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Abstract 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.

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 17β-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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 3β-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.

Objective：To explore the expression and clinical significance of dishevelled-associated activator of morphogenesis 1 (DAAM1)and DAAM2 in pancreatic cancer. Methods：Pancreatic cancer tissue microarray(TMA, HPanA120Su02)was obtained from OUTDO BioTech(Shanghai), including 66 tumor samples and 54 adjacent samples. Immunohistochemistry(IHC)was used to detect the expression levels of DAAM1 and DAAM2 in pancreatic cancer and para-tumor tissues. Expression profiles of DAAM1, DAAM2, PDL1 and other immune checkpoints were downloaded from the TCGA database. Statistical methods, such as t-test, χ2 test, log-rank test and correlation analysis, were used to analyze the differential expression of DAAM1 and DAAM2 between tumor and adjacent samples, the co -expression pattern of DAAM1 and DAAM2, and their association with clinicopathological parameters, prognosis and expression levels of immune checkpoints. Results：Compared with the adjacent tissues, the expression of DAAM1 and DAAM2 in pancreatic cancer were significantly upregulated (P ＜ 0.001), and the expression levels of DAAM1 and DAAM2 in pancreatic cancer tissue were significantly positively correlated (P ＜ 0.001). There was marginally significant in the correlation between DAAM1 expression and tumor differentiation (P=0.062)and survival status (P=0.061), but DAAM1 expression was not significantly correlated with other clinic-pathological parameters (P ＞ 0.05). However, DAAM2 expression was not related to any clinic-pathological parameters (P ＞ 0.05). In the TCGA database, DAAM1 and DAAM2 were positively correlated with the expression levels of multiple immune checkpoints (P ＜ 0.001). Conclusion：The expression of DAAM1 and DAAM2 are significantly upregulated in pancreatic cancer, and positively correlated with the expression levels of multiple immune checkpoints, which may be a critical regulatory factor for the oncogenesis, progression and immune evasion in pancreatic cancer. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 17β-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.

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.

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 TGFβ1 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 TGFβ1 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 β1- and β5-integrin ubiquitination. This resulted in increased αv-, β1-, and β5-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. [Display omitted] • We have elucidated a novel interaction between the formin, DAAM1 and the deubiquitinase, USP10. • DAAM1 binds to and inhibits USP10's DUB activity through the FH2 domain of DAAM1 independent of its actin functions. • DAAM1 inhibition of USP10's DUB activity subsequently affects integrin protein levels and integrin and matrix cell surface recycling. • The USP10/DAAM1 axis regulates integrin homeostasis that is important for a myriad of cellular processes. [ABSTRACT FROM AUTHOR]

[Display omitted] 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. 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. 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. 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. [ABSTRACT FROM AUTHOR]

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 3β-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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Venditti, Ben Hadj Hassine, Messaoudi and Minucci.)

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. [ABSTRACT FROM AUTHOR]

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., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

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.