LASP1 promotes proliferation,metastasis, invasion in head and neck squamous cell carcinoma and through direct interaction with HSPA1A

LIM and SH3 protein 1 (LASP1) is a specific focal adhesion protein that promotes metastasis in a variety of tumours. However, its role in head and neck squamous cell carcinoma (HNSCC) has not been fully validated. The purpose of this study was to analyse the interaction of LASP1 and its binding partner in HNSCC. The expression of LASP1 and HSPA1A in HNSCC was analysed by real‐time PCR and Western blot. The effects of LASP1 on the biology behaviour of HNSCC cell lines were observed in vivo and in vitro. Co‐immunoprecipitation analysis was performed to confirm the interaction between LASP1 and HSPA1A. LASP1 was highly expressed in HNSCC and associated with poor prognosis for patients. LASP1 also promoted cell proliferation, colony formation, invasion and cell cycle G2/M phase transition. Heat shock protein family A member 1A (HSPA1A) is identified as a chaperone protein of LASP1 and co‐localized in the cytoplasm. HSPA1A positively regulates the interaction of LASP1 with P‐AKT and enhances the malignant behaviour of HNSCC cells. LASP1 and HSPA1A are both up‐regulated in HNSCC, and directly binds to each other. Double inhibition of LASP1 and HSPA1A expression may be an effective method for the treatment of HNSCC.     

Increased long noncoding RNA LASP1-AS is critical for hepatocellular carcinoma tumorigenesis via upregulating LASP1

Aberrant long noncoding RNAs (lncRNA) have been proved to be associated with the many types of malignant tumors (including hepatocellular carcinoma [HCC]). In this study, a lncRNAs and mRNAs microarray analysis was performed in three pairs of HCC patitents’ tumor. We found lncRNA LIM and SH3 protein 1 antisense (LASP1-AS) and its sense-cognate gene LIM and SH3 protein 1 (LASP1) were upregulated in HCC and both are correlated with poorer prognosis and lower survival of HCC patients. Meanwhile, the expression of LASP1-AS correlated positively with LASP1 expression in HCC tissues. LASP1-AS promoted the proliferation, migration, and invasion abilities of HCC in vitro and vivo by enhancing LASP1 expression. Our study explored lncRNA LASP1-AS as an oncogene in HCC and promoted proliferation and metastasis capabilities of HCC via increasing the expression of its sense-cognate gene LASP1. LncRNA LASP1-AS might be a potential valuable prognostic biomarker and potential therapeutic target of HCC.     

Knockout of LASP1 in CXCR4 expressing CML cells promotes cell persistence,proliferation and TKI resistance

Chronic myeloid leukaemia (CML) is a clonal myeloproliferative stem cell disorder characterized by the constitutively active BCR-ABL tyrosine kinase. The LIM and SH3 domain protein 1 (LASP1) has recently been identified as a novel BCR-ABL substrate and is associated with proliferation, migration, tumorigenesis and chemoresistance in several cancers. Furthermore, LASP1 was shown to bind to the chemokine receptor 4 (CXCR4), thought to be involved in mechanisms of relapse. In order to identify potential LASP1-mediated pathways and related factors that may help to further eradicate minimal residual disease (MRD), the effect of LASP1 on processes involved in progression and maintenance of CML was investigated. The present data indicate that not only overexpression of CXCR4, but also knockout of LASP1 contributes to proliferation, reduced apoptosis and migration as well as increased adhesive potential of K562 CML cells. Furthermore, LASP1 depletion in K562 CML cells leads to decreased cytokine release and reduced NK cell-mediated cytotoxicity towards CML cells. Taken together, these results indicate that in CML, reduced levels of LASP1 alone and in combination with high CXCR4 expression may contribute to TKI resistance.     

Inhibition of KHSRP sensitizes colorectal cancer to 5-fluoruracil through miR-501-5p-mediated ERRFI1 mRNA degradation

K-homology (KH)-type splicing regulatory protein (KHSRP) is an RNA binding protein that participates in RNA variable splicing and stability, and facilitates the biogenesis of miRNAs that target mRNA. However, to date, the role of KHSRP in colorectal cancer (CRC) progression has not been reported. In this study, the function of KHSRP in CRC proliferation and 5-fluoruracil (5-FU) resistance was investigated. The upregulation of KHSRP expression was confirmed in CRC patient tissues and two CRC cell lines. Manipulating KHSRP expression altered cell proliferation and 5-FU resistance in CRC cells. ERRFI1, a downstream effector of KHSRP in CRC cells, reduced CRC cell proliferation. Sensitivity to 5-FU mediated by KHSRP knockdown was reversed by ERRFI1 knockdown. We found that KHSRP decreased ERRFI1 mRNA expression indirectly. By screening KHSRP-regulated miRNAs, we further found that miR-501-5p directly combines with KHSRP in CRC cells. Mechanistically, the results of a luciferase assay suggested that miR-501-5p directly binds to the ERRFI1 3′-untranslated region. Taken together, our data indicated that modification of ERRFI1 by KHSRP occurs through miR-501-5p, an essential mechanism driving CRC proliferation and 5-FU resistance. Insight into this mechanism may provide novel targets for overcoming drug resistance in CRC.     

ZNF746 plays cardinal roles on colorectal cancer (CRC) cell invasion and migration and regulates mitochondrial dynamics and morphological changes of CRC cells—Role of combined melatonin and 5-FU regimen

The underlying mechanism of colorectal cells developing into cancer cells has been extensively investigated, yet is still not fully delineated, resulting in the treatment of advanced colorectal cancer (CRC) remains regrettably an unmet need. Zinc Finger Protein 746/Parkin-interacting substrate (ZNF746/PARIS) has previously been identified to play a fundamental role on bladder cancer cell proliferation and metastasis that were effectively inhibited by melatonin (Mel). In this study, we utilized ex vivo/in vivo studies to verify whether the ZNF746 signaling was also crucial in CRC growth/invasion/migration. Tissue-bank specimens showed that the protein expression of ZNF746 was significantly increased in CRC than that of healthy colorectal tissues (p < 0.001). Additionally, in vitro study demonstrated that excessive expression of ZNF746 significantly inhibited mitochondrial activity via (1) interfering with the dynamic balance of mitochondrial fusion/fission and (2) inhibiting the protein expression of MFN1/MFN2/PGC1a (all p < 0.001). Furthermore, we identified that inhibition of ZNF746 protein expression significantly reduced the resistance of CRC cell lines to the anticancer drug of 5-FU (p < 0.001), whereas overexpression of ZNF746 significantly augmented resistance of CRC cells to 5-FU (all p < 0.001). Finally, using the cell culture method, we found that combined Mel and 5-FU was superior to merely one on promoting the CRC cell apoptosis (p < 0.001). Our results confirmed that ZNF746 signaling played a cardinal role of CRC cell proliferation/survival and combined Mel and 5-FU treatment attenuated the resistance of CRC cells to the drug mainly through suppressing this signaling.     

A Highlights from MBoC Selection: LIM and SH3 protein 1 localizes to the leading edge of protruding lamellipodia and regulates axon development

The actin cytoskeleton drives cell motility and is essential for neuronal development and function. LIM and SH3 protein 1 (LASP1) is a unique actin-binding protein that is expressed in a wide range of cells including neurons, but its roles in cellular motility and neuronal development are not well understood. We report that LASP1 is expressed in rat hippocampus early in development, and this expression is maintained through adulthood. High-resolution imaging reveals that LASP1 is selectively concentrated at the leading edge of lamellipodia in migrating cells and axonal growth cones. This local enrichment of LASP1 is dynamically associated with the protrusive activity of lamellipodia, depends on the barbed ends of actin filaments, and requires both the LIM domain and the nebulin repeats of LASP1. Knockdown of LASP1 in cultured rat hippocampal neurons results in a substantial reduction in axonal outgrowth and arborization. Finally, loss of the Drosophila homologue Lasp from a subset of commissural neurons in the developing ventral nerve cord produces defasciculated axon bundles that do not reach their targets. Together, our data support a novel role for LASP1 in actin-based lamellipodial protrusion and establish LASP1 as a positive regulator of both in vitro and in vivo axon development.     

LASP1, a Newly Identified Melanocytic Protein with a Possible Role in Melanin Release,but Not in Melanoma Progression

The LIM and SH3 protein 1 (LASP1) is a focal adhesion protein. Its expression is increased in many malignant tumors. However, little is known about the physiological role of the protein. In the present study, we investigated the expression and function of LASP1 in normal skin, melanocytic nevi and malignant melanoma. In normal skin, a distinct LASP1 expression is visible only in the basal epidermal layer while in nevi LASP1 protein is detected in all melanocytes. Melanoma exhibit no increase in LASP1 mRNA compared to normal skin. In melanocytes, the protein is bound to dynamin and mainly localized at late melanosomes along the edges and at the tips of the cell. Knockdown of LASP1 results in increased melanin concentration in the cells. Collectively, we identified LASP1 as a hitherto unknown protein in melanocytes and as novel partner of dynamin in the physiological process of membrane constriction and melanosome vesicle release.     

TUFT1 Facilitates Metastasis,Stemness, and Vincristine Resistance in Colorectal Cancer via Activation of PI3K/AKT Pathway

Since the incidence and mortality of colorectal cancer (CRC) are increasing in recent years, the research on the pathogenesis of colorectal cancer has attracted more and more attention. Here, our results confirmed that the mRNA expression level and proteins accumulation of TUFT1 were significantly increased in CRC tissues from late-stage CRC patients (III?+?IV) (p?<?0.001), indicated by qPCR and IHC assay. The TUFT1 expression was positively correlated with tumor stage by analyzing 126 specimens from CRC patients. Next, we found that up-regulation of TUFT1 enhanced the migration and invasion of LoVo cells, whereas the down-regulation of TUFT1 observably weakened the migration and invasion of SW837 cells, indicating that TUFT1 promotes the metastasis of CRC cells. In addition, TUFT1 overexpression increased the number of mammary spheres and vincristine resistance of LoVo cells by sphere formation assay and measuring the IC50 value, suggesting the TUFT1 promotes stemness and the vincristine resistance of CRC cells. Finally, we found that TUFT1 overexpression increased p-AKT in LoVo cells, while down-regulation of TUFT1 decreased the p-AKT levels in SW837 cells. Therefore, we determined that the function of TUFT1 in CRC depends on PI3K/AKT pathway. Taken together, these data demonstrated that TUFI1 facilitates metastasis, stemness, and vincristine resistance of colorectal cancer cells via activation of PI3K/AKT pathway, which might act as a promising therapeutic target for CRC.

     

LncRNA HAND2-AS1 inhibits 5-fluorouracil resistance by modulating miR-20a/PDCD4 axis in colorectal cancer

BackgroundChemoresistance is one of the main obstacles in the therapy of human cancers, including colorectal cancer (CRC). Long non-coding RNA heart and neural crest derivatives expressed 2-antisense RNA 1 (lncRNA HAND2-AS1) has been demonstrated to be associated with CRC. However, the function of HAND2-AS1 in 5-Fluorouracil (5-FU) resistance of CRC remains unclear.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression of HAND2-AS1, miR-20a and programmed cell death factor 4 (PDCD4) mRNA. 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay was conducted to evaluate IC50 of 5-FU and cell proliferation. Flow cytometry analysis was used to determine cell apoptosis. Transwell assay was carried out to measure cell migration and invasion. Western blot assay was conducted to examine the protein levels of B-cell lymphoma-2 (Bcl-2), BCL2-Associated X (Bax), matrix metalloprotein 2 (MMP2), MMP9 and PDCD4. Dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay and RNA pull down assay were utilized to verify the combination between miR-20a and HAND2-AS1. Dual-luciferase reporter assay was used to analyze the association between miR-20a and PDCD4. Murine xenograft assay was used to confirm the function of HAND2-AS1 in vivo.ResultsHAND2-AS1 and PDCD4 were downregulated and miR-20a was upregulated in 5-FU-resistant CRC tissues and cells. HAND2-AS1 suppressed 5-FU resistance, cell proliferation, migration and invasion and promoted cell apoptosis in 5-FU-resistant CRC cells. HAND2-AS1 acted as a sponge of miR-20a to regulate PDCD4 expression. Moreover, HAND2-AS1 suppressed cell progression and 5-FU resistance by upregulating PDCD4 via sponging miR-20a in 5-FU-resistant CRC cells. Besides, HAND2-AS1 inhibited tumor growth in vivo.ConclusionHAND2-AS1/miR-20a/PDCD4 axis inhibited cell progression and 5-FU resistance in 5-FU-resistant CRC cells.     

A role for ceramide,but not diacylglycerol,in the antagonism of insulin signal transduction by saturated fatty acids

Multiple studies suggest that lipid oversupply to skeletal muscle contributes to the development of insulin resistance, perhaps by promoting the accumulation of lipid metabolites capable of inhibiting signal transduction. Herein we demonstrate that exposing muscle cells to particular saturated free fatty acids (FFAs), but not mono-unsaturated FFAs, inhibits insulin stimulation of Akt/protein kinase B, a serine/threonine kinase that is a central mediator of insulin-stimulated anabolic metabolism. These saturated FFAs concomitantly induced the accumulation of ceramide and diacylglycerol, two products of fatty acyl-CoA that have been shown to accumulate in insulin-resistant tissues and to inhibit early steps in insulin signaling. Preventing de novo ceramide synthesis negated the antagonistic effect of saturated FFAs toward Akt/protein kinase B. Moreover, inducing ceramide buildup recapitulated and augmented the inhibitory effect of saturated FFAs. By contrast, diacylglycerol proved dispensable for these FFA effects. Collectively these results identify ceramide as a necessary and sufficient intermediate linking saturated fats to the inhibition of insulin signaling.     

RFC2, a direct target of miR-744, modulates the cell cycle and promotes the proliferation of CRC cells

Colorectal cancer (CRC) is a common digestive tract malignancy, which is characterized by high mortality, morbidity, and poor prognosis. Replication factor C subunit 2 (RFC2), one RFC family member, was reported to be related to various malignancies and plays an important role in proliferation, invasion, and metastasis. Nonetheless, the RFC2 biological role within CRC is still unknown. RFC2 expression profiles in CRC tissues were collected based on The Cancer Genome Atlas database, whereas miR-744 and RFC2 expression levels were detected in human CRC tissues. miR-744 and RFC2 effects on the proliferation of CRC were assessed both in vivo and in vitro. RFC2 was recognized to be a direct miR-744 target through luciferase reporter assay. RFC2 upregulation was observed within CRC tissues, and a high RFC2 level showed a correlation with poor clinicopathological symptoms. RFC2 knockdown inhibited CRC cell proliferation through promoting cell cycle arrest at the G1 phase, which was achieved by cyclin E2 (CCNE2) downregulation in vivo and in vitro. miR-744 was identified to be the tumor suppressor microRNA, which targeted RFC2 directly for inhibiting the proliferation of CRC cells both in vivo and in vitro. miR-744 downregulation was detected within CRC tissue, and messenger RNA expression showed a negative correlation with RFC2 expression within CRC tissues. Our study demonstrates that the miR-744/RFC2/CCNE2 axis potentially provides a candidate for a treatment strategy for CRC.     

Actin binding of human LIM and SH3 protein is regulated by cGMP- and cAMP-dependent protein kinase phosphorylation on serine 146

Various drugs that elevate cGMP levels and activate cGMP-dependent protein kinase (cGK) inhibit agonist-induced platelet activation. In the present study we identified the LIM and SH3 domain protein (LASP) that was recently cloned from human breast cancer cells (Tomasetto, C., Regnier, C., Moog-Lutz, C., Mattei, M. G., Chenard, M. P., Liderau, R., Basset, P., and Rio, M. C. (1995) Genomics 28, 367-376) as a novel substrate of cGK in human platelets. Recombinant human LASP was phosphorylated by cGMP- and cAMP-dependent protein kinase (cAK) in vitro. Cotransfection of PtK-2 cells with LASP and cGK confirmed phosphorylation of LASP in vivo. Studies with human LASP mutants identified serine 146 as a specific phosphorylation site for cGK and cAK in vivo. LASP is an actin-binding protein, and the phospho-LASP-mimicking mutant S146D showed reduced binding affinity for F-actin in cosedimentation experiments. Immunofluorescence of transfected PtK2 cells demonstrated the localization of LASP in the tips of cell membrane extensions and at cell-cell contacts. Expression of the human LASP mutant S146D resulted in nearly complete relocalization to the cytosol and reduced migration of the cells. Taken together, these data suggest that phosphorylation of LASP by cGK and cAK may be involved in cytoskeletal organization and cell motility.     

Aberrant expression of lncRNAs SNHG6, TRPM2-AS1, MIR4435-2HG,and hypomethylation of TRPM2-AS1 promoter in colorectal cancer

Accumulating evidence has indicated that deregulation of lncRNAs plays essential roles in colorectal cancer (CRC) carcinogenesis. The goal of this study was to analyze the expression of lncRNAs in colorectal cancer and their association with clinicopathological variables. Bioinformatics analysis of published CRC microarray data was performed to identify the important lncRNAs. The expression levels of candidate genes were assessed in the human colon cancer/normal cell lines, CRC, adenomatous colorectal polyps, and their marginal tissues by qRT-PCR. Moreover, the methylation status of the TRPM2-AS1 promoter was studied using qMSP assay. Furthermore, we investigated the molecular mechanisms of these lncRNAs in CRC progression using in silico analysis. Microarray analysis revealed that lncRNAs SNHG6, MIR4435-2HG, and TRPM2-AS1 were upregulated in CRC. These results were validated in colon cell lines. Moreover, qRT-PCR showed that the expression levels of SNHG6 and TRPM2-AS1 were upregulated in the colorectal tumor tissues compared with their paired tissues. Nonetheless, there was no significant increase in MIR4435-2HG expression in CRC samples. Furthermore, we observed a significant hypomethylation of TRPM2-AS1 promoter and its activation in CRC tissues. By in silico analysis, we found that the lncRNAs upregulation could promote proliferation and drug resistance of colorectal cancer cells via miRNAs sponging and modulation of their targets expression. In conclusion, based on our results upregulation of SNHG6 and TRPM2-AS1, and hypomethylation of TRPM2-AS1 promoter might be considered as potential diagnostic biomarkers for CRC initiation and development.     

SLC25A1 promotes tumor growth and survival by reprogramming energy metabolism in colorectal cancer

Abnormal lipid metabolism has been commonly observed in various human cancers, including colorectal cancer (CRC). The mitochondrial citrate carrier SLC25A1 (also known as mitochondrial citrate/isocitrate carrier, CIC), has been shown to play an important role in lipid metabolism regulation. Our bioinformatics analysis indicated that SLC25A1 was markedly upregulated in CRC. However, the role of SLC25A1 in the pathogenesis and aberrant lipid metabolism in CRC remain unexplored. Here, we found that SLC25A1 expression was significantly increased in tumor samples of CRC as compared with paired normal samples, which is associated with poor survival in patients with CRC. Knockdown of SLC25A1 significantly inhibited the growth of CRC cells by suppressing the progression of the G1/S cell cycle and inducing cell apoptosis both in vitro and in vivo, whereas SLC25A1 overexpression suppressed the malignant phenotype. Additionally, we demonstrated that SLC25A1 reprogrammed energy metabolism to promote CRC progression through two mechanisms. Under normal conditions, SLC25A1 increased de novo lipid synthesis to promote CRC growth. During metabolic stress, SLC25A1 increased oxidative phosphorylation (OXPHOS) to protect protects CRC cells from energy stress-induced cell apoptosis. Collectively, SLC25A1 plays a pivotal role in the promotion of CRC growth and survival by reprogramming energy metabolism. It could be exploited as a novel diagnostic marker and therapeutic target in CRC.Subject terms: Colon cancer, Colon cancer     

Hsa_circ_0079662 induces the resistance mechanism of the chemotherapy drug oxaliplatin through the TNF‐α pathway in human colon cancer

The aim of the study was to research the biological functions of circRNA (hsa_circ_0079662) and its underlying mechanism in colorectal cancer. Drug‐resistant cell lines (HT29‐LOHP, HCT116‐LOHP, HCT8‐LOHP) were separately dealt with oxaliplatin concentration gradient (0.1‐10 μmol/L). Real‐time PCR, Western blotting, dual‐luciferase assay, miRNA pull‐down assay, coimmunoprecipitation and ELASA were performed to explore the mechanism of chemotherapy drug oxaliplatin resistance in CRC. The results showed that the expression of hsa_circ_0079662 was increased in drug‐resistant cell lines by RT‐PCR. The expression of HOXA9, TRIP6, Vcam‐1, VEGFC, MMP3, MMP9 and MMP14 was higher by Western blotting. Interaction between HOXA9 and TRIP6 in CO‐IP detection. Additionally, the cytokines TNF‐α, IL‐1 and IL‐6 were also found. In conclusion, hsa_circ_0079662, as a ceRNA binding with hsa‐mir‐324‐5p, can regulate target gene HOXA9 and induced the mechanism of chemotherapy drug oxaliplatin resistance in CRC through the TNF‐α pathway in human colon cancer.     

Lactosylceramide contributes to mitochondrial dysfunction in diabetes

Sphingolipids have been implicated as key mediators of cell-stress responses and effectors of mitochondrial function. To investigate potential mechanisms underlying mitochondrial dysfunction, an important contributor to diabetic cardiomyopathy, we examined alterations of cardiac sphingolipid metabolism in a mouse with streptozotocin-induced type 1 diabetes. Diabetes increased expression of desaturase 1, (dihydro)ceramide synthase (CerS)2, serine palmitoyl transferase 1, and the rate of ceramide formation by mitochondria-resident CerSs, indicating an activation of ceramide biosynthesis. However, the lack of an increase in mitochondrial ceramide suggests concomitant upregulation of ceramide-metabolizing pathways. Elevated levels of lactosylceramide, one of the initial products in the formation of glycosphingolipids were accompanied with decreased respiration and calcium retention capacity (CRC) in mitochondria from diabetic heart tissue. In baseline mitochondria, lactosylceramide potently suppressed state 3 respiration and decreased CRC, suggesting lactosylceramide as the primary sphingolipid responsible for mitochondrial defects in diabetic hearts. Moreover, knocking down the neutral ceramidase (NCDase) resulted in an increase in lactosylceramide level, suggesting a crosstalk between glucosylceramide synthase- and NCDase-mediated ceramide utilization pathways. These data suggest the glycosphingolipid pathway of ceramide metabolism as a promising target to correct mitochondrial abnormalities associated with type 1 diabetes.