The formin DAAM1 regulates the deubiquitinase activity of USP10 and integrin homeostasis.

The differentiation of fibroblasts into pathological myofibroblasts during wound healing is characterized by increased cell surface expression of αv-integrins. Our previous studies found that the deubiquitinase (DUB) USP10 removes ubiquitin from αv-integrins, leading to cell surface integrin accumulation, subsequent TGFß1 activation, and pathological myofibroblast differentiation. In this study, a yeast two-hybrid screen revealed a novel binding partner for USP10, the formin, DAAM1. We found that DAAM1 binds to and inhibits USP10's DUB activity through the FH2 domain of DAAM1 independent of its actin functions. The USP10/DAAM1 interaction was also supported by proximity ligation assay (PLA) in primary human corneal fibroblasts. Treatment with TGFß1 significantly increased USP10 and DAAM1 protein expression, PLA signal, and co-localization to actin stress fibers. DAAM1 siRNA knockdown significantly reduced co-precipitation of USP10 and DAAM1 on purified actin stress fibers, and ß1- and ß5-integrin ubiquitination. This resulted in increased αv-, ß1-, and ß5-integrin total protein levels, αv-integrin recycling, and extracellular fibronectin (FN) deposition. Together, our data demonstrate that DAAM1 inhibits USP10's DUB activity on integrins subsequently regulating cell surface αv-integrin localization and FN accumulation.

D-Aspartate Depletion Perturbs Steroidogenesis and Spermatogenesis in Mice.

High levels of free D-aspartate (D-Asp) are present in vertebrate testis during post-natal development, coinciding with the onset of testosterone production, which suggests that this atypical amino acid might participate in the regulation of hormone biosynthesis. To elucidate the unknown role of D-Asp on testicular function, we investigated steroidogenesis and spermatogenesis in a one-month-old knockin mouse model with the constitutive depletion of D-Asp levels due to the targeted overexpression of D-aspartate oxidase (DDO), which catalyzes the deaminative oxidation of D-Asp to generate the corresponding α-keto acid, oxaloacetate, hydrogen peroxide, and ammonium ions. In the Ddo knockin mice, we found a dramatic reduction in testicular D-Asp levels, accompanied by a significant decrease in the serum testosterone levels and testicular 17ß-HSD, the enzyme involved in testosterone biosynthesis. Additionally, in the testes of these Ddo knockin mice, the expression of PCNA and SYCP3 proteins decreased, suggesting alterations in spermatogenesis-related processes, as well as an increase in the cytosolic cytochrome c protein levels and TUNEL-positive cell number, which indicate an increase in apoptosis. To further investigate the histological and morphometric testicular alterations in Ddo knockin mice, we analyzed the expression and localization of prolyl endopeptidase (PREP) and disheveled-associated activator of morphogenesis 1 (DAAM1), two proteins involved in cytoskeletal organization. Our results showed that the testicular levels of DAAM1 and PREP in Ddo knockin mice were different from those in wild-type animals, suggesting that the deficiency of D-Asp is associated with overall cytoskeletal disorganization. Our findings confirmed that physiological D-Asp influences testosterone biosynthesis and plays a crucial role in germ cell proliferation and differentiation, which are required for successful reproduction.

The novel role of etoposide in inhibiting the migration and proliferation of small cell lung cancer and breast cancer via targeting Daam1.

OBJECTIVES: Daam1 (Dishevelled-associated activator of morphogenesis 1) is a Wnt/PCP signaling protein that engages in cytoskeleton reorganization and is abnormally activated in certain tumors. Daam1 is closely related to cancer metastasis, which is expected to become a target for cancer treatment. However, the natural small molecules targeting Daam1 have not been identified. MATERIALS AND METHODS: We screened several natural small molecules that may bind to Daam1 by Sybyl molecular simulation docking technique. As a first-line drug for the treatment of small cell lung cancer, etoposide was chosen for further investigation. Next, we used Micro Scale Thermophoresis (MST) to verify the interaction of etoposide and Daam1. Small cell lung cancer H446 cells and breast cancer MCF-7 cells were treated with etoposide and subjected to Western blotting to measure the Daam1 expression. The effect of etoposide on cell proliferation was determined by CCK-8 assay in vitro and by a tumor-bearing mouse model in vivo. Wound healing assay and Boyden chamber assay were used to evaluate the role of etoposide in the migration and invasion ability of tumor cells. The effect of etoposide on the microfilament assembly was visualized by immunofluorescence staining with phalloidine. Finally, the possible mechanism of down-regulation of Daam1 expression after etoposide-induced small cell lung cancer cells was detected by a half-life experiment and immunofluorescence staining with lysosomal marker LAMP1. RESULTS: Sybyl molecular modeling docking technique was performed to screen a natural chemical library for molecules that bound to the FH2 domain of Daam1 and found etoposide was virtually interacted with Daam1. MST validated etoposide directly bound to the FH2 domain of Daam1. Etoposide significantly down-regulated the expression of Daam1 in small cell lung cancer H446 cells and breast cancer MCF-7 cells. Moreover, 270 µmol/L etoposide largely inhibited the proliferation, migration, and invasion of H446 cells and MCF-7 cells. Immunofluorescence staining experiments revealed that etoposide induced the disassembly of microfilaments in H446 cells and MCF-7 cells, which were rescued by Daam1 overexpression. In nude mice transplanted with H446 cells, 5, 10, 20 mg/kg etoposide (drug/weight) injected via tail vein largely retarded the proliferation of subcutaneous tumors. Etoposide induced Daam1 to shorten its half-life and enter the lysosome degradation pathway, and eventually leading to the downregulation of Daam1 expression. CONCLUSIONS: Etoposide is a novel natural small molecule targeting Daam1. Etoposide inhibits the proliferation, migration and invasion of small cell lung cancer cells and breast cancer cells, and also suppresses tumor proliferation of small cell lung cancer in vivo.

The simultaneous administration of microplastics and cadmium alters rat testicular activity and changes the expression of PTMA, DAAM1 and PREP.

This paper confirms the damaging effects produced by MP and Cd on testicular activity in the rat. Oral treatment with both chemicals resulted in testicular damage, documented by biomolecular and histological alterations, particularly by impaired morphometric parameters, increased apoptosis, reduced testosterone synthesis, and downregulation of the steroidogenic enzyme 3ß-HSD. We also demonstrated, for the first time, that both MP and Cd can affect the protein level of PTMA, a small peptide that regulates germ cell proliferation and differentiation. Interestingly, the cytoarchitecture of testicular cells was also altered by the treatments, as evidenced by the impaired expression and localization of DAAM1 and PREP, two proteins involved in actin- and microtubule-associated processes, respectively, during germ cells differentiation into spermatozoa, impairing normal spermatogenesis. Finally, we showed that the effect of simultaneous treatment with MP and Cd were more severe than those produced by MP alone and less harmful than those of Cd alone. This could be due to the different ways of exposure of the two substances to rats (in drinking water for Cd and in oral gavage for MP), since being the first contact in the animals' gastrointestinal tract, MP can adsorb Cd, reducing its bioavailability through the Trojan-horse effect.

FOXD3 confers chemo-sensitivity in ovarian cancer through a miR-335/DAAM1/myosin II axis-dependent mechanism.

BACKGROUND: Chemotherapy is among the most common treatment methods for ovarian cancer (OC). However, chemoresistance limits the effectiveness of chemotherapy and leads to treatment failure. We herein investigate the biological effect of forkhead box D3 (FOXD3) in the chemoresistance of OC cells. METHODS: Expression of FOXD3, miR-335 and disheveled-associated activator of morphogenesis 1 (DAAM1) was detected in OC cells and tissues. The regulatory network of FOXD3/miR-335/DAAM1 was validated by dual-luciferase reporter and ChIP assays in vitro. After ectopic expression and depletion experiments in carboplatin/paclitaxel (CP)-resistant (A2780CP) or sensitive (A2780S) OC cells, cell viability, colony formation and apoptosis were tested by CCK-8 assay, colony formation assay and flow cytometry respectively. Effects of FOXD3 on the chemoresistance of OC cells in vivo were evaluated in OC xenografts in nude mice. RESULTS: Overexpression of FOXD3 impaired the proliferation and chemoresistance of OC cells, which was related to the promotion of the miR-335 expression. Functionally, DAAM1 was a putative target of miR-335. Silencing of DAAM1 was responsible for the inhibition of myosin II activation, consequently leading to suppressed OC cell proliferation and chemoresistance. In vivo results further showed that FOXD3 weakened the chemoresistance of OC cells to CP. CONCLUSION: Taken together, we unveil a novel FOXD3/miR-335/DAAM1/myosin II axis that regulates the chemoresistance of OC both in vitro and in vivo.

MicroRNA-137 Inhibits Esophageal Squamous Cell Carcinoma by Downregulating DAAM1.

BACKGROUND: A growing body of evidence demonstrates that miR-137 acts against cancers; however, the biological function of miR-137 in esophageal squamous cell carcinoma (ESCC) remains to be fully understood. OBJECTIVE: The aim of this study is to explore the role of miR-137 in ESCC. METHODS: miR-137 expression was detected by reverse-transcription quantitative polymerase chain reaction (RT-qPCR), and target protein expression was detected by western blot. Cell counting, colony formation and flow cytometry were employed to determine the effects of miR-137 on the growth of ESCC cells. Dual-luciferase reporter assay was performed to validate the binding of miR- 137 with a dishevelled-associated activator of morphogenesis 1 (DAAM1) 3'-UTR. RESULTS: miR-137 was shown to be down-regulated in ESCC. miR-137 expression was inversely correlated with the 5-year survival rate of ESCC patients. Up-regulated miR-137 attenuated ESCC proliferation and promoted ESCC cell apoptosis. Meanwhile, to further reveal how miR-137 regulated the malignant behaviors of ESCC, the downstream mRNA binding targets of miR-137 were explored. miR-137 was demonstrated to bind DAAM1 3'-UTR and repressed the expression of DAAM1. The expression of DAAM1 and miR-137 in ESCC was inversely correlated. Additionally, the reintroduction of DAAM1 had the capacity to reverse the negative role of miR- 137 in ESCC cell growth. CONCLUSION: These findings have uncovered the new function of miR-137 in ESCC via negatively regulating DAAM1, suggesting miR-137 as a potent therapeutic candidate for ESCC treatment.

New Insight on the In Vitro Effects of Melatonin in Preserving Human Sperm Quality.

Spermatozoa (SPZ) are sensitive to stressful conditions, particularly oxidative stress, which alters their quality; thus, the use of protective molecules as an antioxidant is encouraged. Herein, we used melatonin (MLT) to investigate its in vitro effects on human sperm parameters under conditions of oxidative stress induced by cadmium (Cd). Fifteen human semen samples were divided into control, Cd-treated, MLT-treated, and Cd+MLT-treated groups and analyzed after 30 min, 6 h, and 24 h of exposure. Results showed a time-dependent decrease in SPZ motility, DNA integrity, and increased apoptosis induced by oxidative stress, and these effects were counteracted by MLT co-treatment. Based on these data, we further explored additional parameters just at 24 h. The induced oxidative stress, highlighted by the increased lipid peroxidation, reduced the percentage of SPZ able to undertake acrosome reaction and altered the levels and localization of some protein markers of motility (PREP, RSPH6A), morphology (DAAM1), and acrosome membrane (PTMA, IAM38); all these effects were counteracted by MLT co-treatment. Interestingly, MLT alone was able to ameliorate motility at 30 min of incubation compared to the control, while at 24 h, it prevented the physiological alteration in terms of motility, DNA integrity, and apoptosis. Collectively, the data encourage MLT use as an integrative molecule to ameliorate human gamete quality when compromised by stressful conditions.

Prediction of Non-canonical Routes for SARS-CoV-2 Infection in Human Placenta Cells.

The SARS-CoV-2 is the causative agent of the COVID-19 pandemic. The data available about COVID-19 during pregnancy have demonstrated placental infection; however, the mechanisms associated with intrauterine transmission of SARS-CoV-2 is still debated. Intriguingly, while canonical SARS-CoV-2 cell entry mediators are expressed at low levels in placental cells, the receptors for viruses that cause congenital infections such as the cytomegalovirus and Zika virus are highly expressed in these cells. Here we analyzed the transcriptional profile (microarray and single-cell RNA-Seq) of proteins potentially interacting with coronaviruses to identify non- canonical mediators of SARS-CoV-2 infection and replication in the placenta. Despite low levels of the canonical cell entry mediators ACE2 and TMPRSS2, we show that cells of the syncytiotrophoblast, villous cytotrophoblast, and extravillous trophoblast co-express high levels of the potential non-canonical cell-entry mediators DPP4 and CTSL. We also found changes in the expression of DAAM1 and PAICS genes during pregnancy, which are translated into proteins also predicted to interact with coronaviruses proteins. These results provide new insight into the interaction between SARS-CoV-2 and host proteins that may act as non-canonical routes for SARS-CoV-2 infection and replication in the placenta cells.

Daam1 Overexpression Promotes Gastric Cancer Progression and Regulates ERK and AKT Signaling Pathways.

OBJECTIVE: The dishevelled-associated activator of morphogenesis 1 (DAAM1) has been reported to be closely associated with human cancers. However, its involvement in human gastric cancer (GC) remains largely unexplored. This study aimed to investigate the clinical significance and biological roles of Daam1 in human GC. METHODS: Daam1 protein expression was examined in 124 cases of gastric adenocarcinomas using immunohistochemistry. Daam1 plasmid and siRNA transfection were carried out in SGC7901 and AGS cell lines. CCK-8, colony formation, Annexin V/PI, JC-1 staining, and Western blotting were used to explore the biological functions and potential underlying mechanisms of Daam1 in GC cells. RESULTS: Our results showed that Daam1 was overexpressed in GC specimens. A high Daam1 level was associated with tumor-node-metastasis (TNM) stage, T status, nodal metastasis, and poor patient survival. Analysis of the Oncomine dataset revealed upregulation of Daam1 mRNA in GC tissues. Western blot showed that Daam1 protein expression was higher in GC cell lines compared to the normal GES-1 cell line. CCK-8 and colony formation assays showed that ectopic Daam1 expression upregulated the cell growth rate and colony number in SGC-7901 cells, while Daam1 siRNA knockdown downregulated the growth rate and colony number in AGS cells. CCK-8 and Annexin V/PI apoptosis assays demonstrated that Daam1 overexpression decreased cisplatin sensitivity and downregulated cisplatin-induced apoptosis. JC1 staining showed that Daam1 overexpression upregulated, while Daam1 depletion downregulated mitochondrial membrane potential. Mechanistically, Daam1 overexpression downregulated p21 and upregulated p-ERK and p-AKT. The increased proliferation rate and decreased cisplatin sensitivity/apoptosis induced by ectopic Daam1 were reversed after treatment with AKT and ERK inhibitors. CONCLUSION: Taken together, our results showed that Daam1 overexpression was associated with poor prognosis and promoted malignant activity via regulation of ERK and AKT pathways in GC cells, indicating Daam1 is a malignant biomarker and potential therapeutic target in GC.

Preliminary Investigation on the Involvement of Cytoskeleton-Related Proteins, DAAM1 and PREP, in Human Testicular Disorders.

Herein, for the first time, the potential relationships between the cytoskeleton-associated proteins DAAM1 and PREP with different testicular disorders, such as classic seminoma (CS), Leydig cell tumor (LCT), and Sertoli cell-only syndrome (SOS), were evaluated. Six CS, two LCT, and two SOS tissue samples were obtained during inguinal exploration in patients with a suspect testis tumor based on clinical examination and ultrasonography. DAAM1 and PREP protein levels and immunofluorescent localization were analyzed. An increased DAAM1 protein level in CS and SOS as compared to non-pathological (NP) tissue was observed, while LCT showed no significant differences. Conversely, PREP protein level increased in LCT, while it decreased in CS and SOS compared to NP tissue. These results were strongly supported by the immunofluorescence staining, revealing an altered localization and signal intensity of DAAM1 and PREP in the analyzed samples, highlighting a perturbed cytoarchitecture. Interestingly, in LCT spermatogonia, a specific DAAM1 nuclear localization was found, probably due to an enhanced testosterone production, as confirmed by the increased protein levels of steroidogenic enzymes. Finally, although further studies are needed to verify the involvement of other formins and microtubule-associated proteins, this report raised the opportunity to indicate DAAM1 and PREP as new potential markers, supporting the cytoskeleton dynamics changes occurring during normal and/or pathological cell differentiation.

The Wnt/PCP formin Daam1 drives cell-cell adhesion during nephron development.

E-cadherin junctions facilitate assembly and disassembly of cell contacts that drive development and homeostasis of epithelial tissues. In this study, using Xenopus embryonic kidney and Madin-Darby canine kidney (MDCK) cells, we investigate the role of the Wnt/planar cell polarity (PCP) formin Daam1 (Dishevelled-associated activator of morphogenesis 1) in regulating E-cadherin-based intercellular adhesion. Using live imaging, we show that Daam1 localizes to newly formed cell contacts in the developing nephron. Furthermore, analyses of junctional filamentous actin (F-actin) upon Daam1 depletion indicate decreased microfilament localization and slowed turnover. We also show that Daam1 is necessary for efficient and timely localization of junctional E-cadherin, mediated by Daam1's formin homology domain 2 (FH2). Finally, we establish that Daam1 signaling promotes organized movement of renal cells. This study demonstrates that Daam1 formin junctional activity is critical for epithelial tissue organization.

Altered Expression of DAAM1 and PREP Induced by Cadmium Toxicity Is Counteracted by Melatonin in the Rat Testis.

Cadmium (Cd) is one of the most toxic pollutants for health due to its accumulation in several tissues, including testis. This report confirms that Cd increased oxidative stress and apoptosis of germ and somatic cells and provoked testicular injury, as documented by biomolecular and histological alterations, i.e., CAT and SOD activity, the protein level of steroidogenic enzymes (StAR and 3ß-HSD), and morphometric parameters. Additionally, it further documents the melatonin (MLT) coadministration produces affects in mitigating Cd-induced toxicity on adult rat testis, as demonstrated by the reduction of oxidative stress and apoptosis, with reversal of the observed histological changes; moreover, a role of MLT in partially restoring steroidogenic enzymes expression was evidenced. Importantly, the cytoarchitecture of testicular cells was perturbed by Cd exposure, as highlighted by impairment of the expression and localization of two cytoskeleton-associated proteins DAAM1 and PREP, which are involved in the germ cells' differentiation into spermatozoa, altering the normal spermatogenesis. Here, for the first time, we found that the co-treatment with MLT attenuated the Cd-induced toxicity on the testicular DAAM1 and PREP expression. The combined findings provide additional clues about a protective effect of MLT against Cd-induced testicular toxicity by acting on DAAM1 and PREP expression, encouraging further studies to prove its effectiveness in human health.

SNHG15 facilitated malignant behaviors of oral squamous cell carcinoma through targeting miR-188-5p/DAAM1.

BACKGROUND: Long non-coding RNA (lncRNA) small nucleolar RNA host gene 15 (SNHG15) has been discovered and demonstrated to have significant function in multiple cancers. Nevertheless, how it participates in the progression of oral squamous cell carcinoma (OSCC) and its potential regulatory system are still unclear. METHODS: RT-qPCR detected the expression of SNHG15, miR-188-5p, and DAAM1. RNA pull down, RT-qPCR, and bioinformatics were used for finding and selecting downstream targets of SNHG15. RESULTS: SNHG15 presented a high expression in OSCC cells. Moreover, inhibition of SNHG15 exhibited repressive influence on proliferative, migrated, and invasive abilities but induce apoptosis of OSCC cells. Through the search of bioinformatics and RNA pull down assays, we confirmed that miR-188-5p was one target of SNHG15 in OSCC cells. Additionally, miR-188-5p could hamper the growth of OSCC cells. Moreover, it was manifested that DAAM1 was down-regulated by miR-188-5p. DAAM1 was up-regulated in OSCC cells. Furthermore, it exerted oncogenic function in the course of OSCC. Eventually, overexpression of DAAM1 offsets the effects of down-regulation of SNHG15 on the development of OSCC. CONCLUSION: To summarize, our study certified that SNHG15 contributed to the process of OSCC via sponging miR-188-5p to elevate DAAM1 expression. SNHG15 might offer novel sight to improve the results of treatment for OSCC.

Bmp Signal Gradient Modulates Convergent Cell Movement via Xarhgef3.2 during Gastrulation of Xenopus Embryos.

Gastrulation is a critical step in the establishment of a basic body plan during development. Convergence and extension (CE) cell movements organize germ layers during gastrulation. Noncanonical Wnt signaling has been known as major signaling that regulates CE cell movement by activating Rho and Rac. In addition, Bmp molecules are expressed in the ventral side of a developing embryo, and the ventral mesoderm region undergoes minimal CE cell movement while the dorsal mesoderm undergoes dynamic cell movements. This suggests that Bmp signal gradient may affect CE cell movement. To investigate whether Bmp signaling negatively regulates CE cell movements, we performed microarray-based screening and found that the transcription of Xenopus Arhgef3.2 (Rho guanine nucleotide exchange factor) was negatively regulated by Bmp signaling. We also showed that overexpression or knockdown of Xarhgef3.2 caused gastrulation defects. Interestingly, Xarhgef3.2 controlled gastrulation cell movements through interacting with Disheveled (Dsh2) and Dsh2-associated activator of morphogenesis 1 (Daam1). Our results suggest that Bmp gradient affects gastrulation cell movement (CE) via negative regulation of Xarhgef3.2 expression.

LINC01006 facilitates cell proliferation, migration and invasion in prostate cancer through targeting miR-34a-5p to up-regulate DAAM1.

BACKGROUND: Prostate cancer (PCa) is a kind of malignancy occurring in the prostate gland. Substantial researches have proved the major role of long noncoding RNAs (lncRNAs) in PCa. However, the role of long intergenic non-protein coding RNA 1006 (LINC01006) in PCa has not been investigated yet. METHODS: RT-qPCR was used to examine the expression levels of LINC01006 and its downstream targets. The function of LINC01006 in PCa was tested by in vitro and in vivo assays. With application of RNA pull down, RNA immunoprecipitation (RIP) and luciferase reporter assays, the interaction among LINC01006, miR-34a-5p and disheveled associated activator of morphogenesis 1 (DAAM1) were verified. RESULTS: LINC01006 expression presented high in PCa cell lines. LINC01006 silencing suppressed cell proliferative, migratory, invasive capacities while accelerated apoptotic rate. Besides, LINC01006 knockdown also suppressed tumor growth and metastasis in vivo. Furthermore, miR-34a-5p, a tumor suppressor in PCa, was sponged by LINC01006. Moreover, DAAM1 was targeted by miR-34a-5p and promoted PCa progression. More intriguingly, rescue assays suggested that the inhibitory effect of LINC01006 knockdown on PCa development was offset by DAAM1 overexpression. CONCLUSIONS: LINC01006 promoted PCa progression by sponging miR-34a-5p to up-regulate DAAM1, providing a novel target for PCa therapy.

D-Aspartate Upregulates DAAM1 Protein Levels in the Rat Testis and Induces Its Localization in Spermatogonia Nucleus.

Cell differentiation during spermatogenesis requires a proper actin dynamic, regulated by several proteins, including formins. Disheveled-Associated-Activator of Morphogenesis1 (DAAM1) belongs to the formins and promotes actin polymerization. Our results showed that oral D-Aspartate (D-Asp) administration, an excitatory amino acid, increased DAAM1 protein levels in germ cells cytoplasm of rat testis. Interestingly, after the treatment, DAAM1 also localized in rat spermatogonia (SPG) and mouse GC-1 cells nuclei. We provided bioinformatic evidence that DAAM1 sequence has two predicted NLS, supporting its nuclear localization. The data also suggested a role of D-Asp in promoting DAAM1 shuttling to the nuclear compartment of those proliferative cells. In addition, the proliferative action induced by D-Asp is confirmed by the increased levels of PCNA, a protein expressed in the nucleus of cells in the S phase and p-H3, a histone crucial for chromatin condensation during mitosis and meiosis. In conclusion, we demonstrated, for the first time, an increased DAAM1 protein levels following D-Asp treatment in rat testis and also its localization in the nucleus of rat SPG and in mouse GC-1 cells. Our results suggest an assumed role for this formin as a regulator of actin dynamics in both cytoplasm and nuclei of the germ cells.

Wnt5a/ROR1 activates DAAM1 and promotes the migration in osteosarcoma cells.

Receptor tyrosine kinase like orphan receptor 2 (ROR2) regulates Wnt5a-induced cell migration by phosphorylating PI3K/Akt and activating RhoA in osteosarcoma. However, the role of Wnt5a signaling and its corresponding receptors in the regulation of osteosarcoma metastasis remains poorly understood. ROR1 monoclonal antibody (mAb) and short hairpin (sh)RNA targeting ROR2 markedly inhibited the activity of dishevelled associated activator of morphogenesis 1 (DAAM1) and RhoA and retarded cell migration in osteosarcoma. ROR1 mAb and ROR2 shRNA destroyed the microfilament formation of osteosarcoma cells. Silencing of DAAM1 (with DAAM1 shRNA) downregulated RhoA activity and inhibited cell migration. The decrease of cell migration caused by DAAM1 shRNA was rescued by wild-type DAAM1 overexpression. DAAM1 and PI3Kα/Akt were parallel signaling pathways mediating osteosarcoma cell migration in response to Wnt5a. It was concluded that Wnt5a promotes osteosarcoma cell migration via ROR1/2 receptors, and then activates DAAM1 and RhoA.

Overexpressed DAAM1 correlates with metastasis and predicts poor prognosis in breast cancer.

Recent studies have reported that dishevelled-associated activator of morphogenesis 1 (DAAM1) is remarkably essential for mediating cell migration and invasion in breast cancer (BrCa). Nonetheless, the definite expression profile of DAAM1 in BrCa patients and the impact on metastasis of BrCa in vivo have not been explored up to now. The differential expression of DAAM1 in BrCa and adjacent tissues was assessed via immunohistochemistry (IHC) staining. The metastatic capacities of BrCa SUM-1315 cells were examined in BALB/c nude mice. Besides, the prognostic values of DAAM1 mRNA in BrCa were explored based on Kaplan-Meier (KM) plotter. The expression of DAAM1 protein was notably overexpressed in BrCa tissues compared with that in paired normal breast tissues. The high expression of DAAM1 in BrCa tissues was significantly associated with lymph-node metastasis. Furthermore, DAAM1 overexpression promoted the invasive capacity of BrCa cells and stimulated lung metastatic extent in vivo. We also found that overexpressed DAAM1 mRNA was significantly associated with poor relapse-free survival (RFS), overall survival (OS), distance-metastasis-free survival (DMFS), and post-progression survival (PPS). Our findings reveal that DAAM1 might be a novel therapeutic target to manage the deteriorated metastasis of BrCa and identified DAAM1 as a promising biomarker for unfavorable prognosis in BrCa patients.

Wnt5a induces ROR1 and ROR2 to activate RhoA in esophageal squamous cell carcinoma cells.

Background: Wnt5a is a nontransforming Wnt family member and identified as an oncogenic role on cell motility of breast cancer and glioblastoma. However, Wnt5a signaling in esophageal squamous cell carcinoma (ESCC) progression remains poorly defined. Materials and methods: Immunohistochemistry assays were used to measure the Wnt5a expression in ESCC sections. We evaluated the role of receptor tyrosine kinase-like orphan receptor (ROR)1/2 and RhoA on the invasion of ESCC cells by using cell invasion assay, immunoprecipitation, immunofluorescence, and Rho activation assay. Results: Wnt5a was highly expressed in invasive ESCC tissues compared with that in noninvasive and nonmalignant tissues. In vitro assay showed that sfrp2 (Wnt5a antagonist) largely blocked the invasion but not the colony formation of KYSE410 and KYSE520 ESCC cells. Anti-ROR1 mAb and ROR2-shRNA markedly inhibited the disheveled-associated activator of morphogenesis 1 (DAAM1) activity, RhoA activity, microfilament formation and the invasion of ESCC cells. Fluorescent phalloidin staining experiment showed ROR1/ROR2, receptors of Wnt5a signaling, and regulated the reassembly of actin filaments in ESCC cells. Further experiments showed that ROR1 was strongly associated with ROR2 in KYSE410 cells. The activation of RhoA, not Rac1 or Rac2, was involved in ROR1/ROR2 signaling pathway. By using DAAM1 shRNA, we found that RhoA was downstream of DAAM1, which could be rescued by the overexpression of wild-type DAAM1. This could be further proved by a RhoA inhibitor CCG-1423 which could inhibit the invasion of ESCC cells but not DAAM1 activity. Conclusions: Wnt5a promotes ESCC cell invasion via ROR1 and ROR2 receptors and DAAM1/RhoA signaling pathway.

DAAM1-mediated migration and invasion of ovarian cancer cells are suppressed by miR-208a-5p.

Ovarian cancer (OvCa) has the highest morbidity among all gynecologic cancers worldwide, and its distant metastasis is one of main causes for the poor prognosis of OvCa patients. Our previous studies have reported that DAAM1-involved signaling pathways play vital roles in metastasis of breast cancer. However, whether DAAM1 participates in OvCa migration and/or invasion is still unknown. The impact of DAAM1 on cell migration and invasion in OvCa was evaluated by wound healing assay and Boyden chamber assay. The specific miRNA targeting DAAM1 was predicted by bioinformatics methods and verified by dual-luciferase activity assay. The miR-208a-5p expression levels in OvCa tissues and the impacts of miR-208a-5p on cell migration and invasion were also assessed, respectively. High expression of DAAM1 was associated with distant metastasis in OvCa. Silence of DAAM1 by siRNA blocked the migration and invasion of OVCAR-3 cells. MiR-208a-5p directly targeted DAAM1 and was shown a decreased expression in metastatic OvCa tissues. Elevated expression of miR-208a-5p inhibited the migration and invasion of OVCAR-3 cell which can be rescued by DAAM1 overexpression. Our data suggest that miR-208-5p/DAAM1 axis participates in OvCa migration and invasion and may be a novel clinical target to limit OvCa metastasis.