Flotillin-1 Research Articles

Integrative Metabolic and Proteomic Profiling of the Brainstem in Spontaneously Hypertensive Rats.

The brainstem, the core of the central nervous system, plays a vital role in controlling arterial blood pressure and its elevation of hypertension subtypes, especially essential hypertension. Integrative metabolic and proteomic profiling was performed on the brainstem samples of 11 week old spontaneously hypertensive rats (SHRs) and age-matched normotensive Wistar rats, using hydrophilic interaction liquid chromatography quadrupole/time-of-flight mass spectrometry (HILIC-Q/TOFMS) (PeptideAtlas: PASS01621) and nano-liquid chromatography-high-resolution-MS (nano-LC-high-resolution) combined with quantitative tandem mass tags (ProteomeXchange: PXD021210). The results showed a potentially significant measure of metabolic disorders in the brainstem of SHRs, including purine and pyrimidine metabolism and carnitine and acylcarnitine deficiency. By integrating the differential metabolites (VIP > 1 and p < 0.1) with the differentially expressed proteins (>1.2-fold and p < 0.05), the results revealed aberrant insulin signaling in the brainstem of SHRs, including reduced carnitine and acetylcarnitine; increased arginine; and increased flotillin-1 (FLOT1), hemoglobin subunit alpha-1/2, and hemoglobin subunit beta-2 proteins verified by the parallel reaction monitoring analysis (PeptideAtlas: PASS01622). The aberrant insulin signaling pathway in the brainstem of SHRs might help explain the correlation between essential hypertension and insulin resistance. These findings on the brainstem of SHRs could provide new insights into the dysregulation of the central nervous system in hypertension, especially as it relates to metabolite and protein levels.

Abstract 117: Elucidating the role of Flotillin-2 in EGFR signaling and cancer growth

Abstract Epidermal growth factor receptor (EGFR) signaling is frequently dysregulated in a variety of cancers. The ubiquitin ligase Cbl ubiquitinates the activated EGFR, targeting it for degradation, and acts as an adaptor, recruiting proteins required for EGFR trafficking. To study Cbl adaptor function, we used Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) mass spectrometry (MS) to compare Cbl complexes in 3 groups: without or with EGF stimulation, and with EGF stimulation in the presence of an endosomal trafficking inhibitor. This approach identified over a hundred novel Cbl interactors, whose abundance in the complex with Cbl changed with the treatments. Secondary screening with an in cell Elisa assay demonstrated that siRNA mediated knockdown of Flotillin-2 (FLOT2) resulted in increased phosphorylation of FLOT2. Further evaluation of knockdown of FLOT2 in HeLa cells by immunoblotting showed that loss of FLOT2 led to increased phosphorylation, increased Cbl recruitment to the EGFR, increased ubiquitination of the EGFR, and degradation of EGFR upon EGF stimulation compared to negative control siRNA in HeLa cells. CRISPR knockout of FLOT2 in HeLa cells reduced steady-state levels of EGFR, but increased EGF-stimulated phosphorylation and signaling downstream of EGFR. In lung adenocarcinoma H441 cells, knockdown of FLOT2 increased EGF-stimulated EGFR phosphorylation, ubiquitination, and downstream signaling. This effect was reversed by the EGFR inhibitor erlotinib. Conversely, stable overexpression of FLOT2 in HeLa cells decreased EGF-stimulated EGFR phosphorylation and ubiquitination. Overexpression of wild type (WT) FLOT2 inhibited EGFR phosphorylation upon EGF stimulation in HEK293T cells. Conversely, overexpression of a soluble G2A FLOT2 mutant (which does not get recruited to the plasma membrane) did not inhibit EGF induced EGFR phosphorylation. In order to determine the effect of FLOT2 on EGFR-dependent cancer cell growth, both HeLa and H441 cells were treated in the presence or absence of both EGF and erlotinib in FLOT2 knockdown or knockout cells, and growth was monitored by cell counts over 7 days. HeLa cells exhibited increased growth and soft-agar colony formation in response to FLOT2 knockout, however this growth was not reduced by erlotinib treatment suggesting this was EGFR independent. However, EGF treatment greatly increased HeLa growth in the FLOT2 knockout group as compared to the control CRISPR and this increase was abrogated by erlotinib, indicating that FLOT2 knockout sensitizes HeLa cells to increased EGF-induced HeLa cell growth. H441 cells had an increase in growth over time in response to FLOT2 knockdown, and this growth was erlotinib sensitive, showing that the increase in growth was EGFR-dependent. In summary, these data demonstrate that FLOT2 negatively regulates EGFR activation and its subsequent ubiquitination and degradation. Further, we present evidence that FLOT2 negatively regulates cancer cell growth in an EGFR-dependent manner in H441 cells. Citation Format: David John Wisniewski, Mariya S. Liyasova, Xu Zhang, Samuel F. Gilbert, Alexis Catalano, Donna Voeller, Udayan Guha, Stanley Lipkowitz. Elucidating the role of Flotillin-2 in EGFR signaling and cancer growth [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 117.

Circ_001569 regulates FLOT2 expression to promote the proliferation, migration, invasion and EMT of osteosarcoma cells through sponging miR-185-5p.

Osteosarcoma (OS) is a common malignant tumor in the world. Circular RNAs are endogenous non-coding RNAs that have been linked to the development of cancer. However, the role of circ_001569 in OS progression is still unclear. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of circ_001569, microRNA-185-5p (miR-185-5p) and flotillin-2 (FLOT2). The abilities of cell proliferation, migration and invasion were evaluated by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and Transwell assays, respectively. Also, western blot analysis was performed to assess the levels of epithelial–mesenchymal transition (EMT)-related proteins and FLOT2 protein. Besides, the dual-luciferase reporter assay was used to verify the interactions among circ_001569, miR-185-5p and FLOT2. Circ_001569 expression was increased in OS tissues and cells, and its knockdown reduced the proliferation, migration, invasion and EMT of OS cells. MiR-185-5p could interact with circ_001569. Inhibition of miR-185-5p could recover the suppression effects of silenced-circ_001569 on the proliferation and metastasis of OS cells. Furthermore, FLOT2 was a target of miR-185-5p. Overexpressed FLOT2 could restore the inhibition effects of miR-185-5p mimic on the proliferation and metastasis of OS cells. Also, FLOT2 expression was regulated by circ_001569 and miR-185-5p. In addition, circ_001569 knockdown also reduced the OS tumor growth in vivo. Circ_001569 might act as an oncogene in OS progression by regulating the miR-185-5p/FLOT2 axis, which provided a reliable new approach for the treatment of OS patients.

Ex vivo Quantitative Proteomic Analysis of Serotonin Transporter Interactome: Network Impact of the SERT Ala56 Coding Variant.

Altered serotonin (5-HT) signaling is associated with multiple brain disorders, including major depressive disorder (MDD), obsessive-compulsive disorder (OCD), and autism spectrum disorder (ASD). The presynaptic, high-affinity 5-HT transporter (SERT) tightly regulates 5-HT clearance after release from serotonergic neurons in the brain and enteric nervous systems, among other sites. Accumulating evidence suggests that SERT is dynamically regulated in distinct activity states as a result of environmental and intracellular stimuli, with regulation perturbed by disease-associated coding variants. Our lab identified a rare, hypermorphic SERT coding substitution, Gly56Ala, in subjects with ASD, finding that the Ala56 variant stabilizes a high-affinity outward-facing conformation (SERT∗) that leads to elevated 5-HT uptake in vitro and in vivo. Hyperactive SERT Ala56 appears to preclude further activity enhancements by p38α mitogen-activated protein kinase (MAPK) and can be normalized by pharmacological p38α MAPK inhibition, consistent with SERT Ala56 mimicking, constitutively, a high-activity conformation entered into transiently by p38α MAPK activation. We hypothesize that changes in SERT-interacting proteins (SIPs) support the shift of SERT into the SERT∗ state which may be captured by comparing the composition of SERT Ala56 protein complexes with those of wildtype (WT) SERT, defining specific interactions through comparisons of protein complexes recovered using preparations from SERT–/– (knockout; KO) mice. Using quantitative proteomic-based approaches, we identify a total of 459 SIPs, that demonstrate both SERT specificity and sensitivity to the Gly56Ala substitution, with a striking bias being a loss of SIP interactions with SERT Ala56 compared to WT SERT. Among this group are previously validated SIPs, such as flotillin-1 (FLOT1) and protein phosphatase 2A (PP2A), whose functions are believed to contribute to SERT microdomain localization and regulation. Interestingly, our studies nominate a number of novel SIPs implicated in ASD, including fragile X mental retardation 1 protein (FMR1) and SH3 and multiple ankyrin repeat domains protein 3 (SHANK3), of potential relevance to long-standing evidence of serotonergic contributions to ASD. Further investigation of these SIPs, and the broader networks they engage, may afford a greater understanding of ASD as well as other brain and peripheral disorders associated with perturbed 5-HT signaling.

Long Non-Coding RNA TUG1 Promotes Cell Proliferation and Inhibits Cell Apoptosis, Autophagy in Clear Cell Renal Cell Carcinoma via MiR-31-5p/FLOT1 Axis.

PurposeClear cell renal cell carcinoma (ccRCC) is a common urological carcinoma in adults. Long non-coding RNA (lncRNA) taurine-upregulated gene 1 (TUG1) has been reported to be involved in the progression of diverse human cancers, including renal cell carcinoma (RCC). However, the biological mechanism of TUG1 was rarely reported in ccRCC.MethodsThe levels of TUG1, microRNA miR-31-5p and flotillin 1 (FLOT1) in ccRCC tissues and cells were detected by qRT-PCR. The interactions between miR-31-5p and TUG1 or FLOT1 were predicted by starBase v2.0 and TargetScan, respectively, which were further validated by RIP assay and RNA pull-down assay. Cell counting kit-8 (CCK-8), flow cytometry and Western blot were used to assess the effects of TUG1 on cell viability, apoptosis rate and the relative protein expression levels in ccRCC cells. In addition, the xenograft tumor assay was conducted to further verify the functions of TUG1 in ccRCC in vivo.ResultsTUG1 was dramatically up-regulated in ccRCC tissues and cells. TUG1 silencing inhibited cell proliferation and promoted cell apoptosis, autophagy in 786-0 and A498 cells. In addition, TUG1 depletion repressed tumor growth in vivo. Moreover, miR-31-5p was validated as a direct target of TUG1, and microRNA miR-31-5p inhibitor mitigated the effects of TUG1 knockdown on ccRCC progression. Furthermore, FLOT1 was verified to be negatively interacted with miR-31-5p. FLOT1 overexpression attenuated miR-31-5p-mediated inhibitory effect on cell proliferation and promotion effects on cell apoptosis, autophagy. The restoration experiment implicated that TUG1 positively modulated FLOT1 expression by sponging miR-31-5p.ConclusionAll data demonstrated that TUG1 promotes cell proliferation and inhibits cell apoptosis and autophagy in ccRCC by miR-31-5p/FLOT1 axis, which may provide a therapeutic target for ccRCC patients.

Perindopril ameliorates experimental Alzheimer's disease progression: role of amyloid β degradation, central estrogen receptor and hyperlipidemic-lipid raft signaling.

Accumulating evidence indicates that over-stimulation of angiotensin-converting enzyme 1 (ACE1) activity is associated with β-amyloid (Aβ) and phosphorylated tau (p-tau)-induced apoptosis, oxido-nitrosative neuroinflammatory stress and neurodegeneration in Alzheimer's disease (AD). Alternatively, activation of the ACE2, the metalloprotease neprilysin (Neutral Endopeptidase; NEP) and the insulin-degrading enzyme (IDE) could oppose the effects of ACE1 activation. We aim to investigate the relationship between ACE1/ACE2/NEP/IDE and amyloidogenic/hyperlipidemic-lipid raft signaling in hyperlipidemic AD model. Induction of AD was performed in ovariectomized female rats with high-fat high fructose diet (HFFD) feeding after 4weeks following D-galactose injection (150mg/kg). The brain-penetrating ACE1 inhibitor perindopril (0.5mg/kg/day, p.o.) was administered on a daily basis for 30days. Perindopril significantly decreased hippocampal expression of ACE1 and increased expression of ACE2, NEP and IDE. Perindopril markedly decreased Aβ1-42, improved lipid profile and ameliorated the lipid raft protein markers caveolin1 (CAV1) and flotillin 1 (FLOT1). This was accompanied by decreased expression of p-tau and enhancement of cholinergic neurotransmission, coupled with decreased oxido-nitrosative neuroinflammatory stress, enhancement of blood-brain barrier (BBB) functioning and lower expression of the apoptotic markers glial fibrillary acidic protein (GFAP), Bax and β-tubulin. In addition, perindopril ameliorated histopathological damage and improved learning, cognitive and recognition impairment as well as depressive behavior in Morris water maze, Y maze, novel object recognition and forced swimming tests, respectively. Conclusively, perindopril could improve cognitive defects in AD rats, at least through activation of ACE2/NEP/IDE and inhibition of ACE1 and subsequent modulation of amyloidogenic/hyperlipidemic-lipid raft signaling and oxido-nitrosative stress.

Screening of potential inhibitors against flotillin-1 as therapeutics for Alzheimer's disease

Background: Alzheimer's disease (AD) is one of the most common neurological disorders occurring in older people. So far, no specific drug is available for the disease, and only palliative medicines are available for the patients. Accumulation of amyloid beta (Aβ) peptides is considered to play a crucial role in the generation of the disease. Aβ peptide is generated by the proteolysis of amyloid precursor protein by two distinct proteases β and γ-secretase. Flotillin-1 (FLOT1) directly binds to the cytoplasmic tail of β-secretase and affects the sorting and recycling of the enzyme. Increased expression of FLOT1 has been correlated with the progression of AD, while FLOT1 knockdown causes a reduction in Aβ production. Thus, FLOT1 is an attractive therapeutic target for the suppression of beta-secretase 1 (BACE-1 ). Objective: This study aims to screen the potential inhibitors against FLOT1 as therapeutics for AD. Materials and Methodology: In this work, protein–protein interactions, tertiary structure prediction, molecular docking, and molecular dynamics (MD) simulation were performed. Results: Tertiary structure prediction of FLOT1 and screening inhibitors against it helped in finding key molecules with potential therapeutic properties. Protein–protein interaction study of FLOT1 deciphered the interactors playing key role in AD. Pharmacokinetic parameters were quantified for each potential inhibitor. The results of MD simulation analysis revealed that the ZINC67911837 had better inhibitory activities with FLOT1. Conclusion: The analysis suggests that the ZINC67911837 compound could be a novel potential inhibitor of FLOT1 to modulate BACE-1 activity and used as a therapeutic agent for the treatment of AD. This study facilitates the initiation of the natural drug discovery process for the treatment of AD patients.

Different Trafficking Phenotypes of Niemann-Pick C1 Gene Mutations Correlate with Various Alterations in Lipid Storage, Membrane Composition and Miglustat Amenability.

Niemann-Pick Type C (NPC) is an autosomal recessive lysosomal storage disease leading to progressive neurodegeneration. Mutations in the NPC1 gene, which accounts for 95% of the cases, lead to a defect in intra-lysosomal trafficking of cholesterol and an accumulation of storage material including cholesterol and sphingolipids in the endo-lysosomal system. Symptoms are progressive neurological and visceral deterioration, with variable onset and severity of the disease. This study investigates the influence of two different NPC1 mutations on the biochemical phenotype in fibroblasts isolated from NPC patients in comparison to healthy, wild type (WT) cells. Skin derived fibroblasts were cultured from one patient compound-heterozygous for D874V/D948Y mutations, which presented wild-type like intracellular trafficking of NPC1, and a second patient compound- heterozygous for I1061T/P887L mutations, which exhibited a more severe biochemical phenotype as revealed in the delayed trafficking of NPC1. Biochemical analysis using HPLC and TLC indicated that lipid accumulations were mutation-dependent and correlated with the trafficking pattern of NPC1: higher levels of cholesterol and glycolipids were associated with the mutations that exhibited delayed intracellular trafficking, as compared to their WT-like trafficked or wild type (WT) counterparts. Furthermore, variations in membrane structure was confirmed in these cell lines based on alteration in lipid rafts composition as revealed by the shift in flotillin-2 (FLOT2) distribution, a typical lipid rafts marker, which again showed marked alterations only in the NPC1 mutant showing major trafficking delay. Finally, treatment with N-butyldeoxynojirimycin (NB-DNJ, Miglustat) led to a reduction of stored lipids in cells from both patients to various extents, however, no normalisation in lipid raft structure was achieved. The data presented in this study help in understanding the varying biochemical phenotypes observed in patients harbouring different mutations, which explain why the effectiveness of NB-DNJ treatment is patient specific.

Circ-FOXM1 contributes to cell proliferation, invasion, and glycolysis and represses apoptosis in melanoma by regulating miR-143-3p/FLOT2 axis

BackgroundNumerous literatures have demonstrated that circular RNAs (circRNAs) are involved in multiple types of tumors. However, the effects of circRNAs in melanoma are not very clear. In this study, we aimed to investigate the roles and mechanisms of circ-FOXM1 in melanoma.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) was conducted to determine the expression of circ-FOXM1, microRNA-143-3p (miR-143-3p), and Flotillin 2 (FLOT2) mRNA. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, flow cytometry analysis, and transwell assay were employed to test cell proliferation, apoptosis, and invasion, respectively. The glucose consumption and lactate production were examined by specific kits. Western blot assay was utilized for the detection of hexokinase2 (HK2), pyruvate kinase isozyme type M2 (PKM2), and FLOT2. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were employed to verify the targeting association between miR-143-3p and circ-FOXM1 or FLOT2. A murine xenograft model was established to explore the effect of circ-FOXM1 in vivo.ResultsCirc-FOXM1 was elevated and miR-143-3p was reduced in melanoma tissues and cells. Circ-FOXM1 deficiency impeded cell proliferation, invasion, and glycolysis and facilitated cell apoptosis in melanoma in vitro and tumorigenesis in vivo. Circ-FOXM1 acted as a sponge of miR-143-3p and the impacts of circ-FOXM1 silencing on cell proliferation, apoptosis, invasion, and glycolysis were overturned by miR-143-3p deletion. Moreover, FLOT2 was a target gene of miR-143-3p and FLOT2 overexpression rescued the inhibitory effect of miR-143-3p on melanoma progression.ConclusionCirc-FOXM1 facilitated the development of melanoma by upregulating FLOT2 through miR-143-3p.

Moxifloxacin suppresses airway inflammation and modulates expression of caveolin-1 and flotillin-1 in airway smooth muscle cells of asthmatic rats.

Moxifloxacin (MXF) possesses anti-inflammatory properties on asthmatic airway smooth muscle cells (ASMCs) beyond their antimicrobial effects, but the mechanisms are still unknown. This study was to investigate effects of MXF on expression of caveolin-1 (Cav-1) and flotillin-1 (FLOT1) in ASMCs in asthmatic rats. ASMCs were collected from the airway and cultured in vitro. Cells from normal rats were treated with normal saline (Group N); cells from asthmatic rats were incubated with normal saline (Group A) or MXF (20 mg/L) (Group M); Cav-1 expression was up-regulated by transferring Cav-1 expressing lentivirus (Group L) and FLOT1 expression down-regulated by using siRNA in cells from asthmatic rats (Group S). The expressions of Cav-1, FLOT1 and p65 NF-κB were measured by Western blotting and quantificational real-time polymerase chain reaction (qRT-PCR), and interleukin-8 (IL-8) and eotaxin contents were measured by enzyme-linked immunosorbent assay (ELISA). Compared with normal control, Cav-1 expression significantly decreased in asthmatic groups (P<0.01); MXF up-regulated Cav-1 expression in asthmatic groups (P<0.01). However, compared with normal control, the expression of FLOT1 and p65 NF-κB dramatically increased in asthmatic groups (P<0.01); MXF down-regulated the expression of FLOT1 and p65 NF-κB in asthmatic groups (P<0.01); meanwhile, the expressions of FLOT1 and p65 NF-κB decreased after up-regulation of Cav-1 expression in asthmatic groups (P=0.01). Compared with asthmatic groups, the IL-8 and eotaxin contents significantly decreased in MXF Groups, Cav-1 up-regulation asthmatic groups and FLOT1 down-regulation asthmatic groups (P<0.01). MXF can modulate the airway inflammation, upregulate Cav-1 expression, downregulate the expression of FLOT1 and p65 NF-κB in asthmatic rat ASMCs, which may be related to the anti-inflammatory effects of MXF in asthmatic ASMCs.

MicroRNA-351 eases insulin resistance and liver gluconeogenesis via the PI3K/AKT pathway by inhibiting FLOT2 in mice of gestational diabetes mellitus.

Gestational diabetes mellitus (GDM) is known as different degree glucose intolerance that is initially identified during pregnancy. MicroRNAs (miRs) may be a potential candidate for treatment of GDM. Herein, we suggested that miR‐351 could be an inhibitor in the progression of GDM via the phosphoinositide 3‐kinase/protein kinase B (PI3K/AKT) pathway. Microarray analysis was used to identify differentially expressed genes and predict miRs regulating flotillin 2 (FLOT2). Target relationship between miR‐351 and FLOT2 was verified. Gestational diabetes mellitus mice were treated with a series of mimic, inhibitor and small interfering RNA to explore the effect of miR‐351 on insulin resistance (IR), cell apoptosis in pancreatic tissues and liver gluconeogenesis through evaluating GDM‐related biochemical indexes, as well as expression of miR‐351, FLOT2, PI3K/AKT pathway‐, IR‐ and liver gluconeogenesis‐related genes. MiR‐351 and FLOT2 were reported to be involved in GDM. FLOT2 was the target gene of miR‐351. Gestational diabetes mellitus mice exhibited IR and liver gluconeogenesis, up‐regulated FLOT2, activated PI3K/AKT pathway and down‐regulated miR‐351 in liver tissues. Additionally, miR‐351 overexpression and FLOT2 silencing decreased the levels of FLOT2, phosphoenolpyruvate carboxykinase, glucose‐6‐phosphatase, fasting blood glucose, fasting insulin, total cholesterol, triglyceride, glyeosylated haemoglobin and homeostasis model of assessment for IR index (HOMA‐IR), extent of PI3K and AKT phosphorylation, yet increased the levels of HOMA for islet β‐cell function, HOMA for insulin sensitivity index and glucose transporter 2 expression, indicating reduced cell apoptosis in pancreatic tissues and alleviated IR and liver gluconeogenesis. Our results reveal that up‐regulation of miR‐351 protects against IR and liver gluconeogenesis by repressing the PI3K/AKT pathway through regulating FLOT2 in GDM mice, which identifies miR‐351 as a potential therapeutic target for the clinical management of GDM.

Clinical significance and comparison of flotillin 1 expression in left and right colon cancer.

Flotillin 1 (FLOT1) is increasingly implicated in various types of cancer, and has been reported to influence tumorigenesis and cancer progression, leading to poor prognosis for survival time; however, its expression in colorectal cancer (CRC) and its influence on various clinicopathological parameters of this disease remain unknown. In the present study, FLOT1 expression and its effect on different clinicopathological parameters were assessed immunohistochemically and histologically in 81 CRC and 81 non-tumorous colon tissue samples. The immunohistochemical staining was scored semi-quantitatively. The association of FLOT1 expression with various parameters and its effect on overall survival time was also assessed. FLOT1 was upregulated in the CRC tissue, with increased expression in the right colon tissue samples compared with those of left colon. Increased FLOT1 expression in CRC tissue samples was associated with tumor volume, differentiation, tumor grade and poor overall survival time. In the right colon tissue samples in particular, there was a notable association with tumor volume and grade, indicating its effect on proliferation and tumor stage at this site. A multivariate Cox regression hazard analysis revealed that only tumor grade and differentiation were the independent predictors of overall survival time in patients with CRC. Together, the results of the present study suggest that FLOT1 serves important functions in the proliferation and progression of CRC, contributes to decreased survival time, and may serve as a novel therapeutic target for the treatment of CRC.

FLOT2 overexpression is associated with the progression and prognosis of human colorectal cancer

Altered expression of flotillin-2 (FLOT2) has been identified in certain types of cancer, including breast cancer and melanoma; however, to the best of our knowledge, the association between the FLOT2 expression level and colorectal cancer (CRC) remains to be determined. The aim of the present study was to investigate the clinical and prognostic significance of FLOT2 in CRC. The expression of FLOT2 was determined in seven CRC cell lines and one normal colon cell line, and it was identified that FLOT2 was increased in CRC cell lines, suggesting that FLOT2 exhibited an association with CRCs. In addition, FLOT2 protein levels were investigated in primary CRC tissues and corresponding non-cancerous colon tissues from 8 patients. Compared with non-cancerous tissues, FLOT2 protein was apparently upregulated in CRC tissues. To validate this result, an immunohistochemistry assay was performed and it was identified that FLOT2 levels were increased in CRC tissues. Clinical analysis identified that increased expression of FLOT2 was associated with the depth of invasion, lymph node metastasis, distant metastasis and American Joint Committee on Cancer stage of CRCs. Furthermore, multivariate analysis using the Cox regression model indicated that increased FLOT2 expression was an independent prognostic factor in patients with CRC (P=0.013). Taken together, the results of the present study suggest that overexpression of FLOT2 contributes to the progression of CRC and indicate that FLOT2 is a novel target for the treatment of CRC.

MicroRNA-485-5p suppresses the proliferation, migration and invasion of small cell lung cancer cells by targeting flotillin-2

ABSTRACT This study is aimed to elucidate the mechanisms underlying the role of miR-485-5p in small cell lung cancer (SCLC). The expression of miR-485-5p were quantified with real time quantitative PCR and it was found that the level of miR-485-5p was lower in SCLC tissues than normal tissues. In cultured SCLC cell lines, overexpression of miR-485-5p reduced cell proliferation, migration, and invasion in vitro, whereas knockdown of miR-485-5p performed contrary. FLOT2 expression was obviously upregulated and negatively correlated with miR-485-5p expression level in SCLC tissues. Overexpression of miR-485-5p significantly inhibited the protein expression of flotillin-2 (FLOT2) in cultured SCLC cells. Luciferase reporter assay confirmed that FLOT2 was a direct target of miR-485-5p in SCLC cells. It is concluded that miR-485-5p, as a tumor suppressor, inhibits the growth and metastasis in SCLC by targeting FLOT2. Upregulation of miR-485-5p expression may be an attractive strategy for SCLC therapy.

MicroRNA-6809-5p mediates luteolin-induced anticancer effects against hepatoma by targeting flotillin 1

BackgroundLuteolin (3,4,5,7-tetrahydroxy flavone) is a natural flavonoid abundant in fruits and vegetables. Although luteolin has shown pro-apoptotic activity in hepatocellular carcinoma (HCC) cells, the underlying molecular mechanism has not yet been clarified. PurposeThe aim of this study is to identify novel miRNAs involved in the action of luteolin in HCC cells and to explore the biological roles of these miRNAs. MethodsThe effect of luteolin on HCC cell growth was assessed using CCK-8 colony formation assay, flow cytometric analysis in vitro, and a xenograft model in vivo. miRNA expression profiles were assessed using next-generation sequencing. Differentially expressed miRNAs were validated by quantitative PCR. Bioinformatics analysis and luciferase reporter assay were utilized to confirm the binding of miR-6809-5p to the 3′-untranslated region (3′-UTR) of flotillin 1 (FLOT1). Furthermore, the effects of ectopic FLOT1 and miR-6809-5 expression on cell proliferation, colony formation, and cell apoptosis were also assessed. Western blotting analysis was used to detect activation of multiple signaling molecules including Erk1/2, p38, JNK, and NF-κB/p65 in the MAPK pathway. ResultsIt was found that luteolin significantly inhibited HCC growth and caused apoptosis and cell cycle arrest at the G0/G1 phase in Huh7 cells, at the G2/M phase in HepG2 cells in vitro. Tumorigenic studies revealed that luteolin treatment significantly suppressed HCC growth in vivo. miR-6809-5p was upregulated by luteolin. Overexpression of miR-6809-5p suppressed HCC cell growth, while knockdown of miR-6809-5p reversed the anticancer effect of luteolin. With regards to its signaling mechanism, miR-6809-5p directly targets FLOT1in HCC cells. Enforced expression of FLOT1 prevented miR-6809-5p-mediated growth suppression. Downregulation of FLOT1 exerted growth-suppressive effects on HCC cells. Multiple signaling pathways including Erk1/2, p38, JNK, and NF-κB/p65 were inactivated by miR-6809-5p overexpression or FLOT1 downregulation. ConclusionThese findings indicated that miR-6809-5p mediates the growth-suppressive activity of luteolin in HCC, which is causally linked to FLOT1 downregulation. Induction of miR-6809-5p may provide therapeutic benefits in the treatment of HCC.

Effect of flotillin-1 gene small interfering RNA combined with curcumin on apoptosis of renal cell carcinoma in vitro

Objective To investigate the effect of flotillin-1 (FLOT1) small interfering RNA (siRNA) combined with curcumin on the apoptosis of renal cell carcinoma. Methods FLOT1-siRNA was transfected into 786-0 cells of human renal carcinoma by Lipofectamine™ 2000. The protein expression of FLOT1 in the cells transfected with siRNA was detected by Western blotting. Thereafter, FLOT1-siRNA, curcumin and FLOT1-siRNA+ curcumin were used for cell intervention, and a blank control group was set up. The cell viability and apoptosis rate of four groups were detected by cell counting kit-8 (CCK-8) assay and flow cytometry. H2DCFHDA probe was used to detect the content of ROS in four groups. The expression levels of phosphorylated signal transducer and activators of transcription 3 (p-STAT3), proliferating cell nuclear antigen (PCNA) and B cell lymphoma/leukemia-2 associated X protein (bax) protein were detected by Western blotting. Results The expression of FLOT1 (0.087±0.010) was significantly inhibited in 786-0 cells transfected with FLOT1-siRNA, and the difference was statistically significant as compared with the blank control group (0.454±0.038) (t=16.177, P=0.000). Si-FLOT1 (0.491±0.053) or curcumin (0.542±0.058) could inhibit the activity of 786-0 cells, induce 786-0 cell apoptosis [(18.22±1.07)% and (12.39±0.86)%] and ROS production [(189.5±8.2) and (147.7±7.1)], down-regulate the expression of p-STAT3 [(0.118±0.014) and (0.110±0.012)] and PCNA [(0.229±0.025) and (0.302±0.034)] and up-regulate the expression of bax [(0.206±0.021) and (0.123±0.014)], and the difference was statistically significant as compared with NC group [(0.792±0.065), (1.62±0.25)%, (57.7±3.5), (0.202±0.023), (0.577±0.049), (0.069±0.010)] (t=6.736, 7.378, 12.796, 10.112, 7.186, 2.832, P=0.000, 0.000, 0.000, 0.000, 0.000, 0.022). Combined use of si-FLOT1 and curcumin could inhibit the activity of 786-0 cells [(0.378±0.044)], induce apoptosis [(24.18±1.22)%] and ROS production [(249.3±9.4)], down-regulate the expression of p-STAT3 [(0.054±0.008)] and PCNA [(0.135±0.016)] and up-regulate the expression of bax [(0.411±0.038)], with the difference being statistically significant as compared with single si-FLOT1 group or curcumin group (t=5.132, 4.491, 3.456, 6.140, 10.752, 15.106, P=0.001, 0.002, 0.009, 0.000, 0.000, 0.000). Conclusion FLOT1 siRNA or curcumin can inhibit the viability of renal cell carcinoma cells and induce apoptosis, and the combined use of FLOT1 siRNA and curcumin can be more obvious to cell viability and apoptosis. The mechanism may be related to the increases in intracellular ROS and the inhibition of STAT3 signal. Key words: Renal cell carcinoma; Flotillin-1 gene; Curcumin; Apoptosis; Signal transducer and activators of transcription 3 signaling pathway

Abstract 2506: Flotillin 2 negatively regulates EGFR phosphorylation and Cbl-mediated ubiquitination and degradation

Abstract EGF receptor (EGFR) signaling is dysregulated by gene amplification or activating mutations of the EGFR in a variety of human malignancies. Activation of the EGFR by EGF leads to recruitment of the ubiquitin ligase Cbl, which ubiquitinates EGFR targeting it for degradation and acts as an adaptor, recruiting additional proteins required for EGFR internalization and trafficking to the lysosome. However, the detailed molecular mechanisms remain elusive. Complete characterization of proteins involved in EGFR trafficking and degradation may uncover additional mechanisms of EGFR dysregulation in cancer. To characterize the EGFR degradative pathway, we investigated protein complexes formed on Cbl with Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) mass spectrometry. By labeling proteins with stable isotopes in culture, we compared complexes in 3 groups: 1) complexes formed on Cbl without EGF stimulation (control), 2) complexes formed on Cbl with EGF stimulation for 30 min and 3) complexes formed on Cbl with EGF stimulation in the presence of YM201636, a trafficking inhibitor, which was added to prevent degradation of the Cbl/EGFR complex and to enrich it for other proteins important for EGFR trafficking. This approach identified over a hundred novel Cbl interactors, whose abundance in the complex with Cbl changed significantly with the treatments. Fifty of these proteins were selected for RNAi knockdown to examine their effects on EGFR degradation. We found that knockdown of Flotillin 2 (FLOT2) protein, a major scaffold protein within caveolar lipid rafts, led to increased phosphorylation and accelerated degradation of EGFR upon EGF stimulation compared to negative control siRNA in HeLa cells. Conversely, overexpression of FLOT2 in HEK293T inhibited EGFR phosphorylation and formation of the EGFR/Cbl complex upon EGF stimulation and led to decreased EGFR ubiquitination and degradation. CRISPR knockout of FLOT2 in HeLa cells led to decreased steady-state levels of EGFR, increased phosphorylation of EGFR and ERK1/2 compared to non-targeting CRISPR control. Stable overexpression of FLOT2 in HeLa cells increased steady-state levels of EGFR, but decreased EGFR phosphorylation and ubiquitination upon EGF stimulation. FLOT2 is frequently amplified and overexpressed in breast, endometrial and lung cancers. In a lung adenocarcinoma cell line H441, in which FLOT2 gene is amplified, knockdown of FLOT2 increased EGFR phosphorylation and ubiquitination, as well as EGFR signaling, as detected by the phosphorylation of ERK1/2. Our data indicate that FLOT2 negatively regulates EGFR phosphorylation, ubiquitination and degradation. Although it is not clear how FLOT2 amplification could contribute to cancer pathogenesis, we speculate that studying how FLOT2 regulates EGFR signaling could lead to a better understanding of its role in normal physiology and cancer. Citation Format: Mariya Liyasova, Xu Zhang, Udayan Guha, Stanley Lipkowitz. Flotillin 2 negatively regulates EGFR phosphorylation and Cbl-mediated ubiquitination and degradation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2506.

Abstract 5159: FLOT1 is associated with HGF mediated cell invasion and proliferation by increased MMP9

Abstract The flotillin protein family, including flotillin-1 (FLOT1 ) and flotillin-2 (FLOT2), have been known as markers of lipid rafts. Recentely, FLOT1 was reported that FLOT1 overexpression was found in several advanced cancer including breast, colorectal, prostate tumors, and renal cell carcinomas and correlate with poor prognosis in advanced tumors. The purpose of this study is to identify the role of HGF upregulated FLOT1 associated with cancer cell proliferation and invasion in gastric cancer. We used cell culture, western blotting, RT-PCR, MTT assays, CHIP assay, zymography and FLOT 1 knock-down with short hairpin RNA (shRNA). First, we confirmed that the expression level of FLOT1 was up-regulated by HGF(hepatocyte growth factor). To determine the role for FLOT1, we used the knock down cell of FLOT1. FLOT1 -sh RNA cells showed a decreased level of MMP9 and NFkB. We also examined to confirm the role of HGF-mediated FLOT1. HGF-mediated cell proliferation and in vitro invasion was decreased in FLOT1 knock down cell. We perforomed the zymography to evaluate the activity of MMP9 . The activity of MMP9 was decreased in FLOT1 knock down cell. The level of AKT phosphorylation was decreased in the knock down cell of FLOT1. Also, We identified the putative binding site of NFkB in MMP9 promotor region and confirmed the function by CHIP assay. Our study showed that upregulation FLOT1 by HGF increased the level of MMP9 through AKT/NFkB pathway and associated with cell proliferation and invasion in gastric cancer. Citation Format: Sung Ae Koh, Kyung Hee Lee, Jae Ryong Kim, Sang woon Kim, Byung Ik Jang. FLOT1 is associated with HGF mediated cell invasion and proliferation by increased MMP9 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5159.

Flotillin 1 is differentially expressed in human epithelial ovarian tumors.

Although Flotillin 1 (FLOT1) is highly expressed in various human cancers, its relationship with ovarian cancer (OC) remains unknown. This study determines FLOT1 expression in human ovarian tumors and examines its effect on OC cell proliferation. FLOT1 protein expression was assessed in a tissue microarray by immunohistochemical staining. We found that 81.48% malignant and 50% borderline tumors were FLOT1 protein-positive, whereas benign tumors and normal ovarian tissues were negative. The staining was strongest in serous malignant tumor and transitional cell carcinoma and weakest in mucinous tumor. Differentially expressed FLOT1 in freshly isolated serous tumors was confirmed by Western blot and we then evaluated FLOT1 expression association with OC patients' clinical characteristics. Histological typing established that FLOT1 protein expression was significantly associated with serous tumor (P Keywords: cell proliferation, epithelial ovarian cancer, FLOT1, miRNA, therapeutic target, tumorigenesis.

Gene Silencing of Argonaute5 Negatively Affects the Establishment of the Legume-Rhizobia Symbiosis.

The establishment of the symbiosis between legumes and nitrogen-fixing rhizobia is finely regulated at the transcriptional, posttranscriptional and posttranslational levels. Argonaute5 (AGO5), a protein involved in RNA silencing, can bind both viral RNAs and microRNAs to control plant-microbe interactions and plant physiology. For instance, AGO5 regulates the systemic resistance of Arabidopsis against Potato Virus X as well as the pigmentation of soybean (Glycine max) seeds. Here, we show that AGO5 is also playing a central role in legume nodulation based on its preferential expression in common bean (Phaseolus vulgaris) and soybean roots and nodules. We also report that the expression of AGO5 is induced after 1 h of inoculation with rhizobia. Down-regulation of AGO5 gene in P. vulgaris and G. max causes diminished root hair curling, reduces nodule formation and interferes with the induction of three critical symbiotic genes: Nuclear Factor Y-B (NF-YB), Nodule Inception (NIN) and Flotillin2 (FLOT2). Our findings provide evidence that the common bean and soybean AGO5 genes play an essential role in the establishment of the symbiosis with rhizobia.