Proximity labelling identifies pro-migratory endocytic recycling cargo and machinery of the Rab4 and Rab11 families.

Endocytic recycling controls the return of internalised cargoes to the plasma membrane to coordinate their positioning, availability and downstream signalling. The Rab4 and Rab11 small GTPase families regulate distinct recycling routes, broadly classified as fast recycling from early endosomes (Rab4) and slow recycling from perinuclear recycling endosomes (Rab11), and both routes handle a broad range of overlapping cargoes to regulate cell behaviour. We adopted a proximity labelling approach, BioID, to identify and compare the protein complexes recruited by Rab4a, Rab11a and Rab25 (a Rab11 family member implicated in cancer aggressiveness), revealing statistically robust protein-protein interaction networks of both new and well-characterised cargoes and trafficking machinery in migratory cancer cells. Gene ontological analysis of these interconnected networks revealed that these endocytic recycling pathways are intrinsically connected to cell motility and cell adhesion. Using a knock-sideways relocalisation approach, we were further able to confirm novel links between Rab11, Rab25 and the ESCPE-1 and retromer multiprotein sorting complexes, and identify new endocytic recycling machinery associated with Rab4, Rab11 and Rab25 that regulates cancer cell migration in the 3D matrix.

Rab25 is involved in hypospadias via the ß1 integrin/EGFR pathway.

BACKGROUND: Hypospadias is a common congenital abnormality of the penile. Abnormal regulation of critical genes involved in urethral development leads to hypospadias. We used the Rab25-/- mice and foreskin fibroblasts transfected with lentivirus in vitro and in vivo to investigate the role of Rab25 in hypospadias. METHODS: The expression levels of various molecules in tissue samples and foreskin fibroblasts were confirmed using molecular biology methods (western blotting, PCR, immunohistochemistry, etc.). A scanning electron microscope (SEM) was used to visualize the external morphology of genital tubercles (GTs) of gestation day (GD) 18.5 male wild-type (WT) and Rab25-/- mice. RESULTS: An expanded distal cleft and V-shaped urethral opening were observed in GD 18.5 Rab25-/- mice. We demonstrated that Rab25 mediated hypospadias through the ß1 integrin/EGFR pathway. In addition, silencing Rab25 inhibited cell proliferation and migration and promoted apoptosis in the foreskin fibroblasts; Ki-67- and TUNEL-positive cells were mainly concentrated near the urethral seam. CONCLUSION: These findings suggest that Rab25 plays an essential role in hypospadias by activation of ß1 integrin/EGFR pathway, and Rab25 is a critical mediator of urethral seam formation in GD18.5 male fetal mice.

Expression Patterns of Grainyhead-Like 2 and Ovo-Like 2 in Mouse Mammary Gland Development During Pregnancy, Lactation, and Weaning.

Grainyhead-like 2 (Grhl2) is a transcription factor that regulates cell adhesion genes in mammary ductal development and serves as a repressor of the epithelial-mesenchymal transition. Conversely, Ovo-like2 (Ovol2) is a target gene of Grhl2 but functions as a substitute in Grhl2-deficient mice, facilitating successful epithelial barrier formation and lumen expansion in kidney-collecting ductal epithelial cells. Our objective was to examine the expression patterns of Grhl2, Ovol2, and their associated genes during the intricate phases of mouse mammary gland development. The mRNA expression of Grhl2 and Ovol2 increased after pregnancy. We observed Grhl2 protein presence in the epithelial cell's region, coinciding with acini formation, and its signal significantly correlated with E-cadherin (Cdh1) expression. However, Ovol2 was present in the epithelial region without a correlation with Cdh1. Similarly, Zeb1, a mesenchymal transcription factor, showed Cdh1-independent expression. Subsequently, we explored the interaction between Rab25, a small G protein, and Grhl2/Ovol2. The expressions of Grhl2 and Ovol2 exhibited a strong correlation with Rab25 and claudin-4, a tight junction protein. These findings suggest that Grhl2 and Ovol2 may collaborate to regulate genes associated with cell adhesion and are crucial for maintaining epithelial integrity during the different phases of mammary gland development.

RAB25 coordinates filaggrin-containing keratohyalin granule maturation and affects atopic dermatitis severity.

BACKGROUND: Keratohyalin granules (KHGs) supply the critical epidermal protein constituents such as filaggrin for maintaining skin barrier function during epidermal differentiation; however, their regulating mechanism remains largely unelucidated. METHODS: To investigate the role of Ras-related protein Rab-25 (RAB25) expression in skin disease, we utilized skin specimens of patients with moderate-to-severe atopic dermatitis (AD) and healthy controls. To investigate the susceptibility of Rab25 knockout mice to AD, we established an oxazolone-induced AD model. RESULTS: We investigated the role of RAB25 in KHG maturation and AD. RAB25-deficient mice showed a disrupted stratum corneum along with skin barrier dysfunction, decreased KHG production, and abnormal KHG processing. Consistently, in the human keratinocyte cell line HaCaT, RAB25 co-expressed with filaggrin-containing KHG and RAB25 silencing impaired KHG formation, which was attributable to abnormal actin dynamics. Most importantly, RAB25 expression was severely downregulated in the skin lesions of patients with AD, which was strongly correlated with disease severity scores. CONCLUSIONS: RAB25 coordinates KHG homeostasis by regulating actin dynamics and is critical for epidermal differentiation and the pathophysiology of AD.

Notch regulates vascular collagen IV basement membrane through modulation of lysyl hydroxylase 3 trafficking.

Collagen type IV (Col IV) is a basement membrane protein associated with early blood vessel morphogenesis and is essential for blood vessel stability. Defects in vascular Col IV deposition are the basis of heritable disorders, such as small vessel disease, marked by cerebral hemorrhage and drastically shorten lifespan. To date, little is known about how endothelial cells regulate the intracellular transport and selective secretion of Col IV in response to angiogenic cues, leaving a void in our understanding of this critical process. Our aim was to identify trafficking pathways that regulate Col IV deposition during angiogenic blood vessel development. We have identified the GTPase Rab10 as a major regulator of Col IV vesicular trafficking during vascular development using both in vitro imaging and biochemistry as well as in vivo models. Knockdown of Rab10 reduced de novo Col IV secretion in vivo and in vitro. Mechanistically, we determined that Rab10 is an indirect mediator of Col IV secretion, partnering with atypical Rab25 to deliver the enzyme lysyl hydroxylase 3 (LH3) to Col IV-containing vesicles staged for secretion. Loss of Rab10 or Rab25 results in depletion of LH3 from Col IV-containing vesicles and rapid lysosomal degradation of Col IV. Furthermore, we demonstrate that Rab10 is Notch responsive, indicating a novel connection between permissive Notch-based vessel maturation programs and vesicle trafficking. Our results illustrate both a new trafficking-based component in the regulated secretion of Col IV and how this vesicle trafficking program interfaces with Notch signaling to fine-tune basement membrane secretion during blood vessel development.

RAB25 confers resistance to chemotherapy by altering mitochondrial apoptosis signaling in ovarian cancer cells.

Ovarian cancer remains one of the most frequent causes of cancer-related death in women. Many patients with ovarian cancer suffer from de novo or acquired resistance to chemotherapy. Here, we report that RAB25 suppresses chemotherapy-induced mitochondrial apoptosis signaling in ovarian cancer cell lines and primary ovarian cancer cells. RAB25 blocks chemotherapy-induced apoptosis upstream of mitochondrial outer membrane permeabilization by either increasing antiapoptotic BCL-2 proteins or decreasing proapoptotic BCL-2 proteins. In particular, BAX expression negatively correlates with RAB25 expression in ovarian cancer cells. BH3 profiling assays corroborated that RAB25 decreases mitochondrial cell death priming. Suppressing RAB25 by means of RNAi or RFP14 inhibitory hydrocarbon-stapled peptide sensitizes ovarian cancer cells to chemotherapy as well as RAB25-mediated proliferation, invasion and migration. Our data suggest that RAB25 is a potential therapeutic target for ovarian cancer.

Loss of Rab25 promotes the development of skin squamous cell carcinoma through the dysregulation of integrin trafficking.

Rab25 can function as both a tumor suppressor and a tumor promoter across different tissues. This study sought to clarify the role of Rab25 as a tumor suppressor in skin squamous cell carcinoma (SCC). Rab25 loss was closely associated with neoplastic transition in both humans and mice. Rab25 loss was well correlated with increased cell proliferation and poor differentiation in human SCC. While Rab25 knockout (KO) in mice did not induce spontaneous tumor formation, it did significantly accelerate tumor generation and promote malignant transformation in a mouse two-stage skin carcinogenesis model. Xenografting of a Rab25-deficient human keratinocyte cell line, HaCaT, also elicited neoplastic transformation. Notably, Rab25 deficiency led to dysregulation of integrins ß1, ß4, and α6, which matched well with increased epidermal proliferation and impaired desmosome-tight junction formation. Rab25 deficiency induced impairment of integrin recycling, leading to the improper expression of integrins. In line with this, significant attenuation of integrin ß1, ß4, and α6 expression was identified in human SCCs where Rab25 was deficient. Collectively, these results suggest that loss of Rab25 promotes the development and neoplastic transition of SCC through dysregulation of integrin trafficking. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Epidermal growth factor-mediated Rab25 pathway regulates integrin ß1 trafficking in colon cancer.

BACKGROUND: Integrins play a critical role in carcinogenesis. Integrin ß1 localization is regulated by the guanosine-5'-triphosphate hydrolase Rab25 and integrin ß1 levels are elevated in the serum of colon cancer patients; thus, the present study examined the effects of epidermal growth factor (EGF) and Rab25 on integrin ß1 localization in colon cancer cells. METHODS: HCT116 human colon cancer cells were treated with increasing concentrations of EGF, and cell proliferation and protein expression were monitored by MTT and western blot analyses, respectively. Cell fractionation was performed to determine integrin ß1 localization in the membrane and cytosol. Integrin ß1 extracellular shedding was monitored by enzyme-linked immunosorbent assays (ELISAs) with culture supernatants from stimulated cells. HCT116 cells were transfected with Rab25-specific siRNA to determine the significance of Rab25 in integrin ß1 trafficking in the presence of EGF. RESULTS: Total integrin ß1 expression increased in response to EGF and subsequently decreased at 24 h post-stimulation. A similar decrease was observed in purified membrane fractions, whereas no changes were observed in cytosolic levels. ELISAs using media from stimulated cell cultures demonstrated increased integrin ß1 levels corresponding to the decrease observed in membrane fractions, suggesting that EGF induces integrin receptor shedding. EGF stimulation in Rab25-knockdown cells resulted in integrin ß1 accumulation in the membrane, suggesting that Rab25 promotes integrin endocytosis. CONCLUSIONS: Integrin ß1 is shed from colon cancer cells in response to EGF stimulation in a Rab25-dependent manner. These results further the present understanding of the role of integrin ß1 in colon cancer progression.

RIN1 promotes renal cell carcinoma malignancy by activating EGFR signaling through Rab25.

We previously identified the important role of RIN1 expression in the prognosis of clear cell renal cell carcinoma (ccRCC). The role of RIN1 in ccRCC malignancy and underlying molecular mechanisms remain unclear. Here we report that ccRCC cells and tissues expressed more RIN1 than normal controls. Gain-of-function and loss-of-function studies demonstrated that RIN1 enhanced ccRCC cell growth, migration and invasion abilities in vitro and promoted tumor growth and metastasis in vivo. Mechanistic studies revealed that RIN1 has an activating effect on EGFR signaling in ccRCC. In addition, we unveil Rab25, a critical GTPase in ccRCC malignancy, as a functional RIN1 interacting partner. Knockdown of Rab25 eliminated the augmentation of carcinoma cell proliferation, migration and invasion by ectopic RIN1. We also confirmed that RIN1 and Rab25 expression correlates with the overall-survival of ccRCC patients from TCGA. These findings suggest that RIN1 plays an important oncogenic role in ccRCC malignancy by activation of EGFR signaling through interacting with Rab25, and RIN1 could be employed as an effective therapeutic target for ccRCC.

Overexpression of Rab25 promotes hepatocellular carcinoma cell proliferation and invasion.

Rab25 was reported to be associated with several human cancers and malignant biological behavior of cancer cells. The goal of the present study was to determine its expression pattern and biological function in human hepatocellular carcinoma (HCC). We examined Rab25 protein in 92 cases of HCC tissues and 3 HCC cell lines. The results showed that Rab25 was upregulated in HCC tissues and cells compared with normal liver tissues and cell line. Rab25 overexpression correlated with advanced tumor stage and nodal metastasis. Rab25 small interfering RNA (siRNA) was employed in Bel7402 and SK-Hep-1 cell lines. Cell Counting Kit-8 (CCK-8) assay and colony formation assay showed that Rab25 depletion blocked cell growth rate and inhibited colony formation ability. Transwell assay showed that Rab25 depletion negatively regulated the invading ability of HCC cells. To explore the possible mechanisms, we checked several signaling pathways and found that Rab25 depletion downregulated AKT phosphorylation. In addition, luciferase reporter assay showed that Rab25 depletion inhibited the Wnt signaling pathway and its target genes such as cyclin D1, c-myc, and MMP7. In conclusion, Rab25 is overexpressed in human HCC and contributes to cancer cell proliferation and invasion possibly through regulation of the Wnt signaling pathway.

CCN3 overexpression inhibits growth of callosal projections via upregulation of RAB25.

The cysteine-rich 61/connective tissue growth factor 3 (CCN3) is a member of the CCN family of secreted multifunctional proteins involved in a variety of cellular processes including migration, adhesion, and differentiation. Previous studies have shown that CCN3 is expressed in the developing rat central nervous system, and enhanced CCN3 expression is highly correlated with tumorigenesis. However, the expression pattern and influence of abnormal CCN3 expression during mouse cortical development remains to be elucidated. Here, we show that CCN3 expression in mice is first detectable at embryonic day 15 and increases until postnatal day 21. We overexpressed CCN3 in mouse cortical neurons using uni- and bilateral electroporation. Our in vivo overexpression experiments showed that elevated CCN3 expression inhibited the axonal outgrowth of callosal projection neurons. Moreover, we identified the small GTPase RAB25 as a downstream effector molecule of CCN3 using transcriptomic analysis with CCN3 overexpressed in cortical tissue. In vivo ectopic expression of RAB25 or the dominant-negative RAB25-T26N also revealed that the GTPase activity of RAB25 is involved in the CCN3-mediated regulation of neuronal outgrowth. Taken together, our results suggest that tight regulation of CCN3 expression is necessary for normal cortical neuronal connectivity during development, and RAB25 negatively regulates neuronal differentiation as a downstream effector of CCN3.

Rab25 upregulation correlates with the proliferation, migration, and invasion of renal cell carcinoma.

Renal cell carcinoma (RCC) is a common urological cancer with a poor prognosis. A recent cohort study revealed that the median survival of RCC patients was only 1.5 years and that <10% of the patients in the study survived up to 5 years. In tumor development, Rab GTPase are known to play potential roles such as regulation of cell proliferation, migration, invasion, communication, and drug resistance in multiple tumors. However, the correlation between Rabs expression and the occurrence, development, and metastasis of RCC remains unclear. In this study, we analyzed the transcriptional levels of 52 Rab GTPases in RCC patients. Our results showed that high levels of Rab25 expression were significantly correlated with RCC invasion classification (P < 0.01), lymph-node metastasis (P < 0.001), and pathological stage (P < 0.01). Conversely, in 786-O and A-498 cells, knocking down Rab25 protein expression inhibited cell proliferation, migration, and invasion. Our results also demonstrated that Rab25 is a target gene of let-7d, and further suggested that Rab25 upregulation in RCC is due to diminished expression of let-7d. These findings indicate that Rab25 might be a novel candidate molecule involved in RCC development, thus identifying a potential biological therapeutic target for RCC.

Structural and functional analysis of FIP2 binding to the endosome-localised Rab25 GTPase.

Rab small GTPases are the master regulators of intracellular trafficking in eukaryotes. They mediate spatial and temporal recruitment of effector proteins to distinct cellular compartments through GTP-induced changes in their conformation. Despite numerous structural studies, the molecular basis for Rab/effector specificity and subsequent biological activity remains poorly understood. Rab25, also known as Rab11c, which is epithelial-specific, has been heavily implicated in ovarian cancer development and independently appears to act as a tumour suppressor in the context of a distinct subset of carcinomas. Here, we show that Rab25 associates with FIP2 and can recruit this effector protein to endosomal membranes. We report the crystal structure of Rab25 in complex with the C-terminal region of FIP2, which consists of a central dimeric FIP2 coiled-coil that mediates a heterotetrameric Rab25-(FIP2)2-Rab25 complex. Thermodynamic analyses show that, despite a relatively conserved interface, FIP2 binds to Rab25 with an approximate 3-fold weaker affinity than to Rab11a. Reduced affinity is mainly associated with lower enthalpic gains for Rab25:FIP2 complex formation, and can be attributed to subtle differences in the conformations of switch 1 and switch 2. These cellular, structural and thermodynamic studies provide insight into the Rab11/Rab25 subfamily of small GTPases that regulate endosomal trafficking pathways in eukaryotes.

GRAB is a binding partner for the Rab11a and Rab11b GTPases.

Co-ordination of Rab GTPase function has emerged as a crucial mechanism in the control of intracellular trafficking processes in eukaryotic cells. Here, we show that GRAB/Rab3IL1 [guanine nucleotide exchange factor for Rab3A; RAB3A interacting protein (rabin3)-like 1], a protein that has previously be shown to act as a GEF (guanine nucleotide exchange factor) for Rab3a, Rab8a and Rab8b, is also a binding partner for Rab11a and Rab11b, but not the closely related Rab25 GTPase. We demonstrate that exogenous expression of Rab11a and Rab11b shift GRAB's distribution from the cytoplasm onto membranes. We find that the Rab11a/Rab11b-binding region of GRAB lies within its carboxy-terminus, a region distinct from its GEF domain and Rab3a-binding region. Finally, we describe a GRAB deletion mutant (GRABΔ223-228) that is deficient in Rab11-binding ability. These data identify GRAB as a dual Rab-binding protein that could potentially link Rab3 and Rab11 and/or Rab8 and Rab11-mediated intracellular trafficking processes.

Expression of Rab25 correlates with the invasion and metastasis of gastric cancer.

The objective of this study was to determine the expression of the important vesicle trafficking-regulating factor Rab25 in human gastric cancer tissues, to analyze the correlation between Rab25 protein expression with gastric cancer occurrence and development, and to discuss the correlation of Rab25 protein expression with gastric cancer cell metastasis. The overall aim was to provide experimental evidence that can be used to design future biological treatments of human gastric cancer. Human gastric cancer tissue and the adjacent normal gastric tissue were surgically removed, and immunohistochemistry and Western blotting were used to detect Rab25 protein expression. The correlation between Rab25 protein expression with the development and pathological characteristics of gastric cancer was analyzed. Using RNAi, Rab25 expression was reduced in the gastric cancer cell line MGC80-3, and the changes in MGC80-3 cell invasiveness were then monitored. Immunohistochemistry showed that the Rab25 protein expression rates were 78.21% and 23.08% in gastric carcinoma and the adjacent normal gastric tissue, respectively. Immunohistochemistry and Western blot results showed that Rab25 protein expression in gastric cancer was significantly higher than in adjacent normal gastric tissues (P<0.01). Less differentiated gastric cancer cells had higher expression of Rab25 protein (P<0.01). Gastric carcinomas from patients with a late pathological stage (III-IV) had significantly higher Rab25 protein expression than early stage (I-II) patients (P<0.01). Gastric carcinomas from patients with lymph node metastasis had significantly higher Rab25 protein expression than lymph node metastasis-free patients (P<0.01). Gastric carcinomas from patients with distant metastases had significantly higher Rab25 protein expression than the distant metastasis-negative patients (P<0.01). Rab25 protein expression in gastric cancer was not affected by the patients(,) sex, age, or tumor size (P>0.05). MGC80-3 cells transfected with Rab25 siRNA had significantly lower Rab25 protein expression (P<0.01) and a significantly lower number of cells that passed through a Transwell chamber compared with non-transfected controls and the transfected control group (P<0.01). Rab25 protein expression is associated with the development of gastric cancer. siRNA knockdown of Rab25 protein expression in MGC80-3 gastric cancer cells reduced MGC80-3 cell invasiveness and provided experimental evidence for potential future biological treatment strategies of human gastric cancer.

A small Rho GTPase RAB25 with a potential role in chemotherapy resistance in pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDA) is one of the major human health challenges with minimal therapeutic benefits due to its late detection, and de novo - and acquired chemotherapy resistance. OBJECTIVE: In this work we unravel the potential pro-survival role of RAB25 in pancreatic cancer chemotherapy resistance and aim to identify if RAB25 is a prognostic marker of patients' survival in PDA. METHODS: We used RNA sequencing, shRNA mediated gene knockdown, BioGRID open repository of CRISPR screens (ORCS), GEPIA, kmplot.com, and cBioPortal.org databases to identify the role of RAB25 in PDA cell proliferation, chemotherapy response, expression in tumour versus normal tissues, and overall patients' survival. RESULTS: RNA sequencing show Rab25 to be one of the top upregulated genes in gemcitabine resistance mouse PDA cells. Knockdown of Rab25 in these cells enhanced gemcitabine toxicity. In addition, re-analysis of previously published CRISPR/Cas9 data confirm RAB25 to be responsible for chemotherapy resistance in KRASG12D mutant human pancreatic cancer cell line. Finally, we used publicly available TCGA datasets and identify the upregulation of RAB25 in tumour tissues compared to the adjacent normal tissue, co-occurrence of KRASG12 mutations with RAB25 amplifications, and poor patients' survival in cohorts with higher mRNA expression of RAB25. CONCLUSION: RAB25 expression is a prognostic marker for patient's survival and gemcitabine resistance in PDA.

Altered endosomal-lysosomal biogenesis in melanoma.

Cutaneous melanoma is the deadliest form of skin neoplasm and its high mortality rates could be averted by early accurate detection. While the detection of melanoma is currently reliant upon melanin visualisation, research into melanosome biogenesis, as a key driver of pathogenesis, has not yielded technology that can reliably distinguish between atypical benign, amelanotic and melanotic lesions. The endosomal-lysosomal system has important regulatory roles in cancer cell biology, including a specific functional role in melanosome biogenesis. Herein, the involvement of the endosomal-lysosomal system in melanoma was examined by pooled secondary analysis of existing gene expression datasets. A set of differentially expressed endosomal-lysosomal genes was identified in melanoma, which were interconnected by biological function. To illustrate the protein expression of the dysregulated genes, immunohistochemistry was performed on samples from patients with cutaneous melanoma to reveal candidate markers. This study demonstrated the dysregulation of Syntenin-1, Sortilin and Rab25 may provide a differentiating feature between cutaneous melanoma and squamous cell carcinoma, while IGF2R may indicate malignant propensity in these skin cancers.

Expression of Rab25 is down-regulated in the foreskin of children with hypospadias.

BACKGROUND: Hypospadias, a congenital malformation of the penis, is one of the newborns' most common developmental defects. The incidence of hypospadias is increasing yearly, and its pathogenesis is closely related to genetic susceptibility and environmental exposure to endocrine disruptors. Exploring the hypospadias' key molecular regulatory mechanism is crucial to reducing its incidence. OBJECTIVE: To examine the differential expression of Rab25 in hypospadias and normal penile tissue and to identify whether it is a candidate gene for exploring the mechanism of hypospadias. STUDY DESIGN: This study included 18 children aged 1-6 years undergoing hypospadias repair surgery at the Children's Hospital of Chongqing Medical University, and foreskin samples were collected. Children diagnosed with cryptorchidism, intersex status, or endocrine abnormalities were excluded from this study. Another 18 children aged 3-8 years with phimosis were included in the control group. The specimens were used for immunohistochemistry, western blotting, immunofluorescence, and polymerase chain reaction to assess the expression of Rab25. RESULTS: Rab25 protein expression was lower in the hypospadias group than in the control group [ (2.101 ± 0.1845), (0.7506 ± 0.1779), p = 0.0008 < 0.05). The hypospadias group showed decreased expression of Rab25 protein in the epithelial cell layer. Rab25 mRNA levels were downregulated in the foreskin of children with hypospadias compared with controls [(1.697 ± 0.2005), (0.7687 ± 0.2130), p = 0.0053 < 0.05)]. DISCUSSION: Rab25 mRNA and protein expressions in the hypospadias group were significantly downregulated compared with the control group. This was consistent with the results of single-cell sequencing of fetal mice reproductive nodules at 15.5 days of gestation (Zhang Z, Liu Z, Zhang Q, et al., unpublished observations). Our study represents the first report of abnormal Rab25 expression in the foreskin tissue of patients with hypospadias. More detailed research on the relationship between Rab25 and urethral development could be conducted to reveal the molecular mechanism of hypospadias. CONCLUSION: The expression of Rab25 in foreskin tissue was lower in the hypospadias group than in the control group. Rab25 is involved in the formation of the urethral seam and the occurrence of hypospadias. The potential mechanism by which Rab25 affects the canalization of the urethral plate needs to be further investigated.

Methylation-related genes involved in renal carcinoma progression.

Renal carcinomas are a group of malignant tumors often originating in the cells lining the small tubes in the kidney responsible for filtering waste from the blood and urine production. Kidney tumors arise from the uncontrolled growth of cells in the kidneys and are responsible for a large share of global cancer-related morbidity and mortality. Understanding the molecular mechanisms driving renal carcinoma progression results crucial for the development of targeted therapies leading to an improvement of patient outcomes. Epigenetic mechanisms such as DNA methylation are known factors underlying the development of several cancer types. There is solid experimental evidence of relevant biological functions modulated by methylation-related genes, associated with the progression of different carcinomas. Those mechanisms can often be associated to different epigenetic marks, such as DNA methylation sites or chromatin conformation patterns. Currently, there is no definitive method to establish clear relations between genetic and epigenetic factors that influence the progression of cancer. Here, we developed a data-driven method to find methylation-related genes, so we could find relevant bonds between gene co-expression and methylation-wide-genome regulation patterns able to drive biological processes during the progression of clear cell renal carcinoma (ccRC). With this approach, we found out genes such as ITK oncogene that appear hypomethylated during all four stages of ccRC progression and are strongly involved in immune response functions. Also, we found out relevant tumor suppressor genes such as RAB25 hypermethylated, thus potentially avoiding repressed functions in the AKT signaling pathway during the evolution of ccRC. Our results have relevant implications to further understand some epigenetic-genetic-affected roles underlying the progression of renal cancer.

On demand expression control of endogenous genes with DExCon, DExogron and LUXon reveals differential dynamics of Rab11 family members.

CRISPR technology has made generation of gene knock-outs widely achievable in cells. However, once inactivated, their re-activation remains difficult, especially in diploid cells. Here, we present DExCon (Doxycycline-mediated endogenous gene Expression Control), DExogron (DExCon combined with auxin-mediated targeted protein degradation), and LUXon (light responsive DExCon) approaches which combine one-step CRISPR-Cas9-mediated targeted knockin of fluorescent proteins with an advanced Tet-inducible TRE3GS promoter. These approaches combine blockade of active gene expression with the ability to re-activate expression on demand, including activation of silenced genes. Systematic control can be exerted using doxycycline or spatiotemporally by light, and we demonstrate functional knock-out/rescue in the closely related Rab11 family of vesicle trafficking regulators. Fluorescent protein knock-in results in bright signals compatible with low-light live microscopy from monoallelic modification, the potential to simultaneously image different alleles of the same gene, and bypasses the need to work with clones. Protein levels are easily tunable to correspond with endogenous expression through cell sorting (DExCon), timing of light illumination (LUXon), or by exposing cells to different levels of auxin (DExogron). Furthermore, our approach allowed us to quantify previously unforeseen differences in vesicle dynamics, transferrin receptor recycling, expression kinetics, and protein stability among highly similar endogenous Rab11 family members and their colocalization in triple knock-in ovarian cancer cell lines.