Pancreatic RECK inactivation promotes cancer formation, epithelial-mesenchymal transition, and metastasis.

RECK is downregulated in various human cancers; however, how RECK inactivation affects carcinogenesis remains unclear. We addressed this issue in a pancreatic ductal adenocarcinoma (PDAC) mouse model and found that pancreatic Reck deletion dramatically augmented the spontaneous development of PDAC with a mesenchymal phenotype, which was accompanied by increased liver metastases and decreased survival. Lineage tracing revealed that pancreatic Reck deletion induced epithelial-mesenchymal transition (EMT) in PDAC cells, giving rise to inflammatory cancer-associated fibroblast-like cells in mice. Splenic transplantation of Reck-null PDAC cells resulted in numerous liver metastases with a mesenchymal phenotype, whereas reexpression of RECK markedly reduced metastases and changed the PDAC tumor phenotype into an epithelial one. Consistently, low RECK expression correlated with low E-cadherin expression, poor differentiation, metastasis, and poor prognosis in human PDAC. RECK reexpression in the PDAC cells was found to downregulate MMP2 and MMP3, with a concomitant increase in E-cadherin and decrease in EMT-promoting transcription factors. An MMP inhibitor recapitulated the effects of RECK on the expression of E-cadherin and EMT-promoting transcription factors and invasive activity. These results establish the authenticity of RECK as a pancreatic tumor suppressor, provide insights into its underlying mechanisms, and support the idea that RECK could be an important therapeutic effector against human PDAC.

GRK5-mediated inflammation and fibrosis exert cardioprotective effects during the acute phase of myocardial infarction.

During myocardial infarction (MI), cardiac cells at the infarcted area undergo cell death. In response, cardiac myofibroblasts, which are mainly differentiated from resident fibroblasts upon inflammation, produce extracellular matrix proteins such as collagen to fill the damaged areas of the heart to prevent cardiac rupture. In this study, we identified a cardioprotective role of G-protein-coupled receptor kinase 5 (GRK5) in MI. GRK5 expression was found to increase in the mouse heart after MI and was highly expressed in cardiac fibroblasts/myofibroblasts. In fibroblasts/myofibroblasts, GRK5 promoted the expression of inflammation-related genes through nuclear factor-κB activation, leading to an increase in the expression levels of fibrosis-related genes. Bone marrow transfer experiments confirmed that GRK5 in fibroblasts/myofibroblasts, but not in infiltrated macrophages in the infarcted area, is mainly responsible for GRK5-mediated inflammation in infarcted hearts. In addition, inflammation and fibrosis at the infarcted area were significantly suppressed in GRK5 knockout mice, resulting in increased mortality compared with that in wild-type mice. These data indicate that GRK5 in cardiac fibroblasts/myofibroblasts promotes inflammation and fibrosis to ameliorate the damage after MI.

Suppression of tumor metastasis by a RECK-activating small molecule.

RECK encodes a membrane-anchored protease-regulator which is often downregulated in a wide variety of cancers, and reduced RECK expression often correlates with poorer prognoses. In mouse models, forced expression of RECK in tumor xenografts results in suppression of tumor angiogenesis, invasion, and metastasis. RECK mutations, however, are rare in cancer genomes, suggesting that agents that re-activate dormant RECK may be of clinical value. We found a potent RECK-inducer, DSK638, that inhibits spontaneous lung metastasis in our mouse xenograft model. Induction of RECK expression involves SP1 sites in its promoter and may be mediated by KLF2. DSK638 also upregulates MXI1, an endogenous MYC-antagonist, and inhibition of metastasis by DSK638 is dependent on both RECK and MXI1. This study demonstrates the utility of our approach (using a simple reporter assay followed by multiple phenotypic assays) and DSK638 itself (as a reference compound) in finding potential metastasis-suppressing drugs.

Reversion-inducing cysteine-rich protein with Kazal motifs and MT1-MMP promote the formation of robust fibrillin fibers.

Fibrillins (FBNs) form mesh-like structures of microfibrils in various elastic tissues. RECK and FBN1 are co-expressed in many human tissues, suggesting a functional relationship. We found that dermal FBN1 fibers show atypical morphology in mice with reduced RECK expression (RECK-Hypo mice). Dermal FBN1 fibers in mice-lacking membrane-type 1-matrix metalloproteinase (MT1-MMP) show a similar atypical morphology, despite the current notion that MT1-MMP (a membrane-bound protease) and RECK (a membrane-bound protease inhibitor) have opposing functions. Our experiments using dermal fibroblasts indicated that RECK promotes pro-MT1-MMP activation, increases cell-associated gelatinase/collagenase activity, and decreases diffusible gelatinase/collagenase activity, while MT1-MMP stabilizes RECK in these cells. Experiments using purified proteins indicate that RECK and its binding partner ADAMTS10 keep the proteolytic activity of MT1-MMP within a certain range. These findings suggest that RECK, ADAMTS10, and MT1-MMP cooperate to support the formation of robust FBN1 fibers.

Abundant expression of the membrane-anchored protease-regulator RECK in the anterior pituitary gland and its implication in the growth hormone/insulin-like growth factor 1 axis in mice.

The tumor suppressor gene Reversion-inducing cysteine-rich protein with Kazal motifs (Reck) encodes a membrane-anchored protease regulator expressed in multiple tissues in mouse embryos and is essential for embryonic development. In postnatal mice, however, physiological roles for the RECK protein remain unclear. We found in this study that Reck is abundantly expressed in growth hormone (GH)-producing cells (somatotrophs) in the anterior pituitary gland (AP). We also found that two types of viable Reck mutant mice, one with reduced RECK expression (Hypo mice) and the other with induced Reck deficiency from 10 days after birth (iKO mice treated with tamoxifen), exhibit common phenotypes including decreases in body size and plasma levels of insulin-like growth factor-1 (IGF1). To gain insights into the function of RECK in the AP, we characterized several somatotroph-associated molecules in the AP of these mice. Immunoreactivity of GH was greatly reduced in tamoxifen-treated iKO mice; in these mice, two membrane receptors involved in the stimulation of GH secretion [growth hormone secretagogue receptor (GHSR) and growth hormone releasing hormone receptor (GHRHR)] were decreased, however, their mRNAs were increased. Decrease in GHSR immunoreactivity and concomitant increase in its mRNA were also found in the other mutant line, Hypo. Furthermore, reduced immunoreactivity of growth hormone receptor (GHR) and concomitant increase in its mRNA was also found in the liver of Hypo mice. These results raise the possibility that RECK supports proper functioning of the GH/IGF1 axis in mice, thereby affecting their growth and metabolism.

Empagliflozin reduces high glucose-induced oxidative stress and miR-21-dependent TRAF3IP2 induction and RECK suppression, and inhibits human renal proximal tubular epithelial cell migration and epithelial-to-mesenchymal transition.

Proximal tubular epithelial cells (PTEC) in the S1 segment of the kidney abundantly express sodium-glucose co-transporters (SGLT) that play a critical role in whole body glucose homeostasis. We recently reported suppression of RECK (Reversion Inducing Cysteine Rich Protein with Kazal Motifs), a membrane anchored endogenous MMP inhibitor and anti-fibrotic mediator, in the kidneys of db/db mice, a model of diabetic kidney disease (DKD), as well as in high glucose (HG) treated human kidney proximal tubule cells (HK-2). We further demonstrated that empagliflozin (EMPA), an SGLT2 inhibitor, reversed these effects. Little is known regarding the mechanisms underlying RECK suppression under hyperglycemic conditions, and its rescue by EMPA. Consistent with our previous studies, HG (25 mM) suppressed RECK expression in HK-2 cells. Further mechanistic investigations revealed that HG induced superoxide and hydrogen peroxide generation, oxidative stress-dependent TRAF3IP2 upregulation, NF-κB and p38 MAPK activation, inflammatory cytokine expression (IL-1ß, IL-6, TNF-α, and MCP-1), miR-21 induction, MMP2 activation, and RECK suppression. Moreover, RECK gain-of-function inhibited HG-induced MMP2 activation and HK-2 cell migration. Similar to HG, advanced glycation end products (AGE) induced TRAF3IP2 and suppressed RECK, effects that were inhibited by EMPA. Importantly, EMPA treatment ameliorated all of these deleterious effects, and inhibited epithelial-to-mesenchymal transition (EMT) and HK-2 cell migration. Collectively, these findings indicate that hyperglycemia and associated AGE suppress RECK expression via oxidative stress/TRAF3IP2/NF-κB and p38 MAPK/miR-21 induction. Furthermore, these results suggest that interventions aimed at restoring RECK or inhibiting SGLT2 have the potential to treat kidney inflammatory response/fibrosis and nephropathy under chronic hyperglycemic conditions, such as DKD.

RECK in Neural Precursor Cells Plays a Critical Role in Mouse Forebrain Angiogenesis.

RECK in neural precursor cells (NPCs) was previously found to support Notch-dependent neurogenesis in mice. On the other hand, recent studies implicate RECK in endothelial cells (ECs) in WNT7-triggered canonical WNT signaling essential for brain angiogenesis. Here we report that RECK in NPCs is also critical for brain angiogenesis. When Reck is inactivated in Foxg1-positive NPCs, mice die shortly after birth with hemorrhage in the forebrain, with angiogenic sprouts stalling at the periphery and forming abnormal aggregates reminiscent of those in EC-selective Reck knockout mice and Wnt7a/b-deficient mice. The hemorrhage can be pharmacologically suppressed by lithium chloride. An effect of RECK in WNT7-producing cells to enhance canonical WNT-signaling in reporter cells is detectable in mixed culture but not with conditioned medium. Our findings suggest that NPC-expressed RECK has a non-cell-autonomous function to promote forebrain angiogenesis through contact-dependent enhancement of WNT signaling in ECs, implying possible involvement of RECK in neurovascular coupling.

RECK suppresses interleukin-17/TRAF3IP2-mediated MMP-13 activation and human aortic smooth muscle cell migration and proliferation.

Sustained inflammation and matrix metalloproteinase (MMP) activation contribute to vascular occlusive/proliferative disorders. Interleukin-17 (IL-17) is a proinflammatory cytokine that signals mainly via TRAF3 Interacting Protein 2 (TRAF3IP2), an upstream regulator of various critical transcription factors, including AP-1 and NF-κB. Reversion inducing cysteine rich protein with kazal motifs (RECK) is a membrane-anchored MMP inhibitor. Here we investigated whether IL-17A/TRAF3IP2 signaling promotes MMP-13-dependent human aortic smooth muscle cell (SMC) proliferation and migration, and determined whether RECK overexpression blunts these responses. Indeed, IL-17A treatment induced (a) JNK, p38 MAPK, AP-1, NF-κB, and CREB activation, (b) miR-21 induction, (c) miR-27b and miR-320 inhibition, (d) MMP-13 expression and activation, (e) RECK suppression, and (f) SMC migration and proliferation, all in a TRAF3IP2-dependent manner. In fact, gain of TRAG3IP2 function, by itself, induced MMP-13 expression and activation, and RECK suppression. Furthermore, treatment with recombinant MMP-13 stimulated SMC migration in part via ERK activation. Importantly, RECK gain-of-function attenuated MMP-13 activity without affecting its mRNA or protein levels, and inhibited IL-17A- and MMP-13-induced SMC migration. These results indicate that increased MMP-13 and decreased RECK contribute to IL-17A-induced TRAF3IP2-dependent SMC migration and proliferation, and suggest that TRAF3IP2 inhibitors or RECK inducers have the potential to block the progression of neointimal thickening in hyperplastic vascular diseases.

Minocycline inhibits PDGF-BB-induced human aortic smooth muscle cell proliferation and migration by reversing miR-221- and -222-mediated RECK suppression.

Minocycline, a tetracycline antibiotic, is known to exert vasculoprotective effects independent of its anti-bacterial properties; however the underlying molecular mechanisms are not completely understood. Reversion Inducing Cysteine Rich Protein with Kazal Motifs (RECK) is a cell surface expressed, membrane anchored protein, and its overexpression inhibits cancer cell migration. We hypothesized that minocycline inhibits platelet-derived growth factor (PDGF)-induced human aortic smooth muscle cell (SMC) proliferation and migration via RECK upregulation. Our data show that the BB homodimer of recombinant PDGF (PDGF-BB) induced SMC migration and proliferation, effects significantly blunted by pre-treatment with minocycline. Further investigations revealed that PDGF-BB induced PI3K-dependent AKT activation, ERK activation, reactive oxygen species generation, Nuclear Factor-κB and Activator Protein-1 activation, microRNA (miR)-221 and miR-222 induction, RECK suppression, and matrix metalloproteinase (MMP2 and 9) activation, effects that were reversed by minocycline. Notably, minocycline induced RECK expression dose-dependently within the therapeutic dose of 1-100 µM, and silencing RECK partially reversed the inhibitory effects of minocycline on PDGF-BB-induced MMP activation, and SMC proliferation and migration. Further, targeting MMP2 and MMP9 blunted PDGF-BB-induced SMC migration. Together, these results demonstrate that minocycline inhibits PDGF-BB-induced SMC proliferation and migration by restoring RECK, an MMP inhibitor. These results indicate that the induction of RECK is one of the mechanisms by which minocycline exerts vasculoprotective effects.

A RECK-WNT7 Receptor-Ligand Interaction Enables Isoform-Specific Regulation of Wnt Bioavailability.

WNT7A and WNT7B control CNS angiogenesis and blood-brain barrier formation by activating endothelial Wnt/ß-catenin signaling. The GPI-anchored protein RECK and adhesion G protein-coupled receptor GPR124 critically regulate WNT7-specific signaling in concert with FZD and LRP co-receptors. Here, we demonstrate that primarily the GPR124 ectodomain, but not its transmembrane and intracellular domains, mediates RECK/WNT7-induced canonical Wnt signaling. Moreover, RECK is the predominant binding partner of GPR124 in rat brain blood vessels in situ. WNT7A and WNT7B, but not WNT3A, directly bind to purified recombinant soluble RECK, full-length cell surface RECK, and the GPR124:RECK complex. Chemical cross-linking indicates that RECK and WNT7A associate with 1:1 stoichiometry, which stabilizes short-lived, active, monomeric, hydrophobic WNT7A. In contrast, free WNT7A rapidly converts into inactive, hydrophilic aggregates. Overall, RECK is a selective WNT7 receptor that mediates GPR124/FZD/LRP-dependent canonical Wnt/ß-catenin signaling by stabilizing active cell surface WNT7, suggesting isoform-specific regulation of Wnt bioavailability.

The RECK tumor-suppressor protein binds and stabilizes ADAMTS10.

The tumor suppressor protein RECK has been implicated in the regulation of matrix metalloproteinases (MMPs), NOTCH-signaling and WNT7-signaling. It remains unclear, however, how broad the spectrum of RECK targets extends. To find novel RECK binding partners, we took the unbiased approach of yeast two-hybrid screening. This approach detected ADAMTS10 as a RECK-interactor. ADAMTS10 has been characterized as a metalloproteinase involved in fibrillin-rich microfibril biogenesis, and its mutations have been implicated in the connective tissue disorder Weill-Marchesani syndrome. Experiments in vitro using recombinant proteins expressed in mammalian cells indicated that RECK indeed binds ADAMTS10 directly, that RECK protects ADAMTS10 from fragmentation following chemical activation and that ADAMTS10 interferes with the activity of RECK to inhibit MT1-MMP. In cultured cells, RECK increases the amount of ADAMTS10 associated with the cells. Hence, the present study has uncovered novel interactions between two molecules of known clinical importance, RECK and ADAMTS10.This article has an associated First Person interview with the first author of the paper.

Generation and characterization of a mouse line carrying Reck-CreERT2 knock-in allele.

Reck encodes a membrane-anchored glycoprotein implicated in the regulation of extracellular metalloproteinases, Notch-signaling, and Wnt7-signaling and shown to play critical roles in embryogenesis and tumor suppression. Precise mechanisms of its actions in vivo, however, remain largely unknown. By homologous recombination, we generated a new Reck allele, ReckCreERT2 (MGI symbol: Reck). This allele is defective in terms of Reck function but expected to induce loxP-mediated recombination in the cells committed to express Reck. Similarity in the expression patterns of the ReckCreERT2 transgene and the endogenous Reck gene was confirmed in five tissues. In the adult hippocampus, induction of Reck expression after transient cerebral ischemia could be demonstrated using this allele. These results indicate the utility of this Cre-driver allele in further studies.

Cleavage of ß-dystroglycan occurs in sarcoglycan-deficient skeletal muscle without MMP-2 and MMP-9.

BACKGROUND: The dystroglycan complex consists of two subunits: extracellular α-dystroglycan and membrane-spanning ß-dystroglycan, which provide a tight link between the extracellular matrix and the intracellular cytoskeleton. Previous studies showed that 43 kDa ß-dystroglycan is proteolytically cleaved into the 30 kDa fragment by matrix metalloproteinases (MMPs) in various non-muscle tissues, whereas it is protected from cleavage in muscles by the sarcoglycan complex which resides close to the dystroglycan complex. It is noteworthy that cleaved ß-dystroglycan is detected in muscles from patients with sarcoglycanopathy, sarcoglycan-deficient muscular dystrophy. In vitro assays using protease inhibitors suggest that both MMP-2 and MMP-9 contribute to the cleavage of ß-dystroglycan. However, this has remained uninvestigated in vivo. METHODS: We generated triple-knockout (TKO) mice targeting MMP-2, MMP-9 and γ-sarcoglycan to examine the status of ß-dystroglycan cleavage in the absence of the candidate matrix metalloproteinases in sarcoglycan-deficient muscles. RESULTS: Unexpectedly, ß-dystroglycan was cleaved in muscles from TKO mice. Muscle pathology was not ameliorated but worsened in TKO mice compared with γ-sarcoglycan single-knockout mice. The gene expression of MMP-14 was up-regulated in TKO mice as well as in γ-sarcoglycan knockout mice. In vitro assay showed MMP-14 is capable to cleave ß-dystroglycan. CONCLUSIONS: Double-targeting of MMP-2 and MMP-9 cannot prevent cleavage of ß-dystroglycan in sarcoglycanopathy. Thus, matrix metalloproteinases contributing to ß-dystroglycan cleavage are redundant, and MMP-14 could participate in the pathogenesis of sarcoglycanopathy.

Prognostic importance of sodium level trajectory in acute heart failure.

Low sodium levels are strongly associated with poor prognosis in acute heart failure (AHF); however, the prognostic impact of the sodium level trajectory overtime has not been determined. A secondary analysis of the AQUAMARINE study in which patients with AHF and renal impairment were randomized to receive either tolvaptan or conventional treatment was performed. Sodium levels were evaluated at the baseline and at 6, 12, 24, and 48 h. We defined 'sodium dipping' as sodium level falling below the baseline level at any time point. The primary endpoint was the combined event of all-cause death and heart failure rehospitalization during follow-up. The analysis included 184 patients with a median follow-up of 21.1 months. Sodium levels more steeply increased during the 48 h in patients without events as compared to sodium levels in patients with events (P = 0.018 in linear-mixed effect model). The sodium dipping group (n = 100; 54.3%) demonstrated significantly less urine output, less body weight reduction, and poorer diuretic response within 48 h compared to the non-dipping group. The sodium dipping group was also significantly associated with a low combined-event-free survival after adjustment for other prognostic factors (HR 1.97; 95% CI 1.06-3.38; P = 0.033). The trajectory of sodium levels during the acute phase is associated with the prognosis of patients with AHF independently of the baseline sodium level.

Early treatment with tolvaptan improves diuretic response in acute heart failure with renal dysfunction.

BACKGROUND: Poor response to diuretics is associated with worse prognosis in patients with acute heart failure (AHF). We hypothesized that treatment with tolvaptan improves diuretic response in patients with AHF. METHODS: We performed a secondary analysis of the AQUAMARINE open-label randomized study in which a total of 217 AHF patients with renal impairment (eGFR < 60 mL/min/1.73 m2) were randomized to either tolvaptan or conventional treatment. We evaluated diuretic response to 40 mg furosemide or its equivalent based on two different parameters: change in body weight and net fluid loss within 48 h. RESULTS: The mean time from patient presentation to randomization was 2.9 h. Patients with a better diuretic response showed greater relief of dyspnea and less worsening of renal function. Tolvaptan patients showed a significantly better diuretic response measured by diuretic response based both body weight [-1.16 (IQR -3.00 to -0.57) kg/40 mg vs. -0.51 (IQR -1.13 to -0.20) kg/40 mg; P < 0.001] and net fluid loss [2125.0 (IQR 1370.0-3856.3) mL/40 mg vs. 1296.3 (IQR 725.2-1726.5) mL/40 mg; P < 0.001]. Higher diastolic blood pressure and use of tolvaptan were independent predictors of a better diuretic response. CONCLUSIONS: Better diuretic response was associated with greater dyspnea relief and less WRF. Early treatment with tolvaptan significantly improved diuretic response in AHF patients with renal dysfunction.

Efficacy of Tumor-Targeting Salmonella A1-R on a Melanoma Patient-Derived Orthotopic Xenograft (PDOX) Nude-Mouse Model.

Tumor-targeting Salmonella enterica serovar Typhimurium A1-R (Salmonella A1-R) had strong efficacy on a melanoma patient-derived orthotopic xenograft (PDOX) nude-mouse model. GFP-expressing Salmonella A1-R highly and selectively colonized the PDOX melanoma and significantly suppressed tumor growth (p = 0.021). The combination of Salmonella A1-R and cisplatinum (CDDP), both at low-dose, also significantly suppressed the growth of the melanoma PDOX (P = 0.001). Salmonella A1-R has future clinical potential for combination chemotherapy with CDDP of melanoma, a highly-recalcitrant cancer.

Prognostic impact of early treatment with tolvaptan in patients with acute heart failure and renal dysfunction.

BACKGROUND: Renal dysfunction is a common comorbidity in acute heart failure (AHF) patients. The prognostic significance of early treatment with tolvaptan in AHF patients complicated with renal dysfunction has not been elucidated. METHODS: Post hoc analysis was performed on a randomized clinical study for prespecified prognostic endpoints and prespecified subgroups. 217 AHF patients with renal dysfunction (eGFR 15 to 60mL/min/1.73m(2)) were randomized within 6h from hospitalization to either tolvaptan treatment for 2days or conventional treatment. The primary outcome was the combined endpoint of all-cause death and HF readmission. RESULTS: During follow-up (636days, median) 99 patients experienced combined endpoint and 53 patients died. There was no significant difference in event-free survival rate for either the combined events (Log-rank: P=0.197) or all-cause death (Log-rank: P=0.894) between tolvaptan and conventional groups. In prespecified subgroup analysis, in patients whose BUN/creatinine ratio was above the median (>20), tolvaptan significantly reduced the risk of combined events (HR: 0.52, 95% CI: 0.30-0.91, P=0.021) with a significant interaction (P value for interaction=0.045). Likewise, in patients whose eGFR was 30mL/min/1.73m(2) or above, tolvaptan reduced the risk of combined events (HR: 0.54, 95% CI: 0.32-0.90, P=0.017) with a significant interaction (P value for interaction=0.015). CONCLUSION: Short-term use of tolvaptan in acute-phase in AHF with renal dysfunction showed a neutral effect on prognosis. Patients with relatively preserved renal function and relatively high BUN/creatinine ratios are potentially favorable subgroups for treatment with tolvaptan.

Histone deacetyltransferase inhibitors Trichostatin A and Mocetinostat differentially regulate MMP9, IL-18 and RECK expression, and attenuate Angiotensin II-induced cardiac fibroblast migration and proliferation.

Histone acetylation/deacetylation plays a key role in the epigenetic regulation of multiple pro-fibrotic genes. Here we investigated the effects of histone deacetyltransferase (HDAC) inhibition on angiotensin (Ang)-II-induced pro-fibrotic changes in adult mouse cardiac fibroblasts (CF). CF express class I HDACs 1 and 2, and Ang-II induces their activation. Notably, silencing HDAC1 or HDAC2 attenuated Ang-II induced CF proliferation and migration. Under basal conditions, HDAC1 dimerizes with HDAC2 in CF and Ang-II reversed this interaction. Treatment with Trichostatin A (TSA), a broad-spectrum HDAC inhibitor, restored their physical association, and attenuated Ang-II-induced MMP9 expression, IL-18 induction, and extracellular matrix (collagen I, collagen III and fibronectin) production. Further, TSA inhibited Ang-II-induced MMP9 and Il18 transcription by blocking NF-κB and AP-1 binding to their respective promoter regions. By inhibiting Sp1 binding to RECK promoter, TSA reversed Ang-II-induced RECK suppression, collagen and fibronectin expression, and CF migration and proliferation. The class I-specific HDAC inhibitor Mocetinostat (MGCD) recapitulated TSA effects on Ang-II-treated CF. Together, these results demonstrate that targeting HDACs attenuates the pro-inflammatory and pro-fibrotic effects of Ang-II on CF.

Bioinformatic Studies to Predict MicroRNAs with the Potential of Uncoupling RECK Expression from Epithelial-Mesenchymal Transition in Cancer Cells.

RECK is downregulated in many tumors, and forced RECK expression in tumor cells often results in suppression of malignant phenotypes. Recent findings suggest that RECK is upregulated after epithelial-mesenchymal transition (EMT) in normal epithelium-derived cells but not in cancer cells. Since several microRNAs (miRs) are known to target RECK mRNA, we hypothesized that certain miR(s) may be involved in this suppression of RECK upregulation after EMT in cancer cells. To test this hypothesis, we used three approaches: (1) text mining to find miRs relevant to EMT in cancer cells, (2) predicting miR targets using four algorithms, and (3) comparing miR-seq data and RECK mRNA data using a novel non-parametric method. These approaches identified the miR-183-96-182 cluster as a strong candidate. We also looked for transcription factors and signaling molecules that may promote cancer EMT, miR-183-96-182 upregulation, and RECK downregulation. Here we describe our methods, findings, and a testable hypothesis on how RECK expression could be regulated in cancer cells after EMT.