malignant progression of GBM. RNA-seq data showed that VEGF/TNF signaling pathway positively regulated RhoJ. The expression levels of RhoJ was upregulated with the stimulation of VEGF, and reduced by the treatment of JNK inhibitor SP600125. It was also found that the activity of PAK-BRAF-ERK was down-regulated upon RhoJ and JNK knockdown. In conclusion, these results suggested that RhoJ plays an essential role in regulating GBM angiogenesis through the JNK/VEGFR2-PAK-ERK signaling pathway and there might exist a VEGF-JNK/ERK-VEGF circuitry. Thus, RhoJ may be a candidate therapeutic target for anti-angiogenesis treatment in GBM.

Endothelial Focal Adhesions Are Functional Obstacles for Leukocytes During Basolateral Crawling.

An inflammatory response requires leukocytes to migrate from the circulation across the vascular lining into the tissue to clear the invading pathogen. Whereas a lot of attention is focused on how leukocytes make their way through the endothelial monolayer, it is less clear how leukocytes migrate underneath the endothelium before they enter the tissue. Upon finalization of the diapedesis step, leukocytes reside in the subendothelial space and encounter endothelial focal adhesions. Using TIRF microscopy, we show that neutrophils navigate around these focal adhesions. Neutrophils recognize focal adhesions as physical obstacles and deform to get around them. Increasing the number of focal adhesions by silencing the small GTPase RhoJ slows down basolateral crawling of neutrophils. However, apical crawling and diapedesis itself are not affected by RhoJ depletion. Increasing the number of focal adhesions drastically by expressing the Rac1 GEF Tiam1 make neutrophils to avoid migrating underneath these Tiam1-expressing endothelial cells. Together, our results show that focal adhesions mark the basolateral migration path of neutrophils.

Repression of RhoJ expression promotes TGF-ß-mediated EMT in human non-small-cell lung cancer A549cells.

Non-small-cell lung cancer (NSCLC) accounts for most cancer-related deaths because of its strong metastatic ability. It is important to understand NSCLC's molecular mechanisms of metastasis. RhoJ, a protein that belongs to the Rho family of small GTPases, regulates endothelial motility, angiogenesis, and adipogenesis. Recently, bioinformatics analysis showed that NSCLC patients with lower RhoJ expression had a worse survival outcome than those with high RhoJ expression. However, little is known about RhoJ's role in NSCLC. In the present study, we demonstrated that RhoJ knockdown accelerated TGF-ßmediated epithelial-to-mesenchymal transition (EMT), an important cancer metastasis process, in A549 and PC-9 cells. Furthermore, using Matrigel-coated transwell chambers, we showed that RhoJ knockdown enhanced the invasion capacity of A549 cells that had undergone EMT. Also, reduced RhoJ expression increased Smad3 phosphorylation and Snail expression during the EMT process. Our results provide the first evidence of a potential novel role for RhoJ in the inhibition of EMT via modulation of the TGF-ß-Smad signaling pathway, and shed new light on the mechanisms underlying EMT in NSCLC.

Rho GTPases in Retinal Vascular Diseases.

The Rho family of small GTPases (Rho GTPases) act as molecular switches that transduce extrinsic stimuli into cytoskeletal rearrangements. In vascular endothelial cells (ECs), Cdc42, Rac1, and RhoA control cell migration and cell-cell junctions downstream of angiogenic and inflammatory cytokines, thereby regulating vascular formation and permeability. While these Rho GTPases are broadly expressed in various types of cells, RhoJ is enriched in angiogenic ECs. Semaphorin 3E (Sema3E) releases RhoJ from the intracellular domain of PlexinD1, by which RhoJ induces actin depolymerization through competition with Cdc42 for their common effector proteins. RhoJ further mediates the Sema3E-induced association of PlexinD1 with vascular endothelial growth factor receptor (VEGFR) 2 and the activation of p38. Upon stimulation with VEGF-A, RhoJ facilitates the formation of a holoreceptor complex comprising VEGFR2, PlexinD1, and neuropilin-1, leading to the prevention of VEGFR2 degradation and the maintenance of intracellular signal transduction. These pleiotropic roles of RhoJ are required for directional EC migration in retinal angiogenesis. This review highlights the latest insights regarding Rho GTPases in the field of vascular biology, as it will be informative to consider their potential as targets for the treatment of aberrant angiogenesis and hyperpermeability in retinal vascular diseases.

ETV2 (Ets Variant Transcription Factor 2)-Rhoj Cascade Regulates Endothelial Progenitor Cell Migration During Embryogenesis.

OBJECTIVE: Endothelial progenitors migrate early during embryogenesis to form the primary vascular plexus. The regulatory mechanisms that govern their migration are not completely defined. Here, we describe a novel role for ETV2 (Ets variant transcription factor 2) in cell migration and provide evidence for an ETV2-Rhoj network as a mechanism responsible for this process. Approach and Results: Analysis of RNAseq datasets showed robust enrichment of migratory/motility pathways following overexpression of ETV2 during mesodermal differentiation. We then analyzed ETV2 chromatin immunoprecipitation-seq and assay for transposase accessible chromatin-seq datasets, which showed enrichment of chromatin immunoprecipitation-seq peaks with increased chromatin accessibility in migratory genes following overexpression of ETV2. Migratory assays showed that overexpression of ETV2 enhanced cell migration in mouse embryonic stem cells, embryoid bodies, and mouse embryonic fibroblasts. Knockout of Etv2 led to migratory defects of Etv2-EYFP+ angioblasts to their predefined regions of developing embryos relative to wild-type controls at embryonic day (E) 8.5, supporting its role during migration. Mechanistically, we showed that ETV2 binds the promoter region of Rhoj serving as an upstream regulator of cell migration. Single-cell RNAseq analysis of Etv2-EYFP+ sorted cells revealed coexpression of Etv2 and Rhoj in endothelial progenitors at E7.75 and E8.25. Overexpression of ETV2 led to a robust increase in Rhoj in both embryoid bodies and mouse embryonic fibroblasts, whereas, its expression was abolished in the Etv2 knockout embryoid bodies. Finally, shRNA-mediated knockdown of Rhoj resulted in migration defects, which were partially rescued by overexpression of ETV2. CONCLUSIONS: These results define an ETV2-Rhoj cascade, which is important for the regulation of endothelial progenitor cell migration.

MKL1 Mediates TGF-ß Induced RhoJ Transcription to Promote Breast Cancer Cell Migration and Invasion.

Differential regulation of gene transcription contributes to cancer metastasis. We investigated the involvement of a Rho GTPase (RhoJ) in breast cancer metastasis focusing on the mechanism underlying RhoJ trans-activation by pro-metastatic cues. We report that expression of RhoJ was up-regulated in malignant breast cancer cells compared to more benign ones. Higher RhoJ expression was also detected in human breast cancer biopsy specimens of advanced stages. RhoJ depletion attenuated breast cancer cell migration and invasion in vitro and metastasis in vivo. The pro-metastatic stimulus TGF-ß activated RhoJ via megakaryocytic leukemia 1 (MKL1). MKL1 interacted with and was recruited by ETS-related gene 1 (ERG1) to the RhoJ promoter to activate transcription. In conclusion, our data delineate a novel transcriptional pathway that contributes to breast cancer metastasis. Targeting the ERG1-MKL1-RhoJ axis may be considered as a reasonable approach to treat malignant breast cancer.

Rhoj Is a Novel Target for Progression and Invasion of Glioblastoma by Impairing Cytoskeleton Dynamics.

Rho GTPase family members were identified as critical regulators of cell morphology, actin cytoskeleton organization, cell movement, and cell cycle and also contributed to tumor progression, which have been implicated in various types of cancer metastasis and growth. Here, we firstly reported the dysregulation of Rhoj in glioblastoma multiforme (GBM) and aimed to investigate the role and mechanism of Rhoj in GBM. We analyzed the expression of 21 Rho GTPases family members and validated the expression of Rhoj in GBM by immunohistochemistry. We further investigated the role and mechanism of Rhoj in GBM both in vitro and in vivo. We observed that Rhoj is significantly overexpressed in GBM and associated with patients' survival. However, the role and underlying molecular mechanism of Rhoj in GBM are still unclear. We demonstrated that transcription factor c-Jun regulated the expression of Rhoj, and Rhoj interacted with moesin to promote GBM cell proliferation and migration by potentiating the activation of Rac1/PAK pathway and cytoskeletal dynamics. Rhoj may promote migration and invasion of GBM cells by regulating epithelial-mesenchymal transition (EMT)-like process. In conclusion, the Rhoj/Rac1/PAK signaling mediates invasion and progression of GBM and is a potential therapeutic target for GBM treatment. Rhoj may also be a promising biomarker for GBM diagnosis and prognosis.

RhoJ integrates attractive and repulsive cues in directional migration of endothelial cells.

During angiogenesis, VEGF acts as an attractive cue for endothelial cells (ECs), while Sema3E mediates repulsive cues. Here, we show that the small GTPase RhoJ integrates these opposing signals in directional EC migration. In the GTP-bound state, RhoJ interacts with the cytoplasmic domain of PlexinD1. Upon Sema3E stimulation, RhoJ released from PlexinD1 induces cell contraction. PlexinD1-bound RhoJ further facilitates Sema3E-induced PlexinD1-VEGFR2 association, VEGFR2 transphosphorylation at Y1214, and p38 MAPK activation, leading to reverse EC migration. Upon VEGF stimulation, RhoJ is required for the formation of the holoreceptor complex comprising VEGFR2, PlexinD1, and neuropilin-1, thereby preventing degradation of internalized VEGFR2, prolonging downstream signal transductions via PLCγ, Erk, and Akt, and promoting forward EC migration. After conversion to the GDP-bound state, RhoJ shifts from PlexinD1 to VEGFR2, which then terminates the VEGFR2 signals. RhoJ deficiency in ECs efficiently suppressed aberrant angiogenesis in ischemic retina. These findings suggest that distinct Rho GTPases may act as context-dependent integrators of chemotactic cues in directional cell migration and may serve as candidate therapeutic targets to manipulate cell motility in disease or tissue regeneration.

Endothelial cell sprouting driven by RhoJ directly activated by a membrane-anchored Intersectin 1 (ITSN1) RhoGEF module.

Endothelial cell sprouting is a critical event in tumor-induced angiogenesis. In melanoma and lung cancer murine models, targeting RhoJ prevents endothelial sprouting, tumor growth and metastasis and enhances the effects of conventional anti-neoplastic therapy. Aiming to understand how RhoJ is activated, we used a gain of function approach to identify constitutively active Rho guanine nucleotide exchange factors (RhoGEFs) able to promote RhoJ-dependent actin-driven membrane protrusions. We demonstrate that a membrane-anchored Intersectin 1 (ITSN1) DH-PH construct promotes endothelial cell sprouting via RhoJ. Mechanistically, this is controlled by direct interaction between the catalytic ITSN1 DH-PH module and RhoJ, it is sensitive to phosphorylation by focal adhesion kinase (FAK) and to endosomal trapping of the ITSN1 construct by dominant negative RhoJ. This ITSN1/RhoJ signaling axis is independent of Cdc42, a previously characterized ITSN1 target and a RhoJ close homologue. In conclusion, our results elucidate an ITSN1/RhoJ molecular link able to promote endothelial cell sprouting and set the basis to explore this signaling pathway in the context of tumor-induced angiogenesis.

Unraveling a model of TCL/RhoJ allosterism using TC10 reverse chimeras.

In addition to the classical regulation of GTPase activity by effector proteins, investigating the variations in the amino acid sequence and structures of GTPases often provides insights into regulatory mechanisms that are more GTPase-specific. TCL/RhoJ is a Rho GTPase most closely related to Cdc42 and TC10; however, its nucleotide exchange activity is distinctly influenced by N-terminal amino acids 17-20 and the more distal amino acids 121-129. In this short study, we have further explored the differences between TCL and its homolog TC10 and show that its unique mode of allosteric regulation requires broader diversification of its amino acid sequence than previously appreciated.

A Protective Role for RHOJ in NonSmall Cell Lung Cancer Based on Integrated Bioinformatics Analysis.

RHOJ is a small G protein characterized by its abundant expression in endothelial cells. Existing research has documented a link between abnormal RHOJ expression and carcinogenesis. This research aims to investigate the protective role of RHOJ in nonsmall cell lung cancer (NSCLC). In this study, Cancer Genome Atlas database and Gene Expression Omnibus were collected to analyze RHOJ expression and gene regulation networks in NSCLC. Oncomine™ and Gene Expression Profiling Interactive Analysis tools were first utilized to analyze RHOJ expression, and then cBioPortal was employed for identification of RHOJ alterations and associated functional networks. To identify differential RHOJ expression, LinkedOmics was used, which also served to analyze Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways. Besides, the target networks of kinases factors were explored using gene enrichment analysis. Our results suggested that lower expression of RHOJ was observed in patients with NSCLC compared with normal people. Low expression of this gene is linked to functional networks involving cytoskeleton, adhesion, infection, and Ras signaling pathways. Functional network analysis suggested that RHOJ regulates the Staphylococcus aureus infection, AGE-RAGE signaling pathway, and DNA and RNA damage. In conclusion, the results in this study demonstrated that data mining is an effective approach that can uncover information about RHOJ expression and potential regulatory networks in NSCLC, thus laying the groundwork for future studies of a similar kind.