PlexinA1 promotes gastric cancer migration through preventing MICAL1 protein ubiquitin/proteasome-mediated degradation in a Rac1-dependent manner.

Metastasis promotes the development of tumors and is a significant cause of gastric cancer death. For metastasis to proceed, tumor cells must become mobile by modulating their cytoskeleton. MICAL1 (Molecule Interacting with CasL1) is known as an actin cytoskeleton regulator, but the mechanisms by which it drives gastric cancer cell migration are still unclear. Analysis of gastric cancer tissues revealed that MICAL1 expression is dramatically upregulated in stomach adenocarcinoma (STAD) samples as compared to noncancerous stomach tissues. Patients with high MICAL1 expression had shorter overall survival (OS), post-progression survival (PPS) and first-progression survival (FPS) compared with patients with low MICAL1 expression. RNAi-mediated silencing of MICAL1 inhibited the expression of Vimentin, a protein involved in epithelial-mesenchymal transition. This effect correlates with a significant reduction in gastric cancer cell migration. MICAL1 overexpression reversed these preventive effects. Immunoprecipitation experiments and immunofluorescence assays revealed that PlexinA1 forms a complex with MICAL1. Importantly, specific inhibition of PlexinA1 blocked the Rac1 activation and ROS production, which, in turn, impaired MICAL1 protein stability by accelerating MICAL1 ubiquitin/proteasome-dependent degradation. Overexpression of PlexinA1 enhanced Rac1 activation, ROS production, MICAL1 and Vimentin expressions, and favored cell migration. In conclusion, this study identified MICAL1 as an important facilitator of gastric cancer cell migration, at least in part, by affecting Vimentin expression and PlexinA1 promotes gastric cancer cell migration by binding to and suppressing MICAL1 degradation in a Rac1/ROS-dependent manner.

Striatal CDK5 Regulates Cholinergic Neuron Activation and Dyskinesia-like Behaviors through BK Channels.

Disturbance of the cholinergic system plays a crucial role in the pathological progression of neurological diseases that cause dyskinesia-like behaviors. However, the molecular mechanisms underlying this disturbance remain elusive. Here, we showed that cyclin-dependent kinase 5 (Cdk5) was reduced in cholinergic neurons of midbrain according to the single-nucleus RNA sequencing analysis. Serum levels of CDK5 also decreased in patients with Parkinson's disease accompanied by motor symptoms. Moreover, Cdk5 deficiency in cholinergic neurons triggered paw tremors, abnormal motor coordination, and motor balance deficits in mice. These symptoms occurred along with cholinergic neuron hyperexcitability and increases in the current density of large-conductance Ca2+-activated K+ channels (BK channels). Pharmacological inhibition of BK channels restrained the excessive intrinsic excitability of striatal cholinergic neurons in Cdk5-deficient mice. Furthermore, CDK5 interacted with BK channels and negatively regulated BK channel activity via phosphorylation of threonine-908. Restoration of CDK5 expression in striatal cholinergic neurons reduced dyskinesia-like behaviors in ChAT-Cre;Cdk5f/f mice. Together, these findings indicate that CDK5-induced phosphorylation of BK channels involves in cholinergic-neuron-mediated motor function, providing a potential new therapeutic target for treating dyskinesia-like behaviors arising from neurological diseases.

TGFß signaling activation correlates with immune-inflamed tumor microenvironment across human cancers and predicts response to immunotherapy.

Considering the determining role of TGFß signaling in the tumor microenvironment (TME) on immune evasion, the inhibition of signaling is expected to enhance the therapeutic efficacy of immunotherapies, especially immune checkpoint blockade (ICB), which is confirmed in preclinical data. However, successive failures in clinical translation occur at the initial stage. To provide a better understanding of TGFß signaling within the TME and its relation to the individual immunological status, we performed a pan-cancer analysis comparing the activation of TGFß pathway among different TMEs based on multi-omics data. Compared with non-inflamed tumors, increased TGFß signaling activity appeared in four non-cancer cell types within TME in inflamed tumors. Significant correlations were revealed between TGFß signaling and reliable biomarkers for ICB therapy, as well as between TGFß signaling and HPV status. Our findings contribute to explain the inconsistency between preclinical and clinical research, and are crucial to optimizing upcoming clinical trial design and improving patient stratification for personalized prediction.

Comprehensive Analysis of MICALL2 Reveals Its Potential Roles in EGFR Stabilization and Ovarian Cancer Cell Invasion.

Molecules interacting with CasL (MICALs) are critical mediators of cell motility that act by cytoskeleton rearrangement. However, the molecular mechanisms underlying the regulation of cancer cell invasion remain elusive. The aim of this study was to investigate the potential role of one member of MICALs, i.e., MICALL2, in the invasion and function of ovarian cancer cells. We showed by bioinformatics analysis that MICALL2 expression was significantly higher in tissues of advanced-stage ovarian cancer and associated with poor overall survival of patients. MICALL2 was strongly correlated with the infiltration of multiple types of immune cells and T-cell exhaustion markers. Moreover, enrichment analyses showed that MICALL2 was involved in the tumor-related matrix degradation pathway. Mechanistically, MMP9 was identified as the target gene of MICALL2 for the regulation of invadopodium formation and SKOV3, HO-8910PM cell invasion. In addition, EGFR-AKT-mTOR signaling was identified as the downstream pathway of MICALL2 in the regulation of MMP9 expression. Furthermore, MICALL2 silencing promoted EGFR degradation; however, this effect was abrogated by treatment with the autophagy inhibitors acadesine and chloroquine diphosphate. Silencing of MICALL2 resulted in a suppressive activity of Rac1 while suppressing Rac1 activation attenuated the pro-EGFR, pro-MMP9, and proinvasive effects induced by the overexpression of MICALL2. Collectively, our results indicated that MICALL2 participated in the process of immune infiltration and invasion by ovarian cancer cells. Moreover, MICALL2 prevented EGFR degradation in a Rac1-dependent manner, consequently leading to EGFR-AKT-mTOR-MMP9 signaling activation and invadopodia-mediated matrix degradation.

MICAL2 contributes to gastric cancer cell migration via Cdc42-dependent activation of E-cadherin/ß-catenin signaling pathway.

BACKGROUND: Gastric cancer is a common and lethal human malignancy worldwide and cancer cell metastasis is the leading cause of cancer-related mortality. MICAL2, a flavoprotein monooxygenase, is an important regulator of epithelial-to-mesenchymal transition. The aim of this study was to explore the effects of MICAL2 on gastric cancer cell migration and determine the underlying molecular mechanisms. METHODS: Cell migration was examined by wound healing and transwell assays. Changes in E-cadherin/ß-catenin signaling were determined by qPCR and analysis of cytoplasmic and nuclear protein fractions. E-cadherin/ß-catenin binding was determined by co-immunoprecipitation assays. Cdc42 activity was examined by pulldown assay. RESULTS: MICAL2 was highly expressed in gastric cancer tissues. The knockdown of MICAL2 significantly attenuated migratory ability and ß-catenin nuclear translocation in gastric cancer cells while LiCl treatment, an inhibitor of GSK3ß, reversed these MICAL2 knockdown-induced effects. Meanwhile, E-cadherin expression was markedly enhanced in MICAL2-depleted cells. MICAL2 knockdown led to a significant attenuation of E-cadherin ubiquitination and degradation in a Cdc42-dependent manner, then enhanced E-cadherin/ß-catenin binding, and reduced ß-catenin nuclear translocation. CONCLUSIONS: Together, our results indicated that MICAL2 promotes E-cadherin ubiquitination and degradation, leading to enhanced ß-catenin signaling via the disruption of the E-cadherin/ß-catenin complex and, consequently, the promotion of gastric cell migration. Video Abstract.

MICAL2 Contributes to Gastric Cancer Cell Proliferation by Promoting YAP Dephosphorylation and Nuclear Translocation.

Dynamic cytoskeletal rearrangements underlie the changes that occur during cell division in proliferating cells. MICAL2 has been reported to possess reactive oxygen species- (ROS-) generating properties and act as an important regulator of cytoskeletal dynamics. However, whether it plays a role in gastric cancer cell proliferation is not known. In the present study, we found that MICAL2 was highly expressed in gastric cancer tissues, and this high expression level was associated with carcinogenesis and poor overall survival in gastric cancer patients. The knockdown of MICAL2 led to cell cycle arrest in the S phase and attenuated cell proliferation. Concomitant with S-phase arrest, a decrease in CDK6 and cyclin D protein levels was observed. Furthermore, MICAL2 knockdown attenuated intracellular ROS generation, while MICAL2 overexpression led to a decrease in the p-YAP/YAP ratio and promoted YAP nuclear localization and cell proliferation, effects that were reversed by pretreatment with the ROS scavenger N-acetyl-L-cysteine (NAC) and SOD-mimetic drug tempol. We further found that MICAL2 induced Cdc42 activation, and activated Cdc42 mediated the effect of MICAL2 on YAP dephosphorylation and nuclear translocation. Collectively, our results showed that MICAL2 has a promotive effect on gastric cancer cell proliferation through ROS generation and Cdc42 activation, both of which independently contribute to YAP dephosphorylation and its nuclear translocation.

MICAL2 Facilitates Gastric Cancer Cell Migration via MRTF-A-Mediated CDC42 Activation.

Aims and Hypothesis: Cell migration is driven by the reorganization of the actin cytoskeleton. Although MICAL2 is known to mediate the oxidation of actin filaments to regulate F-actin dynamics, relatively few studies have investigated the potential role of MICAL2 during cancer cell migration. Methods: The migratory ability of gastric cancer cells was measured by wound healing and transwell assays. The relationship between MICAL2 expression and MRTF-A nuclear localization was analyzed using gene overexpression and knockdown strategies. The production of reactive oxygen species (ROS) was evaluated by DCFH-DA staining. mRNA and protein levels of MMP9 were measured using qPCR and immunoblotting analysis. The activities of CDC42 and RhoA were assessed using pulldown assays. Results: Depletion of MICAL2 markedly reduced gastric cancer cell migration. Mechanistically, silencing of MICAL2 inhibited the nuclear translocation of MRTF-A in response to EGF and serum stimulation, whereas the contents of MRTF-A remained unchanged. Further analysis showed that silencing of MICAL2 decreased the activation of CDC42 as well as mRNA and protein levels of MMP9. Ectopic expression of MICAL2 augmented MRTF-A levels in the nucleus, and promoted the activation of CDC42, MMP9 expression, and gastric cancer cell migration. Moreover, silencing of MRTF-A inhibited the CDC42 activation induced by overexpression of MICAL2. In addition, MICAL2-induced ROS generation contributed to the effect exerted by MICAL2 on MRTF-A nuclear translocation. Conclusion: Together, these results provide evidence that MICAL2 facilitates gastric cancer cell migration via positive regulation of nuclear translocation of MRTF-A and subsequent CDC42 activation and MMP9 expression.

GRPr-mediated photothermal and thermodynamic dual-therapy for prostate cancer with synergistic anti-apoptosis mechanism.

Conventional prostate cancer treatment strategies, including chemotherapy and radiotherapy, cannot effectively eradicate prostate cancer, especially castration resistance prostate cancer. Herein, we developed a novel nanotherapy platform that consists of synergic photothermal and photodynamic therapy via the unique properties of photothermal conversion by gold nanorods and free radicals generation by encapsulated initiators (AIPH). Mesoporous silica was employed as a coating material, and the bombesin peptide was conjugated onto the mesoporous silica coating layer as the targeting moiety to prostate cancer via its overexpressed gastrin-releasing peptide receptors. An in vitro study with the castration resistance prostate cancer cell exhibited a significant photothermal therapeutic effect as well as enhanced thermodynamic therapy via generating free radicals. P-p38 and p-JNK proteins, as key proteins involved in the cells' stress responses, were found to be upregulated by the synergetic treatment. The in vivo study demonstrated that a significant eradication of prostate tumour could be achieved by the nanoparticle therapeutic platform with a good biocompatibility profile. This work pioneers a novel approach for high-efficient castration resistance prostate cancer treatment by combining photothermal, thermodynamic, and site-specific drug delivery directed by an integrated nanoparticle system.

FLCN Regulates HIF2α Nuclear Import and Proliferation of Clear Cell Renal Cell Carcinoma.

Aims and Hypothesis: This study aims to explore the specific molecular mechanism of folliculin (FLCN)-induced proliferation, migration, and invasion in clear cell renal cell carcinoma (ccRCC) and to investigate the relationship of FLCN and HIF2α. Folliculin was identified as a tumor suppressor gene. Its deletions and mutations are associated with a potential risk of renal cancer. At present, the specific molecular mechanism of FLCN-induced proliferation, invasion, and migration in ccRCC remains elusive. Methods: Cell proliferation was measured by flow cytometry analysis, while cell migration and invasion were measured by wound healing assay and Matrigel invasion assay. The expression of FLCN, HIF2α, MMP9, and p-AKT was examined by Western blotting. The cells were transfected with plasmids or siRNA to upregulate or downregulate the expression of FLCN. Immunofluorescence microscopy was carried out to display the HIF2α location. We also determined the correlation of FLCN and HIF2α in human renal cancer samples. Results: FLCN was combined with HIF2α in renal tubular epithelial and cancer cells, and it effectively alleviates the deterioration of renal cancer cells by degrading HIF2α. The silencing of FLCN showed a promotion of HIF2α protein expression via PI3K/mTORC2 pathway, which in turn led to an increase in downstream target genes Cyclin D1 and MMP9. Moreover, interfering with siFLCN advanced the time of HIF2α entry into the nucleus. Conclusions: Our study illustrated that FLCN could be a new therapeutic target in ccRCC. FLCN combined with HIF2α and identified a novel PI3K/mTORC2/HIF2α signaling in ccRCC cells.

MICAL-L2 Is Essential for c-Myc Deubiquitination and Stability in Non-small Cell Lung Cancer Cells.

Objectives: MICAL-L2, a member of the molecules interacting with the CasL (MICAL) family, was reported to be highly expressed in several types of cancers, however, the roles of MICAL-L2 in NSCLC pathogenesis remain to be explored. This study is designed to clarify the mechanisms by which MICAL-L2 participates in NSCLC cell proliferation. Materials and Methods: The expression levels of MICAL-L2 in human lung cancer samples were assessed by immunohistochemical staining. Cells were transfected with siRNA or plasmids to regulate MICAL-L2 expression. Cell proliferation was measured by EdU staining and CCK-8 assays. MICAL-L2 and phosphorylated/total c-Myc expression were examined by Western blotting analysis. Interaction between MICAL-L2 and c-Myc was assessed by immunofluorescence staining, Western blotting and co-immunoprecipitation assays. Western blotting, polyubiquitylation detection and protein stability assays were used to assess whether MICAL-L2 exerts its oncogenic effect via c-Myc. Results: We found that MICAL-L2 was highly expressed in human NSCLC. While overexpressing MICAL-L2 increased NSCLC cell proliferation, MICAL-L2 depletion decreased the proliferation of NSCLC cells, an effect that was linked to cell cycle arrest. MICAL-L2 physically interacted with the c-Myc protein and functioned to maintain nuclear c-Myc levels and prolonged its half-life. Knockdown of MICAL-L2 expression led to decreased c-Myc protein stability through accelerating polyubiquitylation of c-Myc and gave rise to c-Myc degradation. We further found that MICAL-L2 deubiquitinated c-Myc and blocked its degradation, presumably by inhibiting c-Myc phosphorylation at threonine residue 58. Conclusions: These results indicate that MICAL-L2 is a key regulator of c-Myc deubiquitination and stability in the nucleus, and this activity may be involved in promoting NSCLC cell proliferation.

SOX2 promotes hypoxia-induced breast cancer cell migration by inducing NEDD9 expression and subsequent activation of Rac1/HIF-1α signaling.

BACKGROUND: Hypoxia, a major condition associated with the tumor microenvironment, stimulates the migration of cancer cells. SOX2 is a powerful transcription factor that shows higher expression in several cancers, however, its role in hypoxia-induced breast cancer cell migration remains largely elusive. METHODS: The human breast cancer cell lines MDA-MB-231 and MDA-MB-468 were cultured under hypoxic conditions. The cell migration rate was determined using the wound-healing and transwell assays. The protein levels of SOX2, NEDD9 and HIF-1α were evaluated via western blotting analysis. The NEDD9 mRNA levels were evaluated using qPCR. The activation of Rac1 was detected with the pulldown assay. The binding of SOX2 to the NEDD9 promoter was checked using the luciferase reporter assay. We also transfected breast cancer cells with specific siRNA for SOX2, NEDD9 or the Rac1 inactive mutant (T17 N) to investigate the role of SOX2, NEDD9 and Rac1 in the response to hypoxia. RESULTS: Hypoxia markedly increased SOX2 protein levels in a time-dependent manner. SiRNA-mediated disruption of SOX2 inhibited cell migration under hypoxic conditions. Hypoxia also significantly augmented the NEDD9 mRNA and protein levels. Interestingly, SOX2 is a positive transcriptional regulator of NEDD9. Knockdown of SOX2 inhibited hypoxia-induced NEDD9 mRNA and protein expressions. Furthermore, hypoxia-induced upregulation of Rac1 activity and HIF-1α expression was attenuated by SOX2 or NEDD9 silencing, and Rac1-T17 N abolished HIF-1α expression as well as cell migration in cells subjected to hypoxia. CONCLUSIONS: Our results highlight the essential role of SOX2 in breast cancer cell motility. The upregulation of SOX2 under hypoxic conditions may facilitate NEDD9 transcription and expression, and subsequent activation of Rac1 and HIF-1α expression. This could accelerate breast cancer cell migration.

Non-canonical Notch Signaling Regulates Actin Remodeling in Cell Migration by Activating PI3K/AKT/Cdc42 Pathway.

Tumor cell migration is a critical step in cancer metastasis. Over-activated Notch pathway can promote the migration of cancer cells, especially in the breast cancer. However, the underlying mechanism of non-canonical Notch signaling in modulating the migration has not yet been clearly characterized. Here we demonstrated that DAPT, a gamma secretase inhibitor, inhibited protrusion formation and cell motility, and then reduced the migration of triple-negative breast cancer cells, through increasing the activity of Cdc42 by non-canonical Notch pathway. Phosphorylation of AKT on S473 was surprisingly increased when Notch signaling was inhibited by DAPT. Inhibition of PI3K and AKT by LY294002 and MK2206, respectively, or knockdown of AKT expression by siRNA blocked DAPT-induced activation of Cdc42. Moreover, immunofluorescence staining further showed that DAPT treatment reduced the formation of lamellipodia and induced actin cytoskeleton remodeling. Taken together, these results indicated that DAPT inhibited Notch signaling and consequently activated PI3K/AKT/Cdc42 signaling by non-canonical pathway, facilitated the formation of filopodia and inhibited the assembly of lamellipodia, and finally resulted in the decrease of migration activity of breast cancer cells.

NEDD9 Facilitates Hypoxia-Induced Gastric Cancer Cell Migration via MICAL1 Related Rac1 Activation.

Aims and Hypothesis: NEDD9 is highly expressed in gastric cancer and has a significant involvement in its pathogenesis. However, the mechanism behind hypoxia-promoted cancer cell migration and its regulation because of NEDD9 is still unknown. The aim of this study is to investigate the involvement of NEDD9 in gastric cancer cell migration under hypoxia and explore the underlying potential molecular mechanisms. METHODS: Cell motility was measured by wound healing and transwell assay. NEDD9 and MICAL1 expressions were examined by western blot analysis. Interaction between NEDD9 and MICAL1 was assessed by immunohistochemistry and co-immunoprecipitation assay, respectively. Cells were transfected with plasmids or siRNA to upregulate or downregulate the expression of NEDD9 and MICAL1. Rac1, Cdc42, and RhoA activation was assessed by pulldown assay. RESULTS: The mRNA and protein level of NEDD9 increased as a result of hypoxia in gastric cancer cell lines BGC-823 and SGC-7901 while decreased levels of NEDD9 caused reduced cell migratory potential in response to hypoxia. Hypoxia also caused the enhancement of MICAL1 expression. Furthermore, it was revealed that there is a positive correlation between NEDD9 and MICAL1 protein while hypoxia played role in increasing their interaction. Under hypoxic conditions, silencing of NEDD9 caused reduction in the stability of MICAL1 protein, while depletion of MICAL1 also inhibited the migration of NEDD9-overexpressing gastric cancer cells. In addition, silencing of NEDD9 or MICAL1 expression reversed the increased GTP forms of Rac1 and Cdc42 in hypoxic cells. However, only the upregulation of Rac1-GTP level was observed in gastric cancer cells that were already overexpressed by MICAL1. CONCLUSION: In all, it is concluded that MICAL1 is regulated by NEDD9 that facilitates hypoxia-induced gastric cancer cell migration via Rac1-dependent manner.

MICAL-L2 potentiates Cdc42-dependent EGFR stability and promotes gastric cancer cell migration.

Enhanced migration potential is a common characteristic of cancer cells induced by mechanisms that are incompletely defined. The present study was designed to investigate relationship of a new discovered cytoskeleton regulator MICAL-L2 and the endogenous epidermal growth factor receptor (EGFR) signalling pathways in gastric cancer cell migration. Increased expression of MICAL-L2 in gastric cancer cells up-regulated EGFR protein level, accompanied by the increase of cell migration, whereas silencing MICAL-L2 down-regulated EGFR and inhibited cell migration. Expression of MICAL-L2 was also shown positively correlated with the activation of HSP27/cytoskeleton and HSP27/ß-catenin signalling pathways that provide key mechanisms controlling cell migration. The up-regulating effect of MICAL-L2 on EGFR is mediated through a transcription-independent mechanism that involves inhibiting EGFR protein degradation in lysosome. Further analysis indicated that Cdc42 activation contributed in maintaining the effect of MICAL-L2 on EGFR stability. Furthermore analysis of clinic specimens revealed increased expression of MICAL-L2 in carcinoma tissues and a positive correlation between MICAL-L2 and EGFR expression levels. The above results indicate that MICAL-L2 potentiates gastric cell migration via inhibiting EGFR degradation in lysosome via a Cdc42-dependent manner that leads to the activation of EGFR/HSP27 signalling pathways.