Spry2 Expression Research Articles

Expression and function of microRNA-27b in hepatocellular carcinoma.

It has been reported that microRNAs (miRs) have key roles in tumorigenesis via inhibition of their target genes. Dysregulation of miR‑27b has been detected in numerous types of human cancer, including hepatocellular carcinoma (HCC); however, the detailed role of miR‑27b in HCC has yet to be elucidated. Reverse transcription‑quantitative polymerase chain reaction and western blotting were performed to examine the mRNA and protein expression levels. Transwell assay and wound healing assay were used to determine cell invasion and migration. Luciferase reporter assay was to confirm the targeting relationship. The present study demonstrated that the expression levels of miR‑27b were significantly increased in HCC cell lines, as compared with in normal human liver cells. In addition, miR‑27b was frequently upregulated in HCC tissues, as compared with in normal adjacent tissues; the expression levels of miR‑27b were increased in 77.1% (27/35) of the HCC tissue samples. Furthermore, elevated miR‑27b expression levels were significantly correlated with tumor differentiation, Tumor Node Metastasis stage and vascular invasion (P<0.05). Knockdown of miR‑27b expression inhibited HCC cell migration and invasion. Furthermore, Sprouty 2 (Spry2) was identified as a novel target of miR‑27b in HCC HepG2 cells, and the protein expression levels of Spry2 were negatively regulated by miR‑27b in HepG2 cells. Overexpression of Spry2 suppressed HCC cell migration and invasion, whereas downregulation of Spry2 reversed the suppressive effects of miR‑27b inhibition on HCC cell migration and invasion. The results of the present study suggested that miR‑27b may promote the migration and invasion of HCC cells, at least partially by suppressing Spry2 expression. Therefore, the miR‑27b/Spry2 axis may be considered a potential therapeutic target for HCC.

Sprouty4 mediates amphiregulin-induced down-regulation of E-cadherin and cell invasion in human ovarian cancer cells.

Sprouty (SPRY) proteins are well-characterized factors that inhibit receptor tyrosine kinase (RTK)-mediated activation of cellular signaling pathways. The down-regulation of SPRY4 expression has been reported in human ovarian cancer. However, the specific roles and mechanisms by which SPRY4 affects ovarian cancer progression are completely unknown. Amphiregulin (AREG) binds exclusively to the epidermal growth factor receptor (EGFR) and has been considered to be a dominant autocrine/paracrine EGFR ligand in ovarian cancer. In the present study, we first examined the effects of AREG on SPRY4 expression and the possible underlying molecular mechanisms involved in this process in two human ovarian cancer cell lines. Our results demonstrated that treatment with AREG up-regulated SPRY4 expression by activating the ERK1/2 signaling pathway. In addition, we showed that small interfering RNA (siRNA)-mediated knockdown of SPRY4 attenuated the AREG-induced down-regulation of E-cadherin by inhibiting the expression of SNAIL but not SLUG. In contrast, overexpression of SPRY4 enhanced AREG-induced down-regulation of E-cadherin by increasing the expression of SNAIL. Moreover, SPRY4 knockdown attenuated AREG-induced cell migration and invasion. Overexpression of SPRY4 enhanced AREG-induced cell invasion. This study reveals that SPRY4 is involved in EGFR-mediated human ovarian cancer progression.

Mirna-21 Targets Sprouty2 to Activate SYK Mediated BCR and MAPK-Erk Signaling in Chronic Lymphocytic Leukemia

Abstract INTRODUCTION: Chronic Lymphocytic Leukemia (CLL) represents the most common adult leukemia in the western world. Several reports have demonstrated that the constitutively activated MAPK-Erk signaling pathways promote CLL cell proliferation and survival. However, the precise molecular mechanisms that lead to deregulated MAPK signaling in CLL initiation and progression are not fully understood. We have previously reported that Sprouty2 (Spry2) is a negative regulator of BCR and MAPK-Erk signaling in poor prognosis CLL (American Society of Hematology, 56th Annual Meeting, San Francisco, CA, 2014). Here in follow up studies, we set to further determine molecular basis deregulation of Spry2 in patients with poor prognosis CLL. Spry2 is either epigenetically silenced or targeted by Microrna-21 (miR21) in several malignancies. MiR-21 plays a role of oncomir in CLL, which is significantly upregulated in CLL patients with poor prognosis specifically with Del 17p, High 38 and Zap70 expression. Notably, we observed a decrease in Spry2 expression in CLL cells expressing high levels of miR-21. Also, it is already reported that miR21 is regulated by STAT3 transcription factor in CLL patients with poor prognosis in refractory phase. However the precise molecular mechanism by which the miR21 leads to leukemic progression in poor prognosis CLL is not known. Here we demonstrate the molecular mechanism of miR21 to activate Syk mediated BCR and MAPK-Erk signaling by targeting Spry2 in CLL cells from poor prognosis patients. METHODOLOGY: We isolated CLL cells from peripheral blood of newly diagnosed high CD38 poor prognosis patients. For molecular analysis, in addition to primary CLL cells we use Mec-1 CLL cell line. To measure the levels of miR21 we used Taqman assay qPCR from Invitrogen. To decrease miR21 levels we used miR21 inhibitor (Invitrogen) and STAT3 inhibitor (SantaCruz). To overexpress miR21 we used pcDNAmir-21 construct from Addgene. Activation of BCR and MAPK-Erk signaling pathway was measured by levels of p-Erk and p-Syk. Expression of Spry2, Erk and Syk was measured by western blot analysis. RESULTS: To study if miR21 targets Spry2 in CLL cells, we first overexpressed miR21 in Mec-1 cells. We observed a significant decrease in Spry2 expression in Mec-1 cells expressing high levels of miR-21 compared to empty vector control. In our previous studies we observed Spry2 acts as a negative regulator of BCR and MAPK-Erk signaling. Therefore, we next studied the activity of Spry2 targeting signaling pathways in miR21-overexpressing CLL cells. We observed elevated levels of p-Erk and p-Syk in CLL cells overexpressing miR21. For control we used pcDNA-empty vector. Whereas upon miR21 knockdown in CLL cells resulted in stabilization of Spry2 expression and decrease in BCR and MAPK-Erk signaling. Also, targeting miR21 by STAT3 inhibitor leads to induction of spontaneous apoptosis in CLL cells. We next tested the effect of STAT3 inhibitor on primary CLL cells from 13 different patients. We measured the viability of CLL cells using MTT assay, we observed higher efficacy of STAT3 inhibitor over Syk and BRAF inhibitors on CLL cells. CONCLUSION: We conclude that Spry2 is a target of miR21 in CLL cells from poor prognosis patients. MiR21 targets Spry2 to activate Syk mediated BCR and MAPK signaling in CLL patients. Also, STAT3 can be used as therapeutic target for poor prognosis CLL patients with high miR21 expression. Disclosures No relevant conflicts of interest to declare.

Cooperative and independent functions of FGF and Wnt signaling during early inner ear development.

BackgroundIn multiple vertebrate organisms, including chick, Xenopus, and zebrafish, Fibroblast Growth Factor (FGF) and Wnt signaling cooperate during formation of the otic placode. However, in the mouse, although FGF signaling induces Wnt8a expression during induction of the otic placode, it is unclear whether these two signaling pathways functionally cooperate. Sprouty (Spry) genes encode intracellular antagonists of receptor tyrosine kinase signaling, including FGF signaling. We previously demonstrated that the Sprouty1 (Spry1) and Sprouty2 (Spry2) genes antagonize FGF signaling during induction of the otic placode. Here, we investigate cross talk between FGF/SPRY and Wnt signaling during otic placode induction and assess whether these two signaling pathways functionally cooperate during early inner ear development in the mouse.MethodsEmbryos were generated carrying combinations of a Spry1 null allele, Spry2 null allele, β-catenin null allele, or a Wnt reporter transgene. Otic phenotypes were assessed by in situ hybridization, semi-quantitative reverse transcriptase PCR, immunohistochemistry, and morphometric analysis of sectioned tissue.ResultsComparison of Spry1, Spry2, and Wnt reporter expression in pre-otic and otic placode cells indicates that FGF signaling precedes and is active in more cells than Wnt signaling. We provide in vivo evidence that FGF signaling activates the Wnt signaling pathway upstream of TCF/Lef transcriptional activation. FGF regulation of Wnt signaling is functional, since early inner ear defects in Spry1 and Spry2 compound mutant embryos can be genetically rescued by reducing the activity of the Wnt signaling pathway. Interestingly, we find that although the entire otic placode increases in size in Spry1 and Spry2 compound mutant embryos, the size of the Wnt-reporter-positive domain does not increase to the same extent as the Wnt-reporter-negative domain.ConclusionsThis study provides genetic evidence that FGF and Wnt signaling cooperate during early inner ear development in the mouse. Furthermore, our data suggest that although specification of the otic placode may be globally regulated by FGF signaling, otic specification of cells in which both FGF and Wnt signaling are active may be more tightly regulated.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-015-0083-8) contains supplementary material, which is available to authorized users.

MT1-MMP dependent repression of the tumor suppressor SPRY4 contributes to MT1-MMP driven melanoma cell motility

Metastatic melanoma is the deadliest of all skin cancers. Despite progress in diagnostics and treatment of melanoma, the prognosis for metastatic patients remains poor. We previously showed that Membrane-type 1 Matrix Metalloproteinase (MT1-MMP) is one of the drivers of melanoma metastasis. Classically, MT1-MMP regulates a verity of cellular functions including cell-to-cell interaction and cell-to-matrix communication. Recently, MT1-MMP has been found to also modulate gene expression. To specifically assess MT1-MMP dependent gene regulation in melanoma, microarray gene expression analysis was performed in a melanoma cell line whose metastatic properties depend on the activity of MT1-MMP. We identified the tumor suppressor gene SPRY4 as a new transcriptional target of MT1-MMP that is negatively regulated by the protease. Knockdown of MT1-MMP enhances SPRY4 expression at the mRNA and protein level. SPRY4 expression inversely correlates with that of MT1-MMP in melanoma samples and importantly, correlates with melanoma patient survival. SPRY4 modulates MT1-MMP dependent cell migration such that inhibition of SPRY4 rescues cell migration that has been impaired by MT1-MMP knock down. MT1-MMP decreases SPRY4 in part through an MMP2/RAC1 axis we previously show promotes cell motility downstream of MT1-MMP. These results identify the tumor suppressor SPRY4 as a novel molecular effector of MT1-MMP affecting melanoma cell motility.

Induction of Resistance to BRAF Inhibitor Is Associated with the Inability of Spry2 to Inhibit BRAF-V600E Activity in BRAF Mutant Cells.

The clinical benefits of oncogenic BRAF inhibitor therapies are limited by the emergence of drug resistance. In this study, we investigated the role of a negative regulator of the MAPK pathway, Spry2, in acquired resistance using BRAF inhibitor-resistant derivatives of the BRAF-V600E melanoma (A375P/Mdr). Real-time RT-PCR analysis indicated that the expression of Spry2 was higher in A375P cells harboring the BRAF V600E mutation compared with wild-type BRAF-bearing cells (SK-MEL-2) that are resistant to BRAF inhibitors. This result suggests the ability of BRAF V600E to evade feedback suppression in cell lines with BRAF V600E mutations despite high Spry2 expression. Most interestingly, Spry2 exhibited strongly reduced expression in A375P/Mdr cells with acquired resistance to BRAF inhibitors. Furthermore, the overexpression of Spry2 partially restored sensitivity to the BRAF inhibitor PLX4720 in two BRAF inhibitor-resistant cells, indicating a positive role for Spry2 in the growth inhibition induced by BRAF inhibitors. On the other hand, long-term treatment with PLX4720 induced pERK reactivation following BRAF inhibition in A375P cells, indicating that negative feedback including Spry2 may be bypassed in BRAF mutant melanoma cells. In addition, the siRNA-mediated knockdown of Raf-1 attenuated the rebound activation of ERK stimulated by PLX4720 in A375P cells, strongly suggesting the positive role of Raf-1 kinase in ERK activation in response to BRAF inhibition. Taken together, these data suggest that RAF signaling may be released from negative feedback inhibition through interacting with Spry2, leading to ERK rebound and, consequently, the induction of acquired resistance to BRAF inhibitors.

The inability of Spry2 to inhibit B‐Raf V600E activity is required for induction of resistance to B‐Raf inhibitor in B‐Raf V600E cells

B-Raf is the most frequently mutated protein kinase in human cancer. Thus, inhibition of B-Raf kinase activity could benefit cancer patients. However, the clinical benefit of RAF inhibitor therapies is limited by the emergence of drug resistance. In this study, expression of Spry2, negative regulators of the MAPK pathway, was higher in A375P cells harboring the B-Raf V600E mutation compared to wild-type B-Raf-bearing cells (SK-MEL-2) that is resistant to B-Raf inhibitor. In particular, Spry2 had strongly reduced expression in A375P/Mdr cells with acquired resistance to B-Raf inhibitor. Furthermore, overexpression of Spry2 partially restored sensitivity to B-Raf inhibitor PLX4720 in two B-Raf inhibitor-resistant cells, indicating a positive role for Spry2 in the growth inhibition induced by B-Raf inhibitor. However, Spry2 overexpression in A375P/Mdr cells resulted in no significant change in the levels of pERK or pMEK.On the other hand, when Spry2 is silenced, the A375P cells were significantly more sensitive to B-Raf inhibitor. Moreover, long-term treatment of PLX4720 induced pERK reactivation following B-Raf inhibition in A375P cells, indicating that Spry2 might be bypassed in B-Raf mutant melanoma cells by the presence of B-Raf mutation. Taken together, these data suggest that induction of resistance to B-Raf inhibitor in B-Raf V600E cells can be attributed to the inability of Spry2 to inhibit B-Raf V600E activity. Grant Funding Source: Supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2013R1A1A2A10058897)

P3.04 - Sprouty 2 protein, but not Sprouty 4, is an independent prognostic biomarker for human epithelial ovarian cancer

Sprouty proteins are evolutionary-conserved modulators of receptor tyrosine kinase signaling, deregulation of which has been implicated in the pathophysiology of cancer. In the present study, the expression status of Spry2 and Spry4 proteins and its clinical relevance in human epithelial ovarian cancer (EOC) were investigated retrospectively. We examined the immunohistochemical expression of Spry2 and Spry4 in matched tumor and normal tissue samples from 99 patients. The expression of ERK, p-ERK, Ki67, fibroblast growth factor-2, vascular endothelial growth factor and interleukin-6 and their correlation with Sprouty homologs were also evaluated. Moreover, the correlation between Spry2 and Spry4 and the clinicopathological characteristics were analyzed along with their predictive value for overall survival (OS) and disease-free survival (DFS). Our data indicated significant downregulation of Spry2 and Spry4 in tumor tissues (p < 0.0001). A significant inverse correlation was evident between Spry2 and p-ERK/ERK (p = 0.048), Ki67 (p = 0.011), disease stage (p = 0.013), tumor grade (p = 0.003), recurrence (p < 0.001) and post-treatment ascites (p = 0.001), individually. It was found that Spry2 low-expressing patients had significantly poorer OS (p = 0.002) and DFS (p = 0.004) than those with high expression of Spry2. Multivariate analysis showed that high Spry2 (p = 0.018), low stage (p = 0.049) and no residual tumor (p =0.006) were independent prognostic factors for a better OS. With regard to DFS, high Spry2 (p = 0.044) and low stage (p = 0.046) remained as independent predictors. In conclusion, we report for the first time significant downregulation of Spry2 and Spry4 proteins in human EOC. Spry2 expression was revealed to significantly impact tumor behavior with predictive value as an independent prognostic factor for survival and recurrence.

Sprouty 2 protein, but not Sprouty 4, is an independent prognostic biomarker for human epithelial ovarian cancer.

Sprouty proteins are evolutionary-conserved modulators of receptor tyrosine kinase signaling, deregulation of which has been implicated in the pathophysiology of cancer. In the present study, the expression status of Spry2 and Spry4 proteins and its clinical relevance in human epithelial ovarian cancer (EOC) were investigated retrospectively. We examined the immunohistochemical expression of Spry2 and Spry4 in matched tumor and normal tissue samples from 99 patients. The expression of ERK, p-ERK, Ki67, fibroblast growth factor-2, vascular endothelial growth factor and interleukin-6 and their correlation with Sprouty homologs were also evaluated. Moreover, the correlation between Spry2 and Spry4 and the clinicopathological characteristics were analyzed along with their predictive value for overall survival (OS) and disease-free survival (DFS). Our data indicated significant downregulation of Spry2 and Spry4 in tumor tissues (p < 0.0001). A significant inverse correlation was evident between Spry2 and p-ERK/ERK (p = 0.048), Ki67 (p = 0.011), disease stage (p = 0.013), tumor grade (p = 0.003), recurrence (p < 0.001) and post-treatment ascites (p = 0.001), individually. It was found that Spry2 low-expressing patients had significantly poorer OS (p = 0.002) and DFS (p = 0.004) than those with high expression of Spry2. Multivariate analysis showed that high Spry2 (p = 0.018), low stage (p = 0.049) and no residual tumor (p =0.006) were independent prognostic factors for a better OS. With regard to DFS, high Spry2 (p = 0.044) and low stage (p = 0.046) remained as independent predictors. In conclusion, we report for the first time significant downregulation of Spry2 and Spry4 proteins in human EOC. Spry2 expression was revealed to significantly impact tumor behavior with predictive value as an independent prognostic factor for survival and recurrence.

Mutation of Spry2 induces proliferation and differentiation of osteoblasts but inhibits proliferation of gingival epithelial cells.

Sprouty was identified as an inhibitor of the fibroblast growth factor (FGF) receptor, and Sprouty2 (Spry2) functions as a negative regulator of receptor tyrosine kinase signaling. In this study, we investigated how inhibition of Spry2 affects osteoblasts and gingival epithelial cells in periodontal tissue regeneration in vitro. Transduction of a dominant-negative mutant of Spry2 (Y55A-Spry2) enhanced basic fibroblast growth factor (bFGF)- and epidermal growth factor (EGF)-induced ERK activation in MC3T3-E1 osteoblastic cells. In contrast, it decreased their activation in GE1 cells. Consistent with these observations, Y55A-Spry2 increased osteoblast proliferation with bFGF and EGF stimulation, whereas the proliferation of Y55A-Spry2-introduced GE1 cells was decreased via the ubiquitination and degradation of EGF receptors (EGFRs). In addition, Y55A-Spry2 caused upregulation of Runx2 expression and downregulation of Twist, a negative regulator of Runx2, with treatment of bFGF and EGF, resulting in enhanced osteoblastogenesis accompanied by alkaline phosphatase activation and osteocalcin expression in MC3T3-E1 cells. These data suggest that suppression of Spry2 expression induces proliferation and differentiation of osteoblastic cells after the addition of a bFGF and EGF cocktail but inhibits proliferation in gingival epithelial cells. These in vitro experiments may provide a molecular basis for novel therapeutic approaches in periodontal tissue regeneration. Taken together, our study proposes that combined application of an inhibitor for tyrosine 55 of Spry2, bFGF, and EGF may effectively allow alveolar bone growth and block the ingrowth of gingival epithelial cells toward bony defects, biologically mimicking a barrier effect in guided tissue regeneration, with in vivo investigation in the future.

Sprouty2 mediated tuning of signalling is essential for somite myogenesis.

BackgroundNegative regulators of signal transduction cascades play critical roles in controlling different aspects of normal embryonic development. Sprouty2 (Spry2) negatively regulates receptor tyrosine kinases (RTK) and FGF signalling and is important in differentiation, cell migration and proliferation. In vertebrate embryos, Spry2 is expressed in paraxial mesoderm and in forming somites. Expression is maintained in the myotome until late stages of somite differentiation. However, its role and mode of action during somite myogenesis is still unclear.ResultsHere, we analysed chick Spry2 expression and showed that it overlaps with that of myogenic regulatory factors MyoD and Mgn. Targeted mis-expression of Spry2 led to inhibition of myogenesis, whilst its C-terminal domain led to an increased number of myogenic cells by stimulating cell proliferation.ConclusionsSpry2 is expressed in somite myotomes and its expression overlaps with myogenic regulatory factors. Overexpression and dominant-negative interference showed that Spry2 plays a crucial role in regulating chick myogenesis by fine tuning of FGF signaling through a negative feedback loop. We also propose that mir-23, mir-27 and mir-128 could be part of the negative feedback loop mechanism. Our analysis is the first to shed some light on in vivo Spry2 function during chick somite myogenesis.

MicroRNA-27b inhibits Spry2 expression and promotes cell invasion in glioma U251 cells.

MicroRNA (miR)-27b has been reported to participate in glioma. However, a detailed role of miR-27b and the underlying mechanism remain largely unknown. The present study found that the expression of miR-27b was significantly increased in glioma tissues compared with normal adjacent tissues. In addition, miR-27b was also upregulated in the U87, U251 and SHG44 glioma cell lines compared with normal human astrocytes. Sprouty homolog 2 (Spry2), which has been reported to be associated with invasive glioma, was identified as a novel target of miR-27b in U251 glioma cells, and the protein expression of Spry2 was negatively regulated by miR-27b in U251 cells. Additionally, inhibition of miR-27b and upregulation of Spry2 suppressed glioma cell invasion, while downregulation of Spry2 reversed the suppressive effect of miR-27b inhibition on glioma cell invasion. These data suggest that miR-27b may promote glioma cell invasion through direct inhibition of Spry2 expression. The data also suggest that miR-27b may become a promising molecular target for inhibiting the invasion and metastasis of glioma.

Role of Sprouty2 in the Pathogenesis of Chronic Lymphocytic Leukemia

INTRODUCTION: Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in adults representing about 35% of all adult leukemias in the United States. CLL is a B-cell neoplasm which is characterized by accumulation of long lived mature B-cells in peripheral blood (PB), bone marrow (BM) and lymph nodes (LN). We and others have reported that CLL cells in tissue microenvironments have constitutively activated MAPK-Erk and AKT signaling pathways, which promotes their proliferation and survival. However, the molecular mechanisms and the gene(s) responsible for the activation of these pathways in CLL are not clearly understood. We performed gene expression profiling of CLL cells from PB, BM and LN and transcriptome analyses of CLL cells from PB from good and poor prognosis patients. Our studies identified Sprouty 2 (Spry2), a negative feedback regulator as one of the key genes involved in the regulation of MAPK-Erk and Akt pathways in CLL. We observed that Spry2 levels were down-regulated in poor prognosis CLL cells. Interestingly, Spry2 is epigenetically silenced in most human and mouse lymphomas and ectopic expression of Spry2 induces apoptosis in mouse B cells. We categorized CLL patients into good and poor prognosis to further evaluate Spry2 in CLL biology. Poor prognosis patients were defined by unmutated immunoglobulin variable heavy chain (IgVH), 11q22 deletion, 17p deletion, trisomy 12 or high CD38 and Zap70 expression. Good prognosis patients were defined as having mutated IgVH, 13q14 deletions or low CD38 and Zap70 expression.RESULTS: We found that Spry2 expression was significantly decreased by 3.2 log2-fold, (p=0.0001) at transcript and protein levels, respectively, in CLL cells from patients with poor prognosis compared to patients with good prognosis. Spry2 is a negative feedback regulator which attenuates cell proliferation, migration and survival. To determine the effect of Spry2 on CLL cells we transfected Mec-1 cells (stable human CLL cell line) and primary human CLL cells with a Spry2 cDNA containing vector. Ectopic expression of Spry2 induced spontaneous apoptosis in Mec-1 cells and primary CLL cells from poor prognosis patients. These results indicate that Spry2 negatively regulates the survival of human CLL cells. CLL cells have been shown to have active B-cell receptor (BCR) signaling promoting proliferation in vivo. To study the effect of BCR stimulation on Spry2 we stimulated BCR on Mec-1 and primary CLL cells by BCR cross-linking. Unexpectedly, the rate of proliferation of Mec-1 cells decreased upon BCR stimulation. We were able to demonstrate a transient increase in Spry2 levels that peaked at 12 hours post BCR stimulation, which we suspected was mediating the decrease in proliferation observed. We therefore repeated the BCR stimulation experiment using siRNA to inhibit the transient increase in Spry2 in primary CLL cells from seven good prognosis patients and in the Mec-1 cells. As predicted, we observed a significant increase in proliferation in all primary CLL samples and in the Mec-1 cells upon Spry2 knock down following BCR stimulation. Spry2 attenuates ligand induced MAPK-Erk and AKT signaling leading to suppression of proliferation and survival. We also examined the effect of Spry2 on MAPK-Erk and Akt signaling in CLL cells. Upon induction of exogenous Spry2 expression in Mec-1 cells there was a significant decrease in p-Akt and p-Erk levels. Conversely, Spry2 knock down resulted in an increase in p-Erk and p-Akt and enhanced cell proliferation.CONCLUSIONS: Spry2 is significantly down-regulated in the B-cells from patients with poor prognosis CLL. Down-regulation of Spry2 in CLL cells leads to hyperactivation of MAPK-Erk and Akt signaling and exogenous and endogenous expression of Spry2 leads to spontaneous apoptosis in human CLL cells. These results identify Spry2 as a negative regulator of CLL cell survival and proliferation and provide a potential molecular target for therapeutic intervention. DisclosuresBociek:Seattle Genetics, Inc.: Research Funding.

562 Lysophosphatidic acid-induced breast cancer metastasis depends on LPA1/ZEB1/miR-21-activation pathway

Lysophosphatidic acid (LPA) is a natural bioactive lipid that promotes metastasis of many types of cancer cells. We have shown that blocking LPA receptor type 1 (LPA1) activity inhibits early stage of bone metastasis by inhibiting motility and invasion of breast cancer cells. However, the signaling pathways and gene activation involved in this process have not been well characterized. Micro-RNAs (miR) are well known master regulators of gene expression. Based on the complete miR expression profile in human MDAMB-231 breast cells stimulated by LPA, we found that miR-21 was one of the highest up-regulated miR. Using the Taqman RT-QPCR system, we found that LPA induced miR-21 expression in MDA-MB-231 cells and in their highly osteotropic sub-clone MDA-B02 cells. MiR-21 is well known to act as an oncomiR promoting metastasis in multiple cancers. Also, it is known that miR-21 expression is controlled by several transcription factors. The full transcriptomic analysis of our breast cancer cell lines showed that among those transcription factors, ZEB1, STAT3 and cFos were up regulated by LPA. Interestingly, silencing ZEB1 expression in these cells using synthetic ZEB1-siRNAs abolished LPA-induced miR-21 expression whereas silencing STAT3 or cFos had no effect. Hence, silencing ZEB1 up-regulated the expression of miR-21 target genes PDCD4, PTEN and SPRY2 in MDA-MB-231 cells stimulated by LPA. RT-QPCR analyses also showed that LPA1 was the most abundant LPA receptor in MDA-MB-231 cells (LPA1>LPA2>>LPA6=LPA7>LPA5) whereas LPA3 and LPA4 were not detectable. We found that the treatment of our breast cancer cells with Ki16425, a LPA1/LPA3 antagonist, inhibited LPA-induced ZEB1 and miR-21 expression. Silencing LPA1 expression in these cells using synthetic LPA1siRNAs also abolished LPA-induced ZEB1 and miR-21 expression. MirVana miR-21 inhibitor, silencing LPA1 or ZEB1 totally blocked in vitro LPAinduced cell migration and invasion, and in vivo tumor bone colonization. In all cases the in vitro and in vivo breast cancer functions were rescued with miR-21 mimic. All together our results demonstrate that miR-21 controls the pro-metastatic activity of LPA involving LPA1-dependent activation of ZEB1 in breast cancer cells.

MiR-21 simultaneously regulates ERK1 signaling in HSC activation and hepatocyte EMT in hepatic fibrosis.

BackgroundMicroRNA-21 (miR-21) plays an important role in the pathogenesis and progression of liver fibrosis. Here, we determined the serum and hepatic content of miR-21 in patients with liver cirrhosis and rats with dimethylnitrosamine-induced hepatic cirrhosis and examined the effects of miR-21 on SPRY2 and HNF4α in modulating ERK1 signaling in hepatic stellate cells (HSCs) and epithelial-mesenchymal transition (EMT) of hepatocytes.MethodsQuantitative RT-PCR was used to determine miR-21 and the expression of SPRY2, HNF4α and other genes. Immunoblotting assay was carried out to examine the expression of relevant proteins. Luciferase reporter assay was performed to assess the effects of miR-21 on its predicted target genes SPRY2 and HNF4α. Primary HSCs and hepatocytes were treated with miR-21 mimics/inhibitors or appropriate adenoviral vectors to examine the relation between miR-21 and SPRY2 or HNF4α. ResultsThe serum and hepatic content of miR-21 was significantly higher in cirrhotic patients and rats. SPRY2 and HNF4α mRNA levels were markedly lower in the cirrhotic liver. MiR-21 overexpression was associated with enhanced ERK1 signaling and EMT in liver fibrosis. Luciferase assay revealed suppressed SPRY2 and HNF4α expression by miR-21. Ectopic miR-21 stimulated ERK1 signaling in HSCs and induced hepatocyte EMT by targeting SPRY2 or HNF4α. Downregulating miR-21 suppressed ERK1 signaling, inhibited HSC activation, and blocked EMT in TGFβ1-treated hepatocytes.ConclusionsMiR-21 modulates ERK1 signaling and EMT in liver fibrosis by regulating SPRY2 and HNF4α expression. MiR-21 may serve as a potentially biomarker as well as intervention target for hepatic cirrhosis.

Abstract 1448: Disseminated tumor cell formation promoted by lysophosphatidic acid (LPA) involves ZEB1/miR21-dependent activation pathway

Abstract Lysophosphatidic acid (LPA) is a natural bioactive lipid that promotes metastasis of many types cancer cells. We have shown that blocking LPA receptor type 1 (LPA1) activity inhibits early stage of bone metastasis by inhibiting motility and invasion of breast cancer cells. However, the signaling pathways and gene activation involved in this process have not been well characterized. Micro-ARNs (miR) are well known master regulators of gene expression. Based on the complete miR expression profile in human MDA-MB-231 breast cells stimulated by LPA, we found that miR-21 was one of the most up regulated miR. Using the Taqman RT-QPCR system, we found that LPA induced a rapid increase in miR-21 expression in MDA-MB-231 cells and in their highly osteotropic sub-clone MDA-B02 cells reaching a plateau by two to three folds (EC50 = 0.1μM) after 45 min of stimulation. MiR-21 is well known to act as an oncomiR promoting metastasis in multiple cancers. Also, it is known that miR-21 expression is controlled by several transcription factors. The full transcriptomic analysis of our breast cancer cell lines showed that among those transcription factors, ZEB1, STAT3 and cFos were up regulated by LPA. Interestingly, silencing ZEB1 expression in these cells using synthetic ZEB1-siRNAs abolished LPA-induced miR-21 expression whereas silencing STAT3 or sFos had no effect. Hence, silencing ZEB1 up-regulated the expression of miR-21 target genes PDCD4, PTEN and SPRY2 in MDA-MB-231 cells stimulated by LPA. RT-QPCR analyses also showed that LPA1 was the most abundant LPA receptor in MDA-MB-231 cells (LPA1&amp;gt;LPA2&amp;gt;&amp;gt;LPA6=LPA7&amp;gt;LPA5) whereas LPA3 and LPA4 were not detectable. We found that the treatment of our breast cancer cells with Ki16425, a LPA1/LPA3 antagonist, inhibited LPA-induced miR-21 expression. Silencing LPA1 expression in these cells using synthetic LPA1-siRNAs also abolished LPA-induced miR-21 expression. We showed in animal that Debio0719, another LPA1/LPA3 antagonist, inhibits implantation of disseminated tumor cells (DTC) into the bone marrow. Here, we found that the pretreatment of MDA-B02 cells with a specific blocker of miR-21 (mirVana miR-21 inhibitor) before injection of these cells in BALB/c nude mice decreased the incidence of DTC-positive animals and decreased the number DTCs at the metastatic sites. All together our results demonstrate that miR-21 controls the prometastatic activity of LPA involving LPA1-dependent activation of ZEB1 in breast cancer cells. Citation Format: Debashish Sahay, Raphaël Leblanc, Johnny Ribeiro, Philippe Clézardin, Olivier Peyruchaud. Disseminated tumor cell formation promoted by lysophosphatidic acid (LPA) involves ZEB1/miR21-dependent activation pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1448. doi:10.1158/1538-7445.AM2014-1448

The expression of the Sprouty 1 protein inversely correlates with growth, proliferation, migration and invasion of ovarian cancer cells

BackgroundOur recent study on a panel of human ovarian cancer cells revealed that SKOV-3 cells barely express the Sprouty isoform 1 (Spry1) while 1A9 cells maintain it at a level similar to normal ovarian cells. Here we investigated the functional outcomes of induced alterations in the expression of Spry1 in the two cell lines in vitro.MethodsUsing the Spry1 specific plasmid and siRNA, the expression of Spry1 was induced and conversely silenced in SKOV-3 and 1A9 cells, respectively. The functional outcome was investigated by means of proliferation, MTT, scratch-wound, migration and invasion assays and selection of the stable clones. Mechanism of the effect was explored by Western blot.ResultsIn the Spry1-transfected SKOV-3 cells, a significant reduction in growth and proliferation was evident. Stable clones of the Spry1-transfected SKOV-3 were almost undetectable after day 14. The number of migrated and invaded cells and the percentage of the scratch closure were significantly lower in the Spry1-transfected group. Spry1 silencing in 1A9 cells, on the other hand, led to a significant increase in cell growth and proliferation. The number of migrated and invaded cells and the percentage of the scratch closure significantly increased in Spry1-silenced 1A9 group. Mechanistically, overexpression of Bax, activation of caspases 3, 7, 8 and 9, cleavage of PARP and attenuation of Bcl-2 and Bcl-xl were observed along with reduced activation of Erk and Akt and increased amount and activity of PTEN in the Spry1-transfected SKOV-3 cells.ConclusionsHere, we report the inverse correlation between the expression of Spry1 and growth, proliferation, invasion and migration of ovarian cancer cells.

SPRY4-mediated ERK1/2 signaling inhibition abolishes 17β-estradiol-induced cell growth in endometrial adenocarcinoma cell

Objective: Basic fibroblast growth factor (FGF2)-mediated Extracellular signal-regulated kinases1/2 (ERK1/2) signaling is a critical modulator in angiogenesis. SPRY4 has been reported to be a feedback negative regulator of FGFs-induced ERK1/2 signaling. The aim of this study was to explore the role of SPRY4 in endometrial adenocarcinoma cell.Materials and methods: The effect of SPRY4 expression on FGF2-mediated ERK1/2 signaling was detected by luciferase assay and Western blot analysis. The growth of Ishikawa cells was detected using colony formation assay and cell number counting experiment.Results: We found that plasmid-driven SPRY4 expression efficiently blocked the activity of FGF2-induced ERK1/2 signaling in Ishikawa cells. SPRY4 expression significantly reduced the proliferation and 17β-estradiol-induced proliferation of Ishikawa cells.Conclusion: SPRY4 may function as a tumor suppressor in endometrial adenocarcinoma.

Modulation of Fibroblast Growth Factor Signaling Is Essential for Mammary Epithelial Morphogenesis

Fibroblast growth factor (FGF) signaling is essential for vertebrate organogenesis, including mammary gland development. The mechanism whereby FGF signaling is regulated in the mammary gland, however, has remained unknown. Using a combination of mouse genetics and 3D ex vivo models, we tested the hypothesis that Spry2 gene, which encodes an inhibitor of signaling via receptor tyrosine kinases (RTKs) in certain contexts, regulates FGF signaling during mammary branching. We found that Spry2 is expressed at various stages of the developing mammary gland. Targeted removal of Spry2 function from mammary epithelium leads to accelerated epithelial invasion. Spry2 is up-regulated by FGF signaling activities and its loss sensitizes mammary epithelium to FGF stimulation, as indicated by increased expression of FGF target genes and epithelia invasion. By contrast, Spry2 gain-of-function in the mammary epithelium results in reduced FGF signaling, epithelial invasion, and stunted branching. Furthermore, reduction of Spry2 expression is correlated with tumor progression in the MMTV-PyMT mouse model. Together, the data show that FGF signaling modulation by Spry2 is essential for epithelial morphogenesis in the mammary gland and it functions to protect the epithelium against tumorigenesis.

Clinical and therapeutic implications of Sprouty2 feedback dysregulation in BRAF V600E-mutation-positive papillary thyroid cancer

The BRAF V600E (BRAF+) mutation activates the mitogen-activated protein kinase (MAPK/ERK) pathway and may confer an aggressive phenotype in papillary thyroid cancer (PTC). Clinically, the behavior of BRAF+ PTC, however, varies from an indolent to an aggressive course. SPRY2 is a negative feedback regulator of the MAPK/ERK pathway. We hypothesize that the level of SPRY2 expression contributes to MAPK/ERK pathway output and accounts for BRAF+ and clinical heterogeneity. A tissue microarray with BRAF-positive PTCs (BRAF+ PTCs) was constructed and analyzed for SPRY2 expression and MAPK/ERK output. Data were studied in the context of clinicopathologic factors to develop a risk stratification system predictive of tumor biology. SPRY2 function was studied by silencing SPRY2 in BRAF+ PTC cells. These cells were treated with MAPK/ERK pathway inhibitors and assessed for growth effects. BRAF+ PTCs with an intact MAPK/ERK feedback pathway do not exhibit lymph node metastases. BRAF+ PTCs with dysregulated feedback pathways have nodal metastasis. When SPRY2 is silenced, the BRAF+ PTC cells are significantly more sensitive to MAPK/ERK inhibition. PTC behavior likely is dependent on both the driver of the MAPK/ERK pathway and its regulatory feedback. When the feedback pathway is intact, the tumor phenotype seems to be less aggressive. This observation has direct and important clinical implications and may alter our treatment strategies.