Rho-like GTPases Research Articles

Abstract A115: Collective invasion facilitates liver metastasis in pancreatic ductal adenocarcinoma

Abstract Fifty percent of PDAC patients present with distant metastasis and face a dismal prognosis of 8 to 11 months. However, patients with lung metastases have better prognosis than patients with metastatic spread to other sites. In our PDAC metastasis cohort, patients with lung-only metastasis survived longer than patients with liver metastasis, despite sharing the same tumor subtype. To understand features of the primary tumor linked to metastatic site, we generated a novel gene signature for primary organotropism (pORG) that distinguishes primary tumors that developed liver metastasis from those that developed lung metastasis, independent of known tumor subtypes. Our pORG signature predicts survival independently of transcriptional subtype, genetic alterations and clinicopathological variables and in a validation cohort consisting of TCGA pancreatic cancer. Primary tumors with high pORG scores, i.e., liver-tropic primaries, have increased transcriptomic signatures of proliferation and replication stress whereas low pORG tumors have increased lymphocyte gene expression. To validate our findings, we generated cyclic immunofluorescence multiplex imaging data from 34 primary tumors. As anticipated, single cell image analysis revealed high B and T cell infiltration in low-pORG tumors, and increased proliferation and replication stress foci in high-pORG tumors. Intriguingly, malignant cells from high pORG tumors had an increased fraction KRT19-high tumor cells relative to low pORG tumors and were distinctly epithelial in appearance. To reconcile the metastatic behavior of high pORG tumors with their apparent enrichment for epithelial-state cells, we hypothesized that liver-tropism was associated with a partial epithelial-to-mesenchymal transition (EMT) state and metastasis via collective invasion. Bulk transcriptional analysis of 200 PDAC primary tumors revealed a distinct subset of high pORG tumors have increased expression of markers linked to partial EMT and collective invasion, including RAB11a, Rho-like GTPases, and proteases MMP7 and KLK7. Our results support a model of E-cadherin internalization facilitating partial EMT and collective invasion. Current efforts include in-vitro characterization of migration modes in patient-derived cell lines and organoids. Our work suggests unique and targetable tumor intrinsic features linked to organotropism in PDAC. Citation Format: Jennifer R. Eng, Carl Pelz, Koei Chin, Sam Sivagnanam, Link M. Jason, Dove Keith, Lisa Coussens, Rosalie C. Sears. Collective invasion facilitates liver metastasis in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A115.

MiR-590-5p/Tiam1-mediated glucose metabolism promotes malignant evolution of pancreatic cancer by regulating SLC2A3 stability

BackgroundT lymphoma invasion and metastasis 1 (Tiam1) is a tumor related gene that specifically activates Rho-like GTPases Rac1 and plays a critical role in the progression of various malignancies. Glycolysis plays an important role in cancer progression, it is crucial for supplying energy and producing metabolic end products, which can maintain the survival of tumor cells. As yet, however, the mechanism of Tiam1 in glycolysis reprogramming of pancreatic cancer (PC) remains to be clarified. Here, we investigated the functional role of Tiam1 in PC cell proliferation, metastasis and glycolysis reprogramming. It is expected to provide a new direction for clinical treatment.MethodsThe clinical relevance of Tiam1 was evaluated in 66 patients with PC, the effect of Tiam1 on cell proliferation was detected via 5-Ethynyl-2′-deoxyuridine (EdU) and colony formation. The ability of cell migration was detected by the wound healing and Transwell. Quantitative real time polymerase chain reaction (qRT-PCR) and luciferase reporter gene experiments clarify the regulatory relationship of miR-590-5p inhibiting Tiam1. Detection of the molecular mechanism of Tiam1 regulating glucose metabolism reprogramming in PC by glucose metabolism kit. RNA sequencing and Co-Immunoprecipitation (CoIP) have identified glucose transporter protein 3 (SLC2A3) as a key downstream target gene for miR-590-5p/Tiam1.ResultsWe found that Tiam1 expression increased in PC tissues and was associated with lymph node metastasis. The silencing or exogenous overexpression of Tiam1 significantly altered the proliferation, invasion, and angiogenesis of PC cells through glucose metabolism pathway. In addition, Tiam1 could interact with the crucial SLC2A3 and promote the evolution of PC in a SLC2A3-dependent manner. Moreover, miR-590-5p was found to exacerbate the PC cell proliferation, migration and invasion by targeting Tiam1. Furthermore, the reversing effects on proliferation, migration and invasion were found in PC cells with miR-590-5p/Tiam1 overexpression after applying glucose metabolism inhibition.ConclusionsOur findings demonstrate the critical role of Tiam1 in PC development and the miR-590-5p/Tiam1/SLC2A3 signaling pathway may serve as a target for new PC therapeutic strategies.Graphical

The long non-coding RNA NEAT1 promotes the progression of human ovarian cancer through targeting miR-214-3p and regulating angiogenesis

BackgroundAngiogenesis and metastasis contributes substantially to the poor outcome of patients with ovarian cancer. We aimed to explore the role and mechanisms of the long non-coding RNA NEAT1 (nuclear enriched abundant transcript 1) in regulating angiogenesis and metastasis of human ovarian cancer. NEAT1 expression in human ovarian cancer tissues and cell lines including SKOV-3 and A2780 was investigated through in situ hybridization. Gene knockdown and overexpressing were achieved through lentivirus infection, transfection of plasmids or microRNA mimics. Cell viability was measured with the cell counting kit-8 assay, while apoptosis was determined by flow cytometry. Cell migration and invasion were evaluated by transwell experiments, and protein expression was determined by western blot assays or immunohistochemistry. Duo-luciferase reporter assay was employed to confirm the interaction between NEAT1 and target microRNA. In vivo tumor growth was evaluated in nude mice with xenografted SKOV-3/A2780 cells, and blood vessel formation in tumor was examined by histological staining.ResultsNEAT1 was highly expressed in ovarian cancer tissues of patients and cell lines. MiR-214-3p was identified as a sponging target of NEAT1, and they antagonizedeach other in a reciprocal manner. NEAT1-overexpressing SKOV-3 and A2780 cells had significantly increased proliferation, reduced apoptosis, and augmented abilities of migration and invasion, while cells with NEAT1-knockdown displayed markedly attenuated traits of malignancies. Additionally, the levels of NEAT1 appeared to be positively correlated with the expression levels of angiogenesis-related molecules, including Semaphorin 4D (Sema4D), Sema4D receptor Plexin B1, T-lymphoma invasion and metastasis-inducing protein-1 (Tiam1), and Rho-like GTPases Rac1/2/3. In the xenograft mouse model, more NEAT1 expression resulted in faster in vivo tumor growth, more blood vessel formation in tumor tissues, as well as higher expression levels of angiogenesis-related molecules and CD31.ConclusionsNEAT1 promotes angiogenesis and metastasis in human ovarian cancer. NEAT1 and miR-214-3p are promising targets for developing therapeutics to treat human ovarian cancer.

Comparative functional analysis of PdeNAC2 and AtVND6 in the tracheary element formation.

Tracheary elements (i.e. vessel elements and tracheids) are highly specialized, non-living cells present in the water-conducting xylem tissue. In angiosperms, proteins in the VASCULAR-RELATED NAC-DOMAIN (VND) subgroup of the NAC (NAM, ATAF1,2, and CUC2) transcription factor family (e.g. AtVND6) are required for the differentiation of vessel elements through transcriptional regulation of genes responsible for secondary cell wall formation and programmed cell death. Gymnosperms, however, produce only tracheids, the mechanism of which remains elusive. Here, we report functional characteristics of PdeNAC2, a VND homolog in Pinus densiflora, as a key regulator of tracheid formation. Interestingly, our molecular genetic analyses show that PdeNAC2 can induce the formation of vessel element-like cells in angiosperm plants, demonstrated by transgenic overexpression of either native or NAC domain-swapped synthetic genes of PdeNAC2 and AtVND6 in both Arabidopsis and hybrid poplar. Subsequently, genome-wide identification of direct target (DT) genes of PdeNAC2 and AtVND6 revealed 138 and 174 genes as putative DTs, respectively, but only 17 genes were identified as common DTs. Further analyses have found that PdeNAC2 does not control some AtVND6-dependent vessel differentiation genes in angiosperm plants, such as AtVRLK1, LBD15/30 and pit-forming Rho-like GTPases from plant (ROP) signaling genes. Collectively, our results suggest that different target gene repertoires of PdeNAC2 and AtVND6 may contribute to the evolution of tracheary elements.

ZmRop1 participates in maize defense response to the damage of Spodoptera frugiperda larvae through mediating ROS and soluble phenol production.

As plant-specific molecular switches, Rho-like GTPases (Rops) are vital for plant survival in response to biotic and abiotic stresses. However, their roles in plant defense response to phytophagous insect's damage are largely unknown. In this study, the expression levels of nine maize RAC family genes were analyzed after fall armyworm (FAW) larvae infestation. Among the analyzed genes, ZmRop1 was specifically and highly expressed, and its role in maize response to FAW larvae damage was studied. The results showed that upon FAW larvae infestation, salicylic acid and methyl jasmonate treatment ZmRop1 gene transcripts were all down-regulated. However, upon mechanical injury, the expression level of ZmRop1 was up-regulated. Overexpression of ZmRop1 gene in maize plants could improve maize plant resistance to FAW larvae damage. Conversely, silencing of ZmRop1 increased maize plant susceptibility to FAW larvae damage. The analysis of the potential anti-herbivore metabolites, showed that ZmRop1 promoted the enzyme activities of catalase, peroxidase and the expression levels of ZmCAT, ZmPOD, ZmRBOHA and ZmRBOHB, thereby enhancing the reactive oxygen species (ROS) production, including the content of O2- and H2O2. In addition, overexpression or silencing of ZmRop1 could have influence on the content of the total soluble phenol through mediating the activity of polyphenol oxidase. In summary, the results illuminated our understanding of how ZmRop1 participate in maize defense response to FAW larvae damage as a positive regulator through mediating ROS production and can be used as a reference for the green prevention and control of FAW larvae.

ROP signaling regulates spatial pattern of cell division and specification of meristem notch

The formation of cell polarity is essential for many developmental processes such as polar cell growth and spatial patterning of cell division. A plant-specific ROP (Rho-like GTPases from Plants) subfamily of conserved Rho GTPase plays a crucial role in the regulation of cell polarity. However, the functional study of ROPs in angiosperm is challenging because of their functional redundancy. The Marchantia polymorpha genome encodes a single ROP gene, MpROP, providing an excellent genetic system to study ROP-dependent signaling pathways. Mprop knockout mutants exhibited rhizoid growth defects, and MpROP was localized at the tip of elongating rhizoids, establishing a role for MpROP in the control of polar cell growth and its functional conservation in plants. Furthermore, the Mprop knockout mutant showed defects in the formation of meristem notches associated with disorganized cell division patterns. These results reveal a critical function of MpROP in the regulation of plant development. Interestingly, these phenotypes were complemented not only by MpROP but also Arabidopsis AtROP2, supporting the conservation of ROP's function among land plants. Our results demonstrate a great potential for M. polymorpha as a powerful genetic system for functional and mechanistic elucidation of ROP signaling pathways during plant development.

Comprehensive Analysis of Subcellular Localization, Immune Function and Role in Bacterial wilt Disease Resistance of Solanum lycopersicum Linn. ROP Family Small GTPases.

ROPs (Rho-like GTPases from plants) belong to the Rho-GTPase subfamily and serve as molecular switches for regulating diverse cellular events, including morphogenesis and stress responses. However, the immune functions of ROPs in Solanum lycopersicum Linn. (tomato) is still largely unclear. The tomato genome contains nine genes encoding ROP-type small GTPase family proteins (namely SlRop1–9) that fall into five distinct groups as revealed by phylogenetic tree. We studied the subcellular localization and immune response induction of nine SlRops by using a transient overexpression system in Nicotiana benthamiana Domin. Except for SlRop1 and SlRop3, which are solely localized at the plasma membrane, most of the remaining ROPs have additional nuclear and/or cytoplasmic distributions. We also revealed that the number of basic residues in the polybasic region of ROPs tends to be correlated with their membrane accumulation. Though nine SlRops are highly conserved at the RHO (Ras Homology) domains, only seven constitutively active forms of SlRops were able to trigger hypersensitive responses. Furthermore, we analyzed the tissue-specific expression patterns of nine ROPs and found that the expression levels of SlRop3, 4 and 6 were generally high in different tissues. The expression levels of SlRop1, 2 and 7 significantly decreased in tomato seedlings after infection with Ralstonia solanacearum (E.F. Smith) Yabuuchi et al. (GMI1000); the others did not respond. Infection assays among nine ROPs showed that SlRop3 and SlRop4 might be positive regulators of tomato bacterial wilt disease resistance, whereas the rest of the ROPs may not contribute to defense. Our study provides systematic evidence of tomato Rho-related small GTPases for localization, immune response, and disease resistance.

Functional Characterization of Tomato ShROP7 in Regulating Resistance against Oidium neolycopersici.

ROPs (Rho-like GTPases from plants) are a unique family of small GTP-binding proteins in plants and play vital roles in numerous cellular processes, including growth and development, abiotic stress signaling, and plant defense. In the case of the latter, the role of ROPs as response regulators to obligate parasitism remains largely enigmatic. Herein, we isolated and identified ShROP7 and show that it plays a critical role in plant immune response to pathogen infection. Real-time quantitative PCR analysis revealed that the expression of ShROP7 was significantly increased during incompatible interactions. To establish its requirement for resistance, we demonstrate that virus-induced gene silencing (VIGS) of ShROP7 resulted in increased susceptibility of tomato to Oidium neolycopersici (On) Lanzhou strain (On-Lz). Downstream resistance signaling through H2O2 and the induction of the hypersensitive response (HR) in ShROP7-silenced plants were significantly reduced after inoculating with On-Lz. Taken together, with the identification of ShROP7-interacting candidates, including ShSOBIR1, we demonstrate that ShROP7 plays a positive regulatory role in tomato powdery mildew resistance.

MiR-21-5p/Tiam1-mediated glycolysis reprogramming drives breast cancer progression via enhancing PFKL stabilization.

T lymphoma invasion and metastasis 1 (Tiam1) as a tumor-associated gene specifically activates Rho-like GTPases Rac1 and implicates in the invasive phenotype of many cancers. Altering the glycolytic pathway is foreseen as a sound approach to trigger cancer regression. However, the mechanism of Tiam1 in breast cancer (BC) glycolysis reprogramming remains to be clarified. Here, we reported the Tiam1 high expression and prognostic significance in BC. In vitro and in vivo experimental assays identified the functional role of Tiam1 in promoting BC cell proliferation, metastasis and glycolysis reprogramming. Mechanistically, we showed for the first time that Tiam1 could interact with the crucial glycolytic enzyme phosphofructokinase, liver type (PFKL) and promote the evolution of BC in a PFKL-dependent manner. Moreover, miR-21-5p was found to exacerbate the BC proliferation and aggression by targeting Tiam1. Altogether, our study highlights the critical role of Tiam1 in BC development and that the miR-21-5p/Tiam1/PFKL signaling pathway may serve as a target for new anti-BC therapeutic strategies.

Identification of Kic1p and Cdc42p as Novel Targets to Engineer Yeast Acetic Acid Stress Tolerance

Robust yeast strains that are tolerant to multiple stress environments are desired for an efficient biorefinery. Our previous studies revealed that zinc sulfate serves as an important nutrient for stress tolerance of budding yeast Saccharomyces cerevisiae. Acetic acid is a common inhibitor in cellulosic hydrolysate, and the development of acetic acid-tolerant strains is beneficial for lignocellulosic biorefineries. In this study, comparative proteomic studies were performed using S. cerevisiae cultured under acetic acid stress with or without zinc sulfate addition, and novel zinc-responsive proteins were identified. Among the differentially expressed proteins, the protein kinase Kic1p and the small rho-like GTPase Cdc42p, which is required for cell integrity and regulation of cell polarity, respectively, were selected for further studies. Overexpression of KIC1 and CDC42 endowed S. cerevisiae with faster growth and ethanol fermentation under the stresses of acetic acid and mixed inhibitors, as well as in corncob hydrolysate. Notably, the engineered yeast strains showed a 12 h shorter lag phase under the three tested conditions, leading to up to 52.99% higher ethanol productivity than that of the control strain. Further studies showed that the transcription of genes related to stress response was significantly upregulated in the engineered strains under the stress condition. Our results in this study provide novel insights in exploring zinc-responsive proteins for applications of synthetic biology in developing a robust industrial yeast.

Butyrate Protects against Diet-Induced NASH and Liver Fibrosis and Suppresses Specific Non-Canonical TGF-β Signaling Pathways in Human Hepatic Stellate Cells.

In obesity-associated non-alcoholic steatohepatitis (NASH), persistent hepatocellular damage and inflammation are key drivers of fibrosis, which is the main determinant of NASH-associated mortality. The short-chain fatty acid butyrate can exert metabolic improvements and anti-inflammatory activities in NASH. However, its effects on NASH-associated liver fibrosis remain unclear. Putative antifibrotic effects of butyrate were studied in Ldlr-/-.Leiden mice fed an obesogenic diet (HFD) containing 2.5% (w/w) butyrate for 38 weeks and compared with a HFD-control group. Antifibrotic mechanisms of butyrate were further investigated in TGF-β-stimulated primary human hepatic stellate cells (HSC). HFD-fed mice developed obesity, insulin resistance, increased plasma leptin levels, adipose tissue inflammation, gut permeability, dysbiosis, and NASH-associated fibrosis. Butyrate corrected hyperinsulinemia, lowered plasma leptin levels, and attenuated adipose tissue inflammation, without affecting gut permeability or microbiota composition. Butyrate lowered plasma ALT and CK-18M30 levels and attenuated hepatic steatosis and inflammation. Butyrate inhibited fibrosis development as demonstrated by decreased hepatic collagen content and Sirius-red-positive area. In TGF-β-stimulated HSC, butyrate dose-dependently reduced collagen deposition and decreased procollagen1α1 and PAI1 protein expression. Transcriptomic analysis and subsequent pathway and upstream regulator analysis revealed deactivation of specific non-canonical TGF-β signaling pathways Rho-like GTPases and PI3K/AKT and other important pro-fibrotic regulators (e.g., YAP/TAZ, MYC) by butyrate, providing a potential rationale for its antifibrotic effects. In conclusion, butyrate protects against obesity development, insulin resistance-associated NASH, and liver fibrosis. These antifibrotic effects are at least partly attributable to a direct effect of butyrate on collagen production in hepatic stellate cells, involving inhibition of non-canonical TGF-β signaling pathways.

Membrane receptor-mediated mechano-transduction maintains cell integrity during pollen tube growth within the pistil.

Invasive or penetrative growth is critical for developmental and reproductive processes (e.g., pollen tube penetration of pistils) and disease progression (e.g., cancer metastasis and fungal hyphae invasion). The invading or penetrating cells experience drastic changes in mechanical pressure from the surroundings and must balance growth with cell integrity. Here, we show that Arabidopsis pollen tubes sense and/or respond to mechanical changes via a cell-surface receptor kinase Buddha's Paper Seal 1 (BUPS1) while emerging from compressing female tissues. BUPS1-defective pollen tubes fail to maintain cell integrity after emergence from these tissues. The mechano-transduction function of BUPS1 is established by using a microfluidic channel device mimicking the mechanical features of the invivo growth path. BUPS1-based mechano-transduction activates Rho-like GTPase from Plant 1 (ROP1) GTPase to promote exocytosis that facilitates secretion of BUPS1's ligands for mechanical signal amplification and cell wall rigidification in pollen tubes. These findings uncover a membrane receptor-based mechano-transduction system for cells to cope with the physical challenges during invasive or penetrative growth.

TRP Channels and Small GTPases Interplay in the Main Hallmarks of Metastatic Cancer.

Transient Receptor Potential (TRP) cations channels, as key regulators of intracellular calcium homeostasis, play a central role in the essential hallmarks of cancer. Among the multiple pathways in which TRPs may be involved, here we focus our attention on the ones involving small guanosine triphosphatases (GTPases), summarizing the main processes associated with the metastatic cascade, such as migration, invasion and tumor vascularization. In the last decade, several studies have highlighted a bidirectional interplay between TRPs and small GTPases in cancer progression: TRP channels may affect small GTPases activity via both Ca2+-dependent or Ca2+-independent pathways, and, conversely, some small GTPases may affect TRP channels activity through the regulation of their intracellular trafficking to the plasma membrane or acting directly on channel gating. In particular, we will describe the interplay between TRPC1, TRPC5, TRPC6, TRPM4, TRPM7 or TRPV4, and Rho-like GTPases in regulating cell migration, the cooperation of TRPM2 and TRPV2 with Rho GTPases in increasing cell invasiveness and finally, the crosstalk between TRPC1, TRPC6, TRPM8, TRPV4 and both Rho- and Ras-like GTPases in inducing aberrant tumor vascularization.

Rho GTPase ROP1 Interactome Analysis Reveals Novel ROP1-Associated Pathways for Pollen Tube Polar Growth in Arabidopsis.

ROP (Rho-like GTPases from plants) GTPases are polarly localized key regulators of polar growth in pollen tubes and other cells in plants. However, how ROP GTPases are regulated and how they control polar growth remains to be fully understood. To gain new insights into ROP-dependent mechanisms underlying polar cell growth, we characterized the interactome of ROP1 GTPase that controls Arabidopsis pollen tube (PT) tip growth, an extreme form of polar cell growth. We established an efficient method for culturing Arabidopsis pollen tubes in liquid medium, which was used for immunoprecipitation/mass spectrometry-based identification of ROP1-associated proteins. A total of 654 candidates were isolated from the ROP1 interactome in Arabidopsis pollen tubes, and GO (Gene Ontology) classification and pathway analysis revealed multiple uncharacterized ROP1-dependent processes including translation, cell wall modification, post transcriptional modification, and ion homeostasis, in addition to known ROP1-dependent pathways. The ROP1-interactome data was further supported by the co-expression of the candidate interactors in highly mature pollen with PT germination and growth defects being discovered in 25% (8/32) of the candidate mutant genes. Taken together, our work uncovers valuable information for the identification and functional elucidation of ROP-associated proteins in the regulation of polar growth, and provides a reliable reference to identify critical regulators of polar cell growth in the future.

Roles of FERONIA-like receptor genes in regulating grain size and quality in rice.

Grain yield and quality are critical factors that determine the value of grain crops. In this study, we analyzed the functions of 12 FERONIA-like receptor (FLR) family members in rice and investigated their effects on grain size and quality. We found that FLR1, FLR2 and FLR8 negatively regulated grain size, and FLR15 positively regulated grain size. flr1 mutants had a higher cell number and an accelerated rate of grain filling compared to wild-type plants, which led to grains with greater widths. A mechanism underlying the regulation of grain size by FLR1 is that FLR1 is associated with OsRac1 Rho-like GTPase, a positive regulator of grain size. Regarding grain quality, the flr1 mutant had a higher percentage of chalkiness compared with wild-type plants, and seeds carrying mutations in flr3 and flr14 had endosperms with white floury cores. To elucidate the possible mechanism underlying this phenomenon, we found that FLR1 was constitutively expressed during endosperm development. RNA-seq analysis identified 2,367 genes that were differentially expressed in the flr1 mutant, including genes involved in starch and sucrose metabolism and carbon fixation. In this study, we identified the roles played by several FLR genes in regulating grain size and quality in rice and provided insights into the molecular mechanism governing the FLR1-mediated regulation of grain size.

Pattern of Invasion in Human Pancreatic Cancer Organoids Is Associated with Loss of SMAD4 and Clinical Outcome.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy characterized by extensive local invasion and systemic spread. In this study, we employed a three-dimensional organoid model of human pancreatic cancer to characterize the molecular alterations critical for invasion. Time-lapse microscopy was used to observe invasion in organoids from 25 surgically resected human PDAC samples in collagen I. Subsequent lentiviral modification and small-molecule inhibitors were used to investigate the molecular programs underlying invasion in PDAC organoids. When cultured in collagen I, PDAC organoids exhibited two distinct, morphologically defined invasive phenotypes, mesenchymal and collective. Each individual PDAC gave rise to organoids with a predominant phenotype, and PDAC that generated organoids with predominantly mesenchymal invasion showed a worse prognosis. Collective invasion predominated in organoids from cancers with somatic mutations in the driver gene SMAD4 (or its signaling partner TGFBR2). Reexpression of SMAD4 abrogated the collective invasion phenotype in SMAD4-mutant PDAC organoids, indicating that SMAD4 loss is required for collective invasion in PDAC organoids. Surprisingly, invasion in passaged SMAD4-mutant PDAC organoids required exogenous TGFβ, suggesting that invasion in SMAD4-mutant organoids is mediated through noncanonical TGFβ signaling. The Rho-like GTPases RAC1 and CDC42 acted as potential mediators of TGFβ-stimulated invasion in SMAD4-mutant PDAC organoids, as inhibition of these GTPases suppressed collective invasion in our model. These data suggest that PDAC utilizes different invasion programs depending on SMAD4 status, with collective invasion uniquely present in PDAC with SMAD4 loss. SIGNIFICANCE: Organoid models of PDAC highlight the importance of SMAD4 loss in invasion, demonstrating that invasion programs in SMAD4-mutant and SMAD4 wild-type tumors are different in both morphology and molecular mechanism.

A GTPase-induced switch in phospholipid affinity of collybistin contributes to synaptic gephyrin clustering.

Synaptic transmission between neurons relies on the exact spatial organization of postsynaptic transmitter receptors, which are recruited and positioned by dedicated scaffolding and regulatory proteins. At GABAergic synapses, the regulatory protein collybistin (Cb, also known as ARHGEF9) interacts with small GTPases, cell adhesion proteins and phosphoinositides to recruit the scaffolding protein gephyrin and GABAA receptors to nascent synapses. We dissected the interaction of Cb with the small Rho-like GTPase TC10 (also known as RhoQ) and phospholipids. Our data define a protein-lipid interaction network that controls the clustering of gephyrin at synapses. Within this network, TC10 and monophosphorylated phosphoinositides, particulary phosphatidylinositol 3-phosphate (PI3P), provide a coincidence detection platform that allows the accumulation and activation of Cb in endomembranes. Upon activation, TC10 induces a phospholipid affinity switch in Cb, which allows Cb to specifically interact with phosphoinositide species present at the plasma membrane. We propose that this GTPase-based regulatory switch mechanism represents an important step in the process of tethering of Cb-dependent scaffolds and receptors at nascent postsynapses.

Bioinformatics analysis of potential therapeutic targets among ARHGAP genes in breast cancer.

GTPase activating proteins (RhoGAPs) serve significant roles in multiple aspects of tumor biology. Genes encoding RhoGAPs (ARHGAP), which switch off Rho-like GTPases, are responsible for breast cancer biogenesis. However, the identification of suitable and novel biomarkers for precision treatment and prognosis remains challenging. The present study aimed to evaluate the expression of ARHGAP family genes in breast cancer and investigate the survival data using the Oncomine, Kaplan-Meier Plotter, bcGenExMiner and cBioPortal online databases. The results demonstrated low expression of ARHGAP6, 7, 10, 14, 19, 23 and 24 and high expression of ARHGAP9, 11, 15, 18 and 30 in patients with breast cancer compared with that in healthy individuals. The survival analysis revealed that low expression levels of ARHGAP6, 7 and 19 were associated with poor relapse-free survival (RFS) and overall survival (OS), whereas high expression levels of ARHGAP9, 15 and 30 were associated with preferable RFS and OS. Metastatic relapse data demonstrated that higher expression of ARHGAP9, 15, 18, 19, 25 and 30 were associated with better prognosis and increased expression of ARHGAP11A and 14 exerted negative effects on patient prognosis. The overlapping genes ARHGAP9, 15, 19 and 30 obtained from these bioinformatics analysis tools exhibited significant association with clinical parameters including age, the presence of estrogen receptor, progesterone receptor and epidermal growth factor receptor-2, Scarff-Bloom-Richardson grade and Nottingham prognostic index. In conclusion, bioinformatics analysis revealed that ARHGAP9, 15, 19 and 30, but not other ARHGAP family genes may be promising targets with prognostic value and biological function for precision treatment of breast cancer.

The Rho-family GTPase OsRac1 controls rice grain size and yield by regulating cell division

Grain size is a key factor for determining grain yield in crops and is a target trait for both domestication and breeding, yet the mechanisms underlying the regulation of grain size are largely unclear. Here we show that the grain size and yield of rice (Oryza sativa) is positively regulated by ROP GTPase (Rho-like GTPase from plants), a versatile molecular switch modulating plant growth, development, and responses to the environment. Overexpression of rice OsRac1ROP not only increases cell numbers, resulting in a larger spikelet hull, but also accelerates grain filling rate, causing greater grain width and weight. As a result, OsRac1 overexpression improves grain yield in O. sativa by nearly 16%. In contrast, down-regulation or deletion of OsRac1 causes the opposite effects. RNA-seq and cell cycle analyses suggest that OsRac1 promotes cell division. Interestingly, OsRac1 interacts with and regulates the phosphorylation level of OsMAPK6, which is known to regulate cell division and grain size in rice. Thus, our findings suggest OsRac1 modulates rice grain size and yield by influencing cell division. This study provides insights into the molecular mechanisms underlying the control of rice grain size and suggests that OsRac1 could serve as a potential target gene for breeding high-yield crops.

Recent Advances in Genetic Aspects and Treatments for Steroid-Sensitive Nephrotic Syndrome in Children

This article reviews recent advances in genetic aspects and treatments for steroid-sensitive nephrotic syndrome (SSNS) in children over the previous 5 years. Six genes, which have roles in Rho-like small GTPase activity, have been identified as causes of partially steroid-treatable nephrotic syndrome, and the strongest susceptible gene of SSNS has been located in the HLA DR/DQ locus. For treatment, prolongation of prednisolone therapy beyond 2 to 3 months in the initial episode of SSNS does not reduce the risk of relapse. Rituximab has been established as a treatment for complicated frequently relapsing nephrotic syndrome or steroid-dependent nephrotic syndrome. Rituximab could also be the first-line treatment for early-stage-uncomplicated FRNS/SDNS, although further studies are required to confirm its efficacy and safety. The understanding of pathogenesis and development of new treatments of steroid-sensitive nephrotic syndrome in children made a significant advance over the previous 5 years.