Phosphorylation Of HSP27 Research Articles

Neuroblastoma response to RAS-MAPK inhibitors and APR-246 (eprenetapopt) co-treatment is dependent on SLC7A11

BackgroundWe previously demonstrated that APR-246 (eprenetapopt) could be an efficient treatment option against neuroblastoma (NB), the most common pediatric extracranial solid tumor. APR-246’s mechanism of action is not completely understood and can differ between cell types. Here we investigate the involvement of well-known oncogenic pathways in NB’s response to APR-246.MethodsA proteome profiler kinase assays and western blot analysis were used to identify the molecular pathways involved in the responses to APR-246. Bulk ATP levels were used to determine the viability of cells and the IC50 for APR-246. Cystine-FITC was used to measure the cellular uptake of cysteine. PmRNA5 was used to activate ERK1/2 and pshRNA1 was used to silence HSP27. An IMR-32 xenograft zebrafish embryo model was used to assess APR-246 and sulfasalazine efficacy in vivo.ResultsAfter APR-246 treatment, the most deregulated signaling protein identified was ERK1/2, an end-point kinase of the RAS-MAPK pathway. Induction of phospho-ERK1/2 resulted in increased glutathione (GSH) levels, increased cystine uptake, and increased resistance of NB cells to APR-246. Using ERK1/2 inhibitors in combination with APR-246, we were able to categorize cells into synergistic and antagonistic groups. After co-treatment, these two groups differ by their levels of SLC7A11 and Hsp27 phosphorylation, cystine uptake, and BIM expression. Using erastin and sulfasalazine, both inhibitors of SLC7A11 and activators of ferroptosis, we were able to reverse the antagonistic effects of ERK1/2 inhibitors and demonstrate a strong synergistic action in vitro and in vivo in zebrafish models.ConclusionsThese results demonstrated a pivotal role of the RAS-MAPK pathway in the NB cellular response to APR-246 via the modulation of intracellular concentrations of GSH and the transport of cystine through SLC7A11, phosphorylation of Hsp27, and programmed cell death. Combining APR-246 with RAS-MAPK pathway inhibitors can, in some cases, lead to antagonistic action, which can be reversed by combining APR-246 with the clinically approved drug sulfasalazine.

Lysosomal damage triggers a p38 MAPK-dependent phosphorylation cascade to promote lysophagy via the small heat shock protein HSP27.

Maintenance of lysosomal integrity is essential for cell viability. Upon injury, lysosomes may be targeted for degradation via a selective form of autophagy known as lysophagy. The engulfment of a damaged lysosome by an autophagosome is mediated by the recruitment of adaptor proteins, including SQSTM1/p62. p62 promotes lysophagy via the formation of phase-separated condensates in a mechanism that is regulated by the heat shock protein HSP27. Here, we demonstrate a direct interaction between HSP27 and p62. We used structural modeling to predict the binding interface between HSP27 and p62 and identify several disease-associated mutations that map to this interface. We used proteomics to identify post-translational modifications of HSP27 that regulate HSP27 recruitment to stressed lysosomes, finding robust phosphorylation at several serine residues. Next, we characterized the upstream signaling mechanism leading to HSP27 phosphorylation and found that p38 mitogen-activated protein kinase (MAPK) and its effector kinase MAP kinase-activated protein kinase 2 (MK2) are activated upon lysosomal damage by the kinase mTOR and the production of intracellular reactive oxygen species (ROS). Increased ROS activates p38 MAPK, which in turn allows MK2-dependent phosphorylation of HSP27. Depletion of HSP27 or the inhibition of HSP27 phosphorylation alters the dynamics of p62 condensates on stressed lysosomes, significantly inhibiting p62-dependent lysophagy. Thus, we define a novel lysosomal quality control mechanism in which lysosomal injury triggers a p38 MAPK/MK2 signaling cascade promoting p62-dependent lysophagy. Further, this signaling cascade is activated by many cellular stressors, including oxidative and heat stress, suggesting that other forms of selective autophagy may be regulated by p38 MAPK/MK2/HSP27.

Effect of phosphorylated HSP27 on the proliferation and metastasis of nasopharyngeal carcinoma and its mechanism

Objective:To investigate the effect of phosphorylated HSP27 on the proliferation and metastasis of nasopharyngeal carcinoma and its molecular mechanism. Methods:①Western blot assay was used to detect the expression levels of HSP27 and p-HSP27 in CNE1 and CNE2 cells of nasopharyngeal carcinoma. Inhibited the phosphorylation of HSP27, Transwell assay detected the metastasis ability of nasopharyngeal carcinoma cells. ②Nasopharyngeal carcinoma cell lines（CNE2-OE1 and CNE2-OE2） overexpressing phosphorylated and dephosphorylated mutants of HSP27 were synthesized, empty vector transfected CNE2 cells（CNE2-NC） were used as controls. The proliferation ability of the three groups of cells was detected by CCK8, and the expression levels of CyclinD1, Bax and Bcl-2 were detected by Western blot. Transwell was used to detect the migration and invasion ability of cells, and Western blot was used to detect the expression levels of E-cadherin, Vimentin, MMP2 and MMP9. Results:The expression level of HSP27 in CNE2 was higher than that of CNE1 cells, while the expression level of p-HSP27 was opposite. After inhibition of HSP27 phosphorylation, the invasion and migration ability of CNE1 cells decreased significantly, with no significant change in CNE2 cells. Compared with CNE2-NC, the growth rate of CNE2-OE1 decreased, and the growth rate of CNE2-OE2 increased. The expression level of CyclinD1 was down-regulated in CNE2-OE1 and higher in CNE2-OE2. The expression level of Bax in CNE2-OE1 was increased, while the expression of Bcl-2 was decreased. There was no significant change in the expression of Bax and Bcl-2 in CNE2-OE2. Compared with CNE2-NC, the migration ability of CNE2-OE1 was enhanced and the invasion ability was weakened, while the migration ability of CNE2-OE2 was weakened and the invasion ability was enhanced. There was no significant difference in the expression levels of E-cadherin was decreased in CNE2-OE1 and increased in CNE2-OE2. There was no significant difference in the expression levels of Vimentin among the three groups. The expression levels of MMP2 and MMP9 were up-regulated in CNE2-OE2, and slightly down-regulated in CNE2-OE1. Conclusion:HSP27 and p-HSP27 were differentially expressed in different nasopharyngeal carcinoma cells, and the metastasis ability of nasopharyngeal carcinoma was not only related to the expression level of HSP27, but also related to the level of p-HSP27. The p-HSP27 inhibited CNE 2 cell proliferation and promoted their apoptosis. As p-HSP27 plays different roles in different stages of nasopharyngeal carcinoma metastasis, it can increase the migration ability of CNE2 cells and reduce its invasion ability. p-HSP27 may induce EMT changes in CNE2 by down-regulating E-cadherin, thus promoting CNE2 migration, and may inhibit CNE2 invasion by down-regulating MMPs expression.

Analysis of potential TAK1/Map3k7 phosphorylation targets in hypertrophy and cachexia models of skeletal muscle.

TGFβ-activated kinase-1 (TAK1) is phosphorylated during both muscle growth and muscle wasting. To understand how this can lead to such opposite effects, we first performed multiplex kinase array of mouse embryonic stem cells with and without stimulation of TAK1 to determine its potential downstream targets. The phosphorylation of these targets was then compared in three different models: hypertrophic longissimus muscle of Texel sheep, tibialis anterior muscle of mice with cancer-induced cachexia and C2C12-derived myofibers, with and without blockade of TAK1 phosphorylation. In both Texel sheep and in cancer-induced cachexia, phosphorylation of both TAK1 and p38 was increased. Whereas p90RSK was increased in Texel sheep but not cachexia and the phosphorylation of HSP27 and total Jnk were increased in cachexia but not Texel. To understand this further, we examined the expression of these proteins in C2C12 cells as they differentiated into myotubes, with and without blockade of TAK1 phosphorylation. In C2C12 cells, decreased phosphorylation of TAK1 leads to reduced phosphorylation of p38, JNK, and HSP27 after 16 h and muscle fiber hypertrophy after 3 days. However, continuous blockade of this pathway leads to muscle fiber failure, suggesting that the timing of TAK1 activation controls the expression of context-dependent targets.

Exploring the impact of GSTM1 as a novel molecular determinant of survival in head and neck cancer patients of African descent

BackgroundBlacks/African American (BAA) patients diagnosed with head and neck squamous cell carcinoma (HNSCC) have worse survival outcomes than White patients. However, the mechanisms underlying racial disparities in HNSCC have not been thoroughly characterized.MethodsData on gene expression, copy number variants (CNVs), gene mutations, and methylation were obtained from 6 head and neck cancer datasets. Comparative bioinformatics analysis of the above genomic features was performed between BAAs and Whites. The expression pattern of GSTM1 was validated by immunohistochemistry using tumor tissue microarray (TMA). Effect of GSTM1 knockdown were assessed by cell proliferation, colony formation, and tumor development in an orthotopic mouse model. The changes in protein kinases were determined using the Proteome Profiler Human Phospho-Kinase Array Kit in HNSCC cells with or without GSTM1 knockdown.ResultsWe identified ancestry-related differential genomic profiles in HNSCC. Specifically, in BAA HNSCC, FAT1 mutations were associated with its gene expression, SALL3 gene expression correlated with its gene CNVs, and RTP4 gene expression showed an inverse correlation with its methylation. Notably, GSTM1 emerged as a prognostic risk factor for BAA HNSCC, with high gene CNVs and expression levels correlating with poor overall survival in BAA patients. Immunohistochemistry results from newly developed in-house TMA validated the expression pattern of GSTM1 between BAA HNSCC and White HNSCC. In an orthotopic mouse model, GSTM1 knockdown significantly inhibited malignant progression in tumors derived from BAAs. In contrast, loss of GSTM1 did not affect the development of HNSCC originating in Whites. Mechanistically, GSTM1 knockdown suppressed HSP27 phosphorylation and β-catenin in BAA HNSCC cells, but not in White HNSCC cells. This differential effect at least partially contributes to tumor development in BAA patients.ConclusionThis study identifies GSTM1 as a novel molecular determinant of survival in HNSCC patients of African descent. It also provides a molecular basis for future research focused on identifying molecular determinants and developing therapeutic interventions to improve outcomes for BAA patients with HNSCC.

MiR-3074-5p suppresses non-small cell lung cancer progression by targeting the YWHAZ/Hsp27 axis

Non-small cell lung cancer (NSCLC) accounts for more than 80% of lung cancer cases, and the 5-year survival rate of patients remains unsatisfactory. MicroRNAs (miRNAs) are small endogenous noncoding RNAs that are considered essential posttranscriptional regulators of tumorigenesis, including NSCLC. In this study, we aimed to investigate the biological role of miR-3074-5p in NSCLC cells and the underlying molecular mechanisms. We showed that miR-3074-5p expression was decreased in human NSCLC specimens and cell lines. Moreover, miR-3074-5p overexpression inhibited cell proliferation, migration and invasion and induced apoptosis and cell cycle arrest. In addition, miR-3074-5p overexpression not only suppressed tumor growth but also enhanced the antitumor effect of paclitaxel (PTX) on NSCLC cells in vitro and in vivo. A transcriptome sequencing assay revealed genes that were differentially expressed after miR-3074-5p overexpression, and among the genes whose expression levels were most significantly decreased, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ) was a target of miR-3074-5p. The regulatory effect of miR‐3074-5p on YWHAZ expression was verified by Western blotting and dual‐luciferase reporter assays. The inhibition of A549 cell growth, migration and invasion was reversed by YWHAZ overexpression. Furthermore, we showed that PTX stimulated the expression of the YWHAZ and Hsp27 proteins and promoted the phosphorylation of Hsp27 (at S15 and S78). YWHAZ was confirmed to interact with Hsp27 in A549 cells, and downregulating YWHAZ expression promoted the degradation of the Hsp27 protein. Taken together, these results suggest that the miR‐3074-5p/YWHAZ/Hsp27 axis may be a novel therapeutic target for NSCLC treatment.

Network pharmacology and biochemical experiments reveal the antiapoptotic mechanism of huperzine A for treating diabetic retinopathy

Background/aimsDiabetic retinopathy is the most common eye disease that causes blindness in the working population. Neurodegeneration is the early sign of diabetic retinopathy, but no drug has been approved for...

Targeting Ser78 phosphorylation of Hsp27 achieves potent antiviral effects against enterovirus A71 infection

ABSTRACT A positive-sense (+) single-stranded RNA (ssRNA) virus (e.g. enterovirus A71, EV-A71) depends on viral polypeptide translation for initiation of virus replication after entry. We reported that EV-A71 hijacks Hsp27 to induce hnRNP A1 cytosol redistribution to initiate viral protein translation, but the underlying mechanism is still elusive. Here, we show that phosphorylation-deficient Hsp27-3A (Hsp27S15/78/82A) and Hsp27S78A fail to translocate into the nucleus and induce hnRNP A1 cytosol redistribution, while Hsp27S15A and Hsp27S82A display similar effects to the wild type Hsp27. Furthermore, we demonstrate that the viral 2A protease (2Apro) activity is a key factor in regulating Hsp27/hnRNP A1 relocalization. Hsp27S78A dramatically decreases the IRES activity and viral replication, which are partially reduced by Hsp27S82A. However, Hsp27S15A displays the same activity as the wild-type Hsp27. Peptide S78 potently suppresses EV-A71 protein translation and reproduction through blockage of EV-A71-induced Hsp27 phosphorylation and Hsp27/hnRNP A1 relocalization. A point mutation (S78A) on S78 impairs its inhibitory functions on Hsp27/hnRNP A1 relocalization and viral replication. Taken together, we demonstrate the importance of Ser78 phosphorylation of Hsp27 regulated by virus infection in nuclear translocation, hnRNP A1 cytosol relocation, and viral replication, suggesting a new path (such as peptide S78) for target-based antiviral strategy.

Human Plasma‐Derived Extracellular Vesicles Protect Against Cerebral Ischemia‐Reperfusion Injury via HSP27 Phosphorylation

AbstractIschemic stroke (IS) has become a serious public health problem, with patients undergoing endovascular treatment experiencing ischemia‐reperfusion (I/R) injury, which exacerbates cerebrovascular diseases. Circulating extracellular vesicles (EVs) have shown potential for treating cerebral I/R injury. In this study, the therapeutic effect of human plasma‐derived EVs and protective mechanisms in cerebral I/R injury is explored. An oxygen‐glucose deprivation/reperfusion (OGD/R) model is used to treat SH‐SY5Y cells in vitro, and an I/R injury model is constructed by transient middle cerebral artery occlusion (tMCAO) in mice. Human plasma‐derived EVs are extracted by size exclusion chromatography. Western blot, Immunofluorescence Staining, and 2,3,5‐Triphenyltetrazolium Chloride (TTC) Staining are employed to observe the effects of EVs on the neuroinflammatory response and infarct volumes in tMCAO mice, while TUNEL Staining, Flow Cytometry, and Western Blot are employed to assess cell apoptosis. Human plasma‐derived EVs alleviated apoptosis in SH‐SY5Y cells under OGD/R stress and exerted a protective effect against brain I/R injury in tMCAO mice. Mechanistically, EVs protected against cerebral I/R injury via HSP27 phosphorylation, and the HSP27 phosphorylation inhibitor KRIBB3 attenuated the anti‐apoptotic effects of EVs. Human plasma‐derived EVs activated the phosphorylation of HSP27, thereby inhibiting cell apoptosis and protecting against cerebral I/R injury.

Abstract 1568: Her2 promotes early dissemination of breast cancer by suppressing the p38 pathway through upregulation of Wip1

Abstract (a)Metastasis is the leading cause of cancer-related deaths. Increasing evidence shows that cancer cells can disseminate from early primary lesions that are impalpable, but the pathways are poorly understood. Her2 promotes breast cancer early dissemination by suppressing p38, but how Her2 downregulates p38 is unclear. In this study, we investigated the mechanism underlying suppression of p38 by Her2 in breast cancer early dissemination using cell models and xenograft models of early lesions of breast cancer. (b)We studied the role of Wip1, a p38 phosphatase, in disseminating phenotypes in cell line models of breast cancer early lesions, including cell motility measured in transwell assays, cell invasion measured by percentage of organoids with outward invading cells, and epithelial-to-mesenchymal transition (EMT) measured by levels of EMT markers in Western blotting analysis and immunofluorescence (IF) staining assays. (c)We demonstrate that high levels of Wip1 correlated with Her2+ status, and reduced phosphorylation of p38 and its downstream effectors MK2 and Hsp27 in tissue samples from early lesions of human breast cancer. In addition, ectopic expression of Wip1 reduced phosphorylation of p38, MK2 and Hsp27 in cell models of breast cancer early lesions.We found that Wip1 promoted migration, invasion and EMT, while Wip1 knockdown abrogated the ability of Her2 to induce these disseminating phenotypes, in cell line models of early lesion breast cancer. Thus, Wip1 induction is both sufficient, and necessary for Her2, to promote migration, invasion and EMT in early lesion breast cancer cells. In an attempt to identify the Wip1 downstream effectors involved in disseminating phenotypes, we found that ectopic expression of the constitutively active mutants of p38 reversed the ability of Wip1 to suppress MK2 and Hsp27 phosphorylation and to induce migration, invasion and EMT in early lesion breast cancer cells. These results suggest that Wip1 promotes breast cancer early dissemination by suppressing p38. We investigate the mechanism by which Her2 induces Wip1 expression. Ectopic expression of Her2 did not alter the level of Wip1 mRNA or the Wip1 protein stability, suggesting that Her2 promote Wip1 expression by regulating its translation. We are currently investigating the mechanism by which Her2 induces Wip1 translation.Moreover, inhibitors of Her2, Wip1 and Skp2 can each reduce the disseminating phenotypes in cells models of breast cancer early lesions ectopically expressing Her2, Wip1 or Skp2. (d)Our findings identify the Her2-Wip1-p38-Mk2-Hsp27 cascade as a novel mechanism mediating breast cancer early dissemination and provide a basis for new therapies targeting early metastatic dissemination in Her2+ breast cancer using inhibitors for Her2, Wip1 or Skp2. Citation Format: Nan Wang. Her2 promotes early dissemination of breast cancer by suppressing the p38 pathway through upregulation of Wip1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1568.

Mechanical activation and expression of HSP27 in epithelial ovarian cancer

Understanding the complex biomechanical tumor microenvironment (TME) is of critical importance in developing the next generation of anti-cancer treatment strategies. This is especially true in epithelial ovarian cancer (EOC), the deadliest of the gynecologic cancers due to recurrent disease or chemoresistance. However, current models of EOC progression provide little control or ability to monitor how changes in biomechanical parameters alter EOC cell behaviors. In this study, we present a microfluidic device designed to permit biomechanical investigations of the ovarian TME. Using this microtissue system, we describe how biomechanical stimulation in the form of tensile strains upregulate phosphorylation of HSP27, a heat shock protein implicated in ovarian cancer chemoresistance. Furthermore, EOC cells treated with strain demonstrate decreased response to paclitaxel in the in vitro vascularized TME model. The results provide a direct link to biomechanical regulation of HSP27 as a mediator of EOC chemoresistance, possibly explaining the failure of such therapies in some patients. The work presented here lays a foundation to elucidating mechanobiological regulation of EOC progression, including chemoresistance and could provide novel targets for anti-cancer therapeutics.

Phosphorylation of HSP27 regulates the fluidity of p62 phase condensates in autophagy

Phosphorylation of HSP27 regulates the fluidity of p62 phase condensates in autophagy

Tacrolimus reverses pemphigus vulgaris serum-induced depletion of desmoglein in HaCaT cells via inhibition of heat shock protein 27 phosphorylation

BackgroundGlucocorticoids are the first-line treatment for Pemphigus vulgaris (PV), but its serious side effects can be life-threatening for PV patients. Tacrolimus (FK506) has been reported to have an adjuvant treatment effect against PV. However, the mechanism underlying the inhibitory effect of FK506 on PV-IgG-induced acantholysis is unclear.ObjectiveThe objective of this study was to explore the effect of FK506 on desmoglein (Dsg) expression and cell adhesion in an immortalized human keratinocyte cell line (HaCaT cells) stimulated with PV sera.MethodsA cell culture model of PV was established by stimulating HaCaT cells with 5% PV sera with or without FK506 and clobetasol propionate (CP) treatment. The effects of PV sera on intercellular junctions and protein levels of p38 mitogen-activated protein kinase (p38MAPK), heat shock protein 27 (HSP27), and Dsg were assayed using western blot analysis, immunofluorescence staining, and a keratinocyte dissociation assay.ResultsPV sera-induced downregulation of Dsg3 was observed in HaCaT cells and was blocked by FK506 and/or CP. Immunofluorescence staining revealed that linear deposits of Dsg3 on the surface of HaCaT cells in the PV sera group disappeared and were replaced by granular and agglomerated fluorescent particles on the cell surface; however, this effect was reversed by FK506 and/or CP treatment. Furthermore, cell dissociation assays showed that FK506 alone or in combination with CP increased cell adhesion in HaCaT cells and ameliorated loss of cell adhesion induced by PV sera. Additionally, FK506 noticeably decreased the PV serum-induced phosphorylation of HSP 27, but had no effect on p38MAPK phosphorylation.ConclusionFK506 reverses PV-IgG induced-Dsg depletion and desmosomal dissociation in HaCaT cells, and this effect may be obtained by inhibiting HSP27 phosphorylation.

The heat shock protein Hsp27 controls mitochondrial function by modulating ceramide generation

Sphingolipids have key functions in membrane structure and cellular signaling. Ceramide is the central molecule of the sphingolipid metabolism and is generated by ceramide synthases (CerS) in the de novo pathway. Despite their critical function, mechanisms regulating CerS remain largely unknown. Using an unbiased proteomics approach, we find that the small heat shock protein 27 (Hsp27) interacts specifically with CerS1 but not other CerS. Functionally, our data show that Hsp27 acts as an endogenous inhibitor of CerS1. Wild-type Hsp27, but not a mutant deficient in CerS1 binding, inhibits CerS1 activity. Additionally, silencing of Hsp27 enhances CerS1-generated ceramide accumulation in cells. Moreover, phosphorylation of Hsp27 modulates Hsp27-CerS1 interaction and CerS1 activity in acute stress-response conditions. Biologically, we show that Hsp27 knockdown impedes mitochondrial function and induces lethal mitophagy in a CerS1-dependent manner. Overall, we identify an important mode of CerS1 regulation and CerS1-mediated mitophagy through protein-protein interaction with Hsp27.

Exercise Prevents Glucocorticoid-Induced Myocardial 4-Hydroxynonenal Production.

Long-term administration of glucocorticoids (GCs) increases myocardial oxidative stress. 4-Hydroxynonenal (4-HNE) protein adducts, a marker of oxidative damage, have been associated with several cardiovascular diseases, including atherosclerosis, cardiac hypertrophy, cardiomyopathy, and ischemia-reperfusion injury. Exercise training has been shown to have a protective effect on the heart by lowering the level of oxidative stress in cardiomyocytes. Therefore, we aimed to investigate the effect of long-term dexamethasone treatment and exercise training on myocardial 4-HNE levels. Twenty-four female Wistar albino rats were assigned to sedentary control-saline treated (C, n = 8), sedentary-dexamethasone treated (D, n = 8), and exercise training-dexamethasone treated (DE, n = 8) groups. Daily dexamethasone was injected for 28days at a 1mgkg-1 dose, while C animals were injected with the same volume of saline subcutaneously. DE animals underwent an exercise training protocol of 60min/day, 5days a week, at 25m/min-1 (0% grade) for 28days. Left ventricular 4-HNE, Hsp72 levels, and pHsp25/Hsp25 ratio were determined by Western blot. The administration of dexamethasone led to a significant elevation in 4-HNE levels in the myocardium of adult rats (p < 0.05; D vs. C). The concurrent implementation of exercise training impeded this increase (p > 0.05; DE vs. C). Exercise training induced a threefold increase in myocardial Hsp72 expression (p < 0.001; DE vs. C and D) and attenuated the dexamethasone-induced increase in Hsp25 phosphorylation (p < 0.05; C vs. D) (p < 0.001; DE vs. D). Our results indicate that long-term administration of dexamethasone is associated with an increase in cardiac 4-HNE levels, which is hindered by the addition of exercise training.

Effect of suppressing the p38 pathway through upregulation of Wip1 on Her2 early dissemination of breast cancer.

e13029 Background: Metastasis is the leading cause of cancer-related deaths. While mechanisms for metastasis were extensively studied in cancer cells from patients with detectable tumors, pathways underlying metastatic dissemination from early lesions are poorly understood. Her2 promotes breast cancer early dissemination by suppressing p38, but how it downregulates p38 is unclear. In this study, we investigated the mechanism underlying suppression of p38 by Her2 in breast cancer early dissemination using cell models of early lesions of breast cancer. Methods: We studied the role of Wip1 in disseminating phenotypes in cell line models of breast cancer early lesions. These disseminating phenotypes include cell motility measured in transwell assays, cell invasion measured by percentage of organoids with outwards in organoid-forming medium, and epithelial-to-mesenchymal transition (EMT) measured by E-cadherin and Vimentin levels in Western blotting analysis, percentage of organoids with High E-cadherin junctions and nuclear translocation and activation of β-catenin in immunofluorescence (IF) staining assays. Results: We demonstrate that high levels of Wip1, a p38 phosphatase, correlated with Her2+ status, and reduced phosphorylation of p38 and its downstream effectors MK2 and Hsp27 in tissue samples and cell models of breast cancer early lesions. We found that Wip1 promoted migration, invasion and EMT, while Wip1 knockdown abrogated the ability of Her2 to induce these disseminating phenotypes, in cell line models of early lesion of breast cancer. In contrast to the wild type Wip1, the catalytically inactive mutants of Wip1 failed to induce the disseminating phenotypes. Thus, Wip1 induction is both sufficient, and necessary for Her2, to promote migration, invasion and EMT in breast cancer early lesion cells. In an attempt to identify the Wip1 downstream effectors involved in disseminating phenotypes, we found that ectopic expression of the constitutively active mutants of p38γ and δ, but not that of p38α and β, reversed the ability of Wip1 to suppress MK2 and Hsp27 phosphorylation and to induce migration, invasion and EMT. It suggests that Wip1 promotes the early dissemination by suppressing p38γ and δ. We investigate the mechanism by which Her2 induces Wip1 expression. Ectopic expression of Her2 did not alter the level of Wip1 mRNA or the Wip1 protein stability, suggesting that Her2 may promote Wip1 expression by regulating its translation. Moreover, inhibitors of Her2, Wip1 and Skp2 can each reduce the disseminating phenotypes in cells models of breast cancer early lesions ectopically expressing Her2, Wip1 or Skp2. Conclusions: Our findings identify the Her2-Wip1-p38-Mk2-Hsp27 cascade as a novel mechanism mediating breast cancer early dissemination and provide a basis for new therapies targeting early metastatic dissemination in Her2+ breast cancer using inhibitors for Her2, Wip1 or Skp2.

Overexpression of DDX49 in prostate cancer is associated with poor prognosis

BackgroundThere is increasing evidence that DEAD-box helicases (DDX) can act either as promoters or suppressors in various cancer types. Nevertheless, the function of DDX49 in prostate cancer (PCa) is unknown. This study reveals the prognostic and predictive value of DDX49 in PCa.MethodsFirst, we evaluated the expression of DDX49 between PCa and normal tissues based on TCGA and GEO databases. Univariate and multivariate regression analyses were conducted to reveal the risk factors for PCa recurrence. A K–M curve was employed to assess the relationship between DDX49 and recurrence-free survival. In vitro, DDX49 expression was evaluated in PCa and normal prostate cell lines. Furthermore, we constructed a shDDX49 lentivirus to knock down the expression of DDX49. Celigo® Image Cytometer and MTT assay were performed to analyse cell proliferation in PC-3 cells. Cell cycle distribution was detected with flow cytometry analysis. Apoptosis affected by the lack of DDX49 was metred with the PathScan® Stress and Apoptosis Signalling Antibody Array Kit.ResultsThis study shows a high increase in DDX49 in PCa tissues in comparison with normal tissues and that increased DDX49 indicates a poor prognosis among PCa patients. Meanwhile, DDX49 knockdown suppressed the proliferation and migration of PC-3 cells, causing cell cycle arrest in the G1 phase. Stress and apoptosis pathway analysis revealed that the phosphorylation of HSP27, p53, and SAPK/JNK was reduced in the DDX49 knockdown group compared with the control group.ConclusionsIn summary, these results suggest that high expression of DDX49 predicts a poor prognosis among PCa patients. Downregulation of DDX49 can suppress cell proliferation, block the cell cycle, and facilitate cell apoptosis. Therefore, knockdown of DDX49 is a promising novel therapy for treating patients with PCa.

Akkermansia muciniphila improves heat stress-impaired intestinal barrier function by modulating HSP27 in Caco-2 cells

ObjectiveHeat stress causes an elevation of intestinal epithelial barrier permeability and leads to multiple organ dysfunction in heatstroke. Akkermansia muciniphila (A. muciniphila) plays a role in maintaining intestinal integrity and improving the inflammatory state. This study aimed to investigate whether A. muciniphila could alleviate heat stress-induced dysfunction of intestinal permeability in Caco-2 monolayers and have the preventive effects on heatstroke. MethodsHuman intestinal epithelial Caco-2 cells were preincubated with live or pasteurized A. muciniphila then exposed to heat stress at 43 °C. Transepithelial electrical resistance (TEER) and the flux of horseradish peroxidase (HRP) across cell monolayers were measured to determine intestinal permeability. The levels of the tight junction proteins Occludin, ZO-1 and HSP27 were analyzed by Western blotting. These proteins were immunostained and localized by fluorescence microscopy. TJ morphology was observed using transmission electron microscopy (TEM). ResultsBoth live and pasteurized A. muciniphila effectively attenuated the decrease in TEER and impairment of intestinal permeability in HRP flux induced by heat exposure. A. muciniphila significantly elevated the expression of Occludin and ZO-1 by promoting HSP27 phosphorylation. The distortion and redistribution of tight junction proteins and disruption of morphology were also effectively prevented by pretreatment with A. muciniphila. ConclusionThis study indicates for the first time that both live and pasteurized A. muciniphila play an important protective role against heat-induced permeability dysfunction and epithelial barrier damage.

Tramadol regulates the activation of human platelets via Rac but not Rho/Rho-kinase.

Tramadol is a useful analgesic which acts as a serotonin and noradrenaline reuptake inhibitor in addition to μ-opioid receptor agonist. Cytoplasmic serotonin modulates the small GTPase activity through serotonylation, which is closely related to the human platelet activation. We recently reported that the combination of subthreshold collagen and CXCL12 synergistically activates human platelets. We herein investigated the effect and the mechanism of tramadol on the synergistic effect. Tramadol attenuated the synergistically stimulated platelet aggregation (300 μM of tramadol, 64.3% decrease, p<0.05). Not morphine or reboxetine, but duloxetine, fluvoxamine and sertraline attenuated the synergistic effect of the combination on the platelet aggregation (30 μM of fluvoxamine, 67.3% decrease, p<0.05; 30 μM of sertraline, 67.8% decrease, p<0.05). The geranylgeranyltransferase inhibitor GGTI-286 attenuated the aggregation of synergistically stimulated platelet (50 μM of GGTI-286, 80.8% decrease, p<0.05), in which GTP-binding Rac was increased. The Rac1-GEF interaction inhibitor NSC23766 suppressed the platelet activation and the phosphorylation of p38 MAPK and HSP27 induced by the combination of collagen and CXCL12. Tramadol and fluvoxamine almost completely attenuated the levels of GTP-binding Rac and the phosphorylation of both p38 MAPK and HSP27 stimulated by the combination. Suppression of the platelet aggregation after the duloxetine administration was observed in 2 of 5 patients in pain clinic. These results suggest that tramadol negatively regulates the combination of subthreshold collagen and CXCL12-induced platelet activation via Rac upstream of p38 MAPK.

FAM83G-based Peptide Induces Apoptosis on Cultured Liver Cancer Cell.

Previously, AF-956, which contains S356 of FAM83G and an N-terminal antenna peptide for entry into colon cancer cells, is markedly antiproliferative compared to a control peptide (AF-859), which lacks the N-terminal antenna peptide, by inducing apoptosis via the inhibition of HSP27 phosphorylation at residues S15 and S82. Because FAM83G-derived peptides are promising lead compounds for colon cancer treatment, we reanalyzed the effect of AG-066, which contains S356 of FAM83G and an N-terminal antenna peptide for entry into the liver cancer cells. HepG2 liver cancer cells were incubated with either AF-859 or AG-066 at a concentration of 54 μM at 37 °C for 24, 48, and 72 h. The effects of AF-859 and AG-066 on the cultured HepG2 cells were estimated using an inverted light microscope. Furthermore, the DNA ladder method and the dead cell assay were performed by applying Live/Dead Cell Staining Kit II. Erk phosphorylation was estimated by western blotting. Treatment with AG-066 markedly reduced HepG2 viable cell counts compared to the AF- 859-treated HepG2 cells, as evident from the significantly increased number of dead cells in the culture medium. Additionally, AG-066 treatment increased cellular DNA laddering. We found no difference in Erk phosphorylation status between the AG-066- and AF-859-treated groups. This study illustrated that the peptide with a structure based on FAM83G functions as a spontaneous apoptosis inducer for liver cancer cells. Hence, it is a promising lead compound for the treatment of liver cancer.