IL-12 improves the anti-HCC efficacy of dendritic cells loaded with exosomes from overexpressing Rab27a tumor cells.

Determining the appropriate source of antigens for optimal antigen presentation to T cells is a major challenge in designing dendritic cell (DC) -based therapeutic strategies against hepatocellular carcinoma (HCC). Tumor-derived exosomes (Tex) express a wide range of tumor antigens, making them a promising source of antigens for DC vaccines. As reported, the exosomes secreted by tumor cells can inhibit the antitumor function of immune cells. In this study, we transfected hepatocellular carcinoma cells with Rab27a to enhance the yield of exosomes, which were characterized using transmission electron microscopy and Western blot analysis. We found that Tex secreted by overexpressing Rab27a Hepatocellular carcinoma cell lines pulsed DC is beneficial for the differentiation and maturation of DCs but inhibits the secretion of the IL-12 cytokine. Consequently, we developed a complementary immunotherapy approach by using Tex as an antigen loaded onto DCs, in combination with the cytokine IL-12 to induce antigen-specific cytotoxic T lymphocytes （CTLs）. The results indicated that the combination of DC-Tex and IL-12 was more effective in stimulating T lymphocyte proliferation, releasing IFN-γ， and enhancing cytotoxicity compared to using exosomes or IL-12 alone. Additionally, the inclusion of IL-12 also compensated for the reduced IL-2 secretion by DCs caused by Tex. Moreover, in a BALB/c nude mice model of hepatocellular carcinoma, CTLs induced by DC-Tex combined with IL-12 maximized the tumor-specific T-cell immune effect and suppressed tumor growth. Thus, Tex provides a novel and promising source of antigens, with cytokines compensating for the shortcomings of Tex as a tumor antigen. This work helps to clarify the role of exosomes in tumor immunotherapy and may offer a safe and effective prospective strategy for the clinical application of exosome-based cellular immunotherapy.

Campothecin suppresses cell proliferation and migration in head and neck squamous cell carcinoma by blocking RAB27A-mediated phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) pathway.

Camptothecin (CPT), a naturally occurring alkaloid derived from the Camptotheca acuminate plant, exerts anti-tumor properties. However, its specific impact on head and neck squamous cell carcinoma (HNSCC) remains uncertain. The study was to explore the action and mechanism of CPT on HNSCC cells. First, two HNSCC cell lines (FaDu and TU686) and a normal immortalized keratinocyte (HEK001) cell line, were exposed to a spectrum of CPT concentrations (ranging from 10 to 50 µM) for durations of 24 h and 48 h. Cell viability, proliferation, migration, and invasion were assessed by CCK-8 assay, EdU incorporation assay, wound healing assay and transwell assay. Subsequently, si-RAB27A or negative control (NC) was introduced into FaDu and TU686 cells through transfection, and the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway was manipulated with L740Y-P, an activator of this pathway. The expression of proliferating cell nuclear antigen (PCNA), E-cadherin, PI3K/AKT signaling factors and RAB27A were determined by Western blot analysis. RAB27A was detected by immunofluorescence assay. It was found that CPT significantly hindered the viability, proliferation (p<0.01), migration (p<0.001), and invasion (p<0.001) of FaDu and TU686 cells. At the molecular level, administration of CPT caused a decline in the expression of PCNA, P-PI3K, P-AKT, and RAB27A, alongside an elevation in E-cadherin levels within HNSCC cells (p<0.05, p<0.01 and p<0.001). Reducing RAB27A expression enhanced the suppressive impacts of CPT on HNSCC cell viability (p<0.05 and p<0.01), migration (p<0.001) and invasion (p<0.01), these effects that were reversed upon treatment with L740Y-P in HNSCC cells (p<0.001). In summary, our study highlights the efficacy of CPT in HNSCC, demonstrating its influence on cell processes via the RAB27A-mediated PI3K/AKT pathway.

Effects of a Global Rab27a Null Mutation on Murine PVAT and Cardiovascular Function.

BACKGROUND: RAB27A is a member of the RAS oncogene superfamily of GTPases and regulates cell secretory function. It, is expressed within blood vessels and perivascular adipose tissue. We hypothesized that loss of RAB27A would alter cardiovascular function. METHODS: Body weight of Rab27aash mice was measured from 2 to 18 months of age, along with glucose resorption at 6 and 12 months of age and glucose sensitivity at 18 months of age. Body weight and cellular and molecular features of perivascular adipose tissue and aortic tissue were examined in a novel C57BL/6J Rab27a null strain. Analyses included morphometric quantification and proteomic analyses. Wire myography measured vasoreactivity, and echocardiography measured cardiac function. Comparisons across ages and genotypes were evaluated via 2-way ANOVA with multiple comparison testing. Significance for myography was determined via 4-parameter nonlinear regression testing. RESULTS: Genome-wide association data linked rare human RAB27A variants with body mass index and glucose handling. Changes in glucose tolerance were observed in Rab27aash male mice at 18 months of age. In WT (wild-type) and Rab27a null male mice, body weight, adipocyte lipid area, and aortic area increased with age. In female mice, only body weight increased with age, independent of RAB27A presence. Protein signatures from male Rab27a null mice suggested greater associations with cardiovascular and metabolic phenotypes compared with female tissues. Wire myography results showed Rab27a null males exhibited increased vasoconstriction and reduced vasodilation at 8 weeks of age. Rab27a null females exhibited increased vasoconstriction and vasodilation at 20 weeks of age. Consistent with these vascular changes, male Rab27a null mice experienced age-related cardiomyopathy, with severe differences observed by 21 weeks of age. CONCLUSIONS: Global RAB27A loss impacted perivascular adipose tissue and thoracic aorta proteomic signatures, altered vasocontractile responses, and decreased left ventricular ejection fraction in mice.

The role of Rab27 in tick extracellular vesicle biogenesis and pathogen infection.

BACKGROUND: The blacklegged tick, Ixodes scapularis, transmits most vector-borne diseases in the US. It vectors seven pathogens of public health relevance, including the emerging human pathogen Anaplasma phagocytophilum. Nevertheless, it remains critically understudied compared to other arthropod vectors. Ixodes scapularis releases a variety of molecules that assist in the modulation of host responses. Recently, it was found that extracellular vesicles (EVs) carry several of these molecules and may impact microbial transmission to the mammalian host. EV biogenesis has been studied in mammalian systems and is relatively well understood, but the molecular players important for the formation and secretion of EVs in arthropods of public health relevance remain elusive. RabGTPases are among the major molecular players in mammalian EV biogenesis. They influence membrane identity and vesicle budding, uncoating, and motility. METHODS: Using BLAST, an in silico pathway for EV biogenesis in ticks was re-constructed. We identified Rab27 for further study. EVs were collected from ISE6 tick cells after knocking down rab27 to examine its role in tick EV biogenesis. Ixodes scapularis nymphs were injected with small interfering RNAs to knock down rab27 and then fed on naïve and A. phagocytophilum-infected mice to explore the importance of rab27 in tick feeding and bacterial acquisition. RESULTS: Our BLAST analysis identified several of the proteins involved in EV biogenesis in ticks, including Rab27. We show that silencing rab27 in I. scapularis impacts tick fitness. Additionally, ticks acquire less A. phagocytophilum after rab27 silencing. Experiments in the tick ISE6 cell line show that silencing of rab27 causes a distinct range profile of tick EVs, indicating that Rab27 is needed to regulate EV biogenesis. CONCLUSIONS: Rab27 is needed for successful tick feeding and may be important for acquiring A. phagocytophilum during a blood meal. Additionally, silencing rab27 in tick cells results in a shift of extracellular vesicle size. Overall, we have observed that Rab27 plays a key role in tick EV biogenesis and the tripartite interactions among the vector, the mammalian host, and a microbe it encounters.

GDP-bound Rab27a regulates clathrin disassembly through HSPA8 after insulin secretion.

Clathrin-dependent endocytosis is a key process for secretory cells, in which molecules on the plasma membrane are both degraded and recycled in a stimulus-dependent manner. There are many reports showing that disruption of endocytosis is involved in the onset of various diseases. Recently, it has been reported that such disruption in pancreatic ß-cells causes impaired insulin secretion and might be associated with the pathology of diabetes mellitus. Compared with exocytosis, there are few reports on the molecular mechanism of endocytosis in pancreatic ß-cells. We previously reported that GDP-bound Rab27a regulates endocytosis through its GDP-dependent effectors after insulin secretion. In this study, we identified heat shock protein family A member 8 (HSPA8) as a novel interacting protein for GDP-bound Rab27a. HSPA8 directly bound GDP-bound Rab27a via the ß2 region of its substrate binding domain (SBD). The ß2 fragment was capable of inhibiting the interaction between HSPA8 and GDP-bound Rab27a, and suppressed glucose-induced clathrin-dependent endocytosis in pancreatic ß-cells. The region also affected clathrin dynamics on purified clathrin-coated vesicles (CCVs). These results suggest that the interaction between GDP-bound Rab27a and HSPA8 regulates clathrin disassembly from CCVs and subsequent vesicle transport. The regulatory stages in endocytosis by HSPA8 differ from those for other GDP-bound Rab27a effectors. This study shows that GDP-bound Rab27a dominantly regulates each stage in glucose-induced endocytosis through its specific effectors in pancreatic ß-cells.

Overexpression of RAB27A in Oral Squamous Cell Carcinoma Promotes Tumor Migration and Invasion via Modulation of EGFR Membrane Stability.

Oral squamous cell carcinoma (OSCC) is the most prevalent subtype of head and neck tumors, highly prone to lymph node metastasis. This study aims to examine the expression pattern of Ras-related protein Rab-27A (RAB27A) and explore its potential implications in OSCC. The expression of RAB27A was assessed through immunohistochemical analysis utilizing tissue microarrays. In vitro experiments were conducted using RAB27A-knockdown cells to investigate its impact on OSCC tumor cells. Additionally, transcriptome sequencing was performed to elucidate potential underlying mechanisms. RAB27A was significantly overexpressed in OSCC, and particularly in metastatic lymph nodes. It was positively correlated with the clinical progression and poor survival prognosis. Silencing RAB27A notably decreased the proliferation, migration, and invasion abilities of OSCC cells in vitro. A Gene Ontology (GO) enrichment analysis indicated a strong association between RAB27A and the epidermal growth factor receptor (EGFR) signaling pathway. Further investigations revealed that RAB27A regulated the palmitoylation of EGFR via zinc finger DHHC-type containing 13 (ZDHHC13). These findings provide insights into OSCC progression and highlight RAB27A as a potential therapeutic target for combating this aggressive cancer.

SMC3 epigenetic silencing regulates Rab27a expression and drives pancreatic cancer progression.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is expected to soon surpass colorectal cancer as a leading cause of cancer mortality in both males and females in the US, only lagging behind lung cancer. The lethality of PDAC is driven by late diagnosis and inefficient therapies. The complex biology of PDAC involves various cellular components, including exosomes that carry molecular information between cells. Thus, recipient cells can be reprogrammed, impacting tumorigenesis. Rab27a is a GTPase responsible for the last step of exosomes biogenesis. Hence, dissecting the mechanisms that regulate the expression of Rab27a and that control exosomes biogenesis can provide fundamental insights into the molecular underpinnings regulating PDAC progression. METHODS: To assess the mechanism that regulates Rab27a expression in PDAC, we used PDAC cell lines. The biological significance of these findings was validated in PDAC genetically engineered mouse models (GEMMs) and human samples. RESULTS: In this work we demonstrate in human PDAC samples and GEMMs that Rab27a expression decreases throughout the development of the disease, and that Rab27a knockout promotes disease progression. What is more, we demonstrate that Rab27a expression is epigenetically regulated in PDAC. Treatment with demethylating agents increases Rab27a expression specifically in human PDAC cell lines. We found that SMC3, a component of the cohesin complex, regulates Rab27a expression in PDAC. SMC3 methylation is present in human PDAC specimens and treatment with demethylating agents increases SMC3 expression in human PDAC cell lines. Most importantly, high levels of SMC3 methylation are associated with a worse prognosis in PDAC. Mechanistically, we identified an enhancer region within the Rab27a gene that recruits SMC3, and modulates Rab27a expression. CONCLUSION: Overall, we dissected a mechanism that regulates Rab27a expression during PDAC progression and impacts disease prognosis.

Rab27a-mediated exosome secretion in anterior cingulate cortex contributes to colorectal visceral pain in adult mice with neonatal maternal deprivation.

Chronic visceral pain is a common symptom of irritable bowel syndrome (IBS). Exosomes are involved in the development of pain. Rab27a can mediate the release of exosomes. The purpose of this study is to investigate how Rab27a-mediated exosome secretion in the anterior cingulate cortex (ACC) regulates visceral hyperalgesia induced with neonatal maternal deprivation (NMD) in adult mice. The colorectal distension method was adopted to measure visceral pain. The BCA protein assay kit was applied to detect the exosome protein concentration. Western blotting, quantitative PCR, and immunofluorescence technique were adopted to detect the expression of Rab27a and the markers of exosomes. Exosomes extracted from ACC were more in NMD mice than in control (CON) mice. Injection of the exosome-specific inhibitor GW4869 in ACC attenuated colorectal visceral pain of NMD mice. Injection of NMD-derived exosomes produced colorectal visceral pain in CON mice. Rab27a was upregulated in ACC of NMD mice. Rab27a was highly expressed in ACC neurons of NMD mice, rather than astrocytes and microglia. Injection of Rab27a-siRNA reduced the release of exosomes and attenuated the colorectal visceral pain in NMD mice. This study suggested that overexpression of Rab27a increased exosome secretion in ACC neurons, thus contributing to visceral hyperalgesia in NMD mice.NEW & NOTEWORTHY This work demonstrated that the expression of Rab27a in the anterior cingulate cortex was upregulated, which mediated multivesicular bodies trafficking to the plasma membrane and led to the increased release of neuronal exosomes, thus contributing to colorectal visceral pain in neonatal maternal deprivation (NMD) mice. Blocking the release of exosomes or downregulation of Rab27a could alleviate colorectal visceral pain in NMD mice. These data may provide a promising strategy for the treatment of visceral pain in irritable bowel syndrome patients.

Immunodeficiency with susceptibility to lymphoma with complex genotype affecting energy metabolism (FBP1, ACAD9) and vesicle trafficking (RAB27A).

Introduction: Inborn errors of immunity (IEI) are characterized by a dysfunction of the immune system leading to increased susceptibility to infections, impaired immune regulation and cancer. We present a unique consanguineous family with a history of Hodgkin lymphoma, impaired EBV control and a late onset hemophagocytic lymphohistiocytosis (HLH). Methods and results: Overall, family members presented with variable impairment of NK cell and cytotoxic T cell degranulation and cytotoxicity. Exome sequencing identified homozygous variants in RAB27A, FBP1 (Fructose-1,6-bisphosphatase 1) and ACAD9 (Acyl-CoA dehydrogenase family member 9). Variants in RAB27A lead to Griscelli syndrome type 2, hypopigmentation and HLH predisposition. Discussion: Lymphoma is frequently seen in patients with hypomorphic mutations of genes predisposing to HLH. We hypothesize that the variants in FBP1 and ACAD9 might aggravate the clinical and immune phenotype, influence serial killing and lytic granule polarization by CD8 T cells. Understanding of the interplay between the multiple variants identified by whole exome sequencing (WES) is essential for correct interpretation of the immune phenotype and important for critical treatment decisions.

Molecular findings and clinical manifestations of 18 Iranian children with Griscelli syndrome type 2: Two novel homozygote mutations in RAB27A gene in a patient.

Griscelli syndrome type 2 (GS2) is an autosomal recessive immunodeficiency characterized by hair hypopigmentation, recurrent fever, hepatosplenomegaly and pancytopenia. This study aims to find new genetic changes and clinical features in 18 children with GS2 caused by the RAB27A gene defect. In all, 18 Iranian children with GS2 who presented with silver grey hair and frequent pyogenic infection were included in this study. After recording demographic and clinical data, PCR sequencing of the RAB27A gene was performed for all exons and exon-intron boundaries. Two patients in this study were subjected to whole-exome sequencing followed by Sanger sequencing. Light microscopy study of hair showed large irregular clumps of pigment with the absence of giant granules on the blood smear. Mutation analysis of the RAB27A gene identified two novel missense mutations as homozygous in a patient, one in exon 2, c.140G>C and another in exon 4, c.328G>T. In addition, for 17 other patients, 6 reported mutations were obtained including c.514_518delCAAGC, c.150_151delAGinsC, c.400_401delAA, c.340delA, c.428T>C and c.221A>G. The mutation c.514_518delCAAGC was the most frequent and found in 10 patients; this mutation may be considered a hotspot in Iran. Early diagnosis and treatment of RAB27A deficiency can contribute to better disease outcomes. In affected families, genetic results could be urgently needed to make a timely decision about haematopoietic stem cell transplantation and prenatal diagnosis.

Tumour extracellular vesicles and particles induce liver metabolic dysfunction.

Cancer alters the function of multiple organs beyond those targeted by metastasis1,2. Here we show that inflammation, fatty liver and dysregulated metabolism are hallmarks of systemically affected livers in mouse models and in patients with extrahepatic metastasis. We identified tumour-derived extracellular vesicles and particles (EVPs) as crucial mediators of cancer-induced hepatic reprogramming, which could be reversed by reducing tumour EVP secretion via depletion of Rab27a. All EVP subpopulations, exosomes and principally exomeres, could dysregulate hepatic function. The fatty acid cargo of tumour EVPs-particularly palmitic acid-induced secretion of tumour necrosis factor (TNF) by Kupffer cells, generating a pro-inflammatory microenvironment, suppressing fatty acid metabolism and oxidative phosphorylation, and promoting fatty liver formation. Notably, Kupffer cell ablation or TNF blockade markedly decreased tumour-induced fatty liver generation. Tumour implantation or pre-treatment with tumour EVPs diminished cytochrome P450 gene expression and attenuated drug metabolism in a TNF-dependent manner. We also observed fatty liver and decreased cytochrome P450 expression at diagnosis in tumour-free livers of patients with pancreatic cancer who later developed extrahepatic metastasis, highlighting the clinical relevance of our findings. Notably, tumour EVP education enhanced side effects of chemotherapy, including bone marrow suppression and cardiotoxicity, suggesting that metabolic reprogramming of the liver by tumour-derived EVPs may limit chemotherapy tolerance in patients with cancer. Our results reveal how tumour-derived EVPs dysregulate hepatic function and their targetable potential, alongside TNF inhibition, for preventing fatty liver formation and enhancing the efficacy of chemotherapy.

[Silencing RAB27a inhibits proliferation, invasion and adhesion of triple-negative breast cancer cells].

OBJECTIVE: To investigate the effect of inhibition of RAB27 protein family, which plays a pivotal role in exosome secretion, on biological behaviors of triple-negative breast cancer cells. METHODS: Quantitative real-time PCR and Western blotting were used to examine the expressions of RAB27 family and exosome secretion in 3 triple-negative breast cancer cell lines (MDA-MB-231, MDA-MB-468, and Hs578T) and a normal breast epithelial cell line (MCF10A). The effect of small interfering RNA (siRNA)-mediated silencing of RAB27a and RAB27b on exosome secretion in the 3 breast cancer cell lines was detected using Western blotting, and the changes in cell proliferation, invasion and adhesion were evaluated. RESULTS: Compared with normal breast epithelial cells, the 3 triple-negative breast cancer cell lines exhibited more active exosome secretion (P < 0.001) and showed significantly higher expressions of RAB27a and RAB27b at both the mRNA and protein levels (P < 0.01). Silencing of RAB27a in the breast cancer cells significantly down-regulated exosome secretion (P < 0.001), while silencing of RAB27b did not significantly affect exosome secretion. The 3 breast cancer cell lines with RAB27a silencing-induced down-regulation of exosome secretion showed obvious inhibition of proliferation, invasion and adhesion (P < 0.01) as compared with the cell lines with RAB27b silencing. CONCLUSION: RAB27a plays central role in the exosome secretion in triple-negative breast cancer cells, and inhibiting RAB27a can inhibit the proliferation, invasion and adhesion of the cells.

Expression and significant roles of the lncRNA NEAT1/miR-493-5p/Rab27A axis in ulcerative colitis.

BACKGROUND: Long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) have been reported to play regulatory roles in ulcerative colitis (UC). In this study, we aimed to determine the specific roles and action mechanism of the nuclear paraspeckle assembly transcript 1 (NEAT1) in UC. METHODS: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to determine the lncRNA NEAT1 and miR-493-5p expression levels in patients with UC and healthy volunteers. We determine the forecast linkage points of NEAT1 and miR-493-5p using Starbase and those of miR-493-5p and Rab27A using TargetScan, and further verified them using a double luciferase gene reporter kit. RT-qPCR and Western blot analysis were used to determine the lncRNA NEAT1, miR-493-5p, and Rab27A expression levels in lipopolysaccharide (LPS)-induced Caco-2 cells. Flow cytometry and cell counting kit-8 were used to assess Caco-2 cell viability. Tumor necrosis factor-α, interleukin (IL)-6, IL-8, and IL-1ß levels were determined via an enzyme-linked immunosorbent assay. RESULTS: Expression levels of NEAT1 were upregulated and those of miR-493-5p were downregualted in 10 ng/mL LPS-treated Caco-2 cells and patients with UC. Dual-luciferase gene reporter assay revealed that miR-493-5p is linked to NEAT1, and Rab27A is a downstream target of miR-493-5p. Overexpression of miR-493-5p inhibited the apoptosis and inflammation in LPS-treated Caco-2 cells. Moreover, downregulation of lncRNA NEAT1 expression also inhibited the apoptosis and inflammation in LPS-treated Caco-2 cells, which was reversed by Rab27A plasmid cotransfection. CONCLUSION: Our results revealed that NEAT1 participates in UC progression by inhibiting miR-493-5p expression.

Lack of Rab27a attenuates foam cell formation and macrophage inflammation in uremic apolipoprotein E knockout mice.

As the most common cardiovascular disease, atherosclerosis (AS), is a leading cause of high mortality in patients with chronic renal failure. Rab27a has been reported to regulate the progression of cardiovascular and renal diseases. Nevertheless, little studies investigated the role and mechanism of Rab27a in uremic-accelerated AS (UAAS). An animal model of UAAS was established in apolipoprotein E knockout (apoE-/-) mice using 5/6 nephrectomy (NX). We conducted in vitro and in vivo functional experiments to explore the role of Rab27a in UAAS, including the presence of oxidized low-density lipoprotein (ox-LDL). Rab27a expression was upregulated in the plaque tissues of NX apoE-/- mice. The knockout of Rab27a (Rab27a-/-) reduced AS-induced artery injury, as manifested by the reductions of plaque area, collagen deposition, inflammation and lipid droplet. Besides, cholesterol efflux was increased, while the expression of lipid metabolism-related proteins and the secretions of pro-inflammatory factors were decreased in ox-LDL-induced NX Rab27a-/- apoE-/- mice group. Further, Rab27a deletion inhibited the activation of nuclear factor κB (NF-κB) pathway. In conclusion, our study indicated that Rab27a deficiency attenuated foam cell formation and macrophage inflammation, depending on the NF-κB pathway activation, to inhibit AS progression in uremic apoE-/- mice. This finding may provide a new targeting strategy for UAAS therapy.

Multiple pathways and independent functional pools in insulin granule exocytosis.

In contrast to synaptic vesicle exocytosis, secretory granule exocytosis follows a much longer time course, and thus allows for different prefusion states prior to stimulation. Indeed, total internal reflection fluorescence microscopy in living pancreatic ß cells reveals that, prior to stimulation, either visible or invisible granules fuse in parallel during both early (first) and late (second) phases after glucose stimulation. Therefore, fusion occurs not only from granules predocked to the plasma membrane but also from those translocated from the cell interior during ongoing stimulation. Recent findings suggest that such heterogeneous exocytosis is conducted by a specific set of multiple Rab27 effectors that appear to operate on the same granule; namely, exophilin-8, granuphilin, and melanophilin play differential roles in distinct secretory pathways to final fusion. Furthermore, the exocyst, which is known to tether secretory vesicles to the plasma membrane in constitutive exocytosis, cooperatively functions with these Rab27 effectors in regulated exocytosis. In this review, the basic nature of insulin granule exocytosis will be described as a representative example of secretory granule exocytosis, followed by a discussion of the means by which different Rab27 effectors and the exocyst coordinate to regulate the entire exocytic processes in ß cells.

Serotonin/5-HT7 receptor provides an adaptive signal to enhance pigmentation response to environmental stressors through cAMP-PKA-MAPK, Rab27a/RhoA, and PI3K/AKT signaling pathways.

Serotonin (5-HT), a neurotransmitter, is essential for normal and pathological pigmentation processing, and its receptors may be therapeutical targets. The effect and behavior of the 5-HT7 receptor (5-HT7R) in melanogenesis in high vertebrates remain unknown. Herein, we examine the role and molecular mechanism of 5-HT7R in the pigmentation of human skin cells, human tissue, mice, and zebrafish models. Firstly, 5-HT7R protein expression decreased significantly in stress-induced depigmentation skin and vitiligo epidermis. Stressed mice received transdermal serotonin 5-HT7R selective agonists (LP-12, 0.01%) for 12 or 60 days. Mice might recover from persistent stress-induced depigmentation. The downregulation of tyrosinase (Tyr), microphthalmia-associated transcription factor (Mitf) expression, and 5-HT7R was consistently restored in stressed skin. High-throughput RNA sequencing showed that structural organization (dendrite growth and migration) and associated pathways were activated in the dorsal skin of LP-12-treated animals. 5-HT7R selective agonist, LP-12, had been demonstrated to enhance melanin production, dendrite growth, and chemotactic motility in B16F10 cells, normal human melanocytes (NHMCs), and zebrafish. Mechanistically, the melanogenic, dendritic, and migratory functions of 5-HT7R were dependent on the downstream signaling of cAMP-PKA-ERK1/2, JNK MAPK, RhoA/Rab27a, and PI3K/AKT pathway activation. Importantly, pharmacological inhibition and genetic siRNA of 5-HT7R by antagonist SB269970 partially/completely abolished these functional properties and the related activated pathways in both NHMCs and B16F10 cells. Consistently, htr7a/7b genetic knockdown in zebrafish could blockade melanogenic effects and abrogate 5-HT-induced melanin accumulation. Collectively, we have first identified that 5-HT7R regulates melanogenesis, which may be a targeted therapy for pigmentation disorders, especially those worsened by stress.

Rab27b, a Regulator of Exosome Secretion, Is Associated With Peritoneal Metastases in Gastric Cancer.

BACKGROUND/AIM: Peritoneal metastasis (PM) of gastric cancer (GC) leads to poor clinical outcomes. Tumor-derived exosomes promote metastasis via communication between tumor cells and host cells. In this study, we investigated the effect of Rab27, which is required for exosome secretion, on the PM of GC. MATERIALS AND METHODS: We established a stable knockdown of two Rab27 homologs, Rab27a and Rab27b, in human GC cells (58As9) with a high potential of PM. We examined the level of exosome secretion from Rab27-knockdown 58As9 cells by Western blotting and the ability of Rab27b knockdown to suppress PM in 58As9 cells using a mouse xenograft model. In vitro proliferation and invasion assays were performed in the Rab27b-knockdown cells. Next, Rab27b expression was evaluated in human GC tissues by immunohistochemistry. Finally, we assessed the clinicopathological and prognostic significance of Rab27b expression by RT-qPCR in both our and other TCGA datasets of GC. RESULTS: Rab27a and Rab27b knockdown in 58As9 cells decreased the secretion of exosomes, characterized by the endocytic marker CD63. Rab27b knockdown decreased PM in vivo without affecting the in vitro proliferation or invasion ability of 58As9 cells. In human GC tissues, Rab27b was overexpressed in tumor cells. The overall and recurrence-free survival rates were significantly lower in GC patients with high compared to low Rab27b mRNA expression in our and other TCGA datasets. CONCLUSION: Rab27b expression potentially serves as a poor prognostic biomarker, possibly affecting PM via exosome secretion from GC cells.

Silencing RAB27a inhibits proliferation, invasion and adhesion of triple-negative breast cancer cells / 南方医科大学学报

OBJECTIVE@#To investigate the effect of inhibition of RAB27 protein family, which plays a pivotal role in exosome secretion, on biological behaviors of triple-negative breast cancer cells.@*METHODS@#Quantitative real-time PCR and Western blotting were used to examine the expressions of RAB27 family and exosome secretion in 3 triple-negative breast cancer cell lines (MDA-MB-231, MDA-MB-468, and Hs578T) and a normal breast epithelial cell line (MCF10A). The effect of small interfering RNA (siRNA)-mediated silencing of RAB27a and RAB27b on exosome secretion in the 3 breast cancer cell lines was detected using Western blotting, and the changes in cell proliferation, invasion and adhesion were evaluated.@*RESULTS@#Compared with normal breast epithelial cells, the 3 triple-negative breast cancer cell lines exhibited more active exosome secretion (P < 0.001) and showed significantly higher expressions of RAB27a and RAB27b at both the mRNA and protein levels (P < 0.01). Silencing of RAB27a in the breast cancer cells significantly down-regulated exosome secretion (P < 0.001), while silencing of RAB27b did not significantly affect exosome secretion. The 3 breast cancer cell lines with RAB27a silencing-induced down-regulation of exosome secretion showed obvious inhibition of proliferation, invasion and adhesion (P < 0.01) as compared with the cell lines with RAB27b silencing.@*CONCLUSION@#RAB27a plays central role in the exosome secretion in triple-negative breast cancer cells, and inhibiting RAB27a can inhibit the proliferation, invasion and adhesion of the cells.

RAB27A promotes the proliferation and invasion of colorectal cancer cells.

Colorectal cancer (CRC) is one of the most commonly diagnosed cancer types worldwide. Despite significant advances in prevention and diagnosis, CRC is still one of the leading causes of cancer-related mortality globally. RAB27A, the member of RAB27 family of small GTPases, is the critical protein for intracellular secretion and has been reported to promote tumor progression. However, it is controversial for the role of RAB27A in CRC progression, so we explored the exact function of RAB27A in CRC development in this study. Based on the stable colon cancer cell lines of RAB27A knockdown and ectopic expression, we found that RAB27A knockdown inhibited proliferation and clone formation of SW480 colon cancer cells, whereas ectopic expression of RAB27A in RKO colon cancer cells facilitated cell proliferation and clone formation, indicating that RAB27A is critical for colon cancer cell growth. In addition, our data demonstrated that the migration and invasion of colon cancer cells were suppressed by RAB27A knockdown, but promoted by RAB27A ectopic expression. Therefore, RAB27A is identified as an onco-protein in mediating CRC development, which may be a valuable prognostic indicator and potential therapeutic target for CRC.

Clinical, immunological, molecular and therapeutic findings in monogenic immune dysregulation diseases: Middle East and North Africa registry.

Monogenic immune dysregulation diseases (MIDD) are caused by defective immunotolerance. This study was designed to increase knowledge on the prevalence and spectrum of MIDDs, genetic patterns, and outcomes in Middle East and North Africa (MENA). MIDD patients from 11 MENA countries (Iran, Turkey, Kuwait, Oman, Algeria, Egypt, United Arab Emirates, Tunisia, Jordan, Qatar, and Azerbaijan) were retrospectively evaluated. 343 MIDD patients (58% males and 42% female) at a median (IQR) age of 101 (42-192) months were enrolled. The most common defective genes were LRBA (23.9%), LYST (8.2%), and RAB27A (7.9%). The most prevalent initial and overall manifestations were infections (32.2% and 75.1%), autoimmunity (18.6% and 41%), and organomegaly (13.3% and 53.8%), respectively. Treatments included immunoglobulin replacement therapy (53%), hematopoietic stem cell transplantation (HSCT) (14.3%), immunosuppressives (36.7%), and surgery (3.5%). Twenty-nine (59.2%) patients survived HSCT. Along with infectious complications, autoimmunity and organomegaly may be the initial or predominant manifestations of MIDD.