Association of Hsp90 with p53 and Fizzy related homolog (Fzr) synchronizing Anaphase Promoting Complex (APC/C): An unexplored ally towards oncogenic pathway.

The intricate molecular interactions leading to the oncogenic pathway are the consequence of cell cycle modification controlled by a bunch of cell cycle regulatory proteins. The tumor suppressor and cell cycle regulatory proteins work in coordination to maintain a healthy cellular environment. The integrity of this cellular protein pool is perpetuated by heat shock proteins/chaperones, which assist in proper protein folding during normal and cellular stress conditions. Among these versatile groups of chaperone proteins, Hsp90 is one of the significant ATP-dependent chaperones that aid in stabilizing many tumor suppressors and cell cycle regulator protein targets. Recently, studies have revealed that in cancerous cell lines, Hsp90 stabilizes mutant p53, 'the guardian of the genome.' Hsp90 also has a significant impact on Fzr, an essential regulator of the cell cycle having an important role in the developmental process of various organisms, including Drosophila, yeast, Caenorhabditis elegans, and plants. During cell cycle progression, p53 and Fzr coordinately regulate the Anaphase Promoting Complex (APC/C) from metaphase to anaphase transition up to cell cycle exit. APC/C mediates proper centrosome function in the dividing cell. The centrosome acts as the microtubule organizing center for the correct segregation of the sister chromatids to ensure perfect cell division. This review examines the structure of Hsp90 and its co-chaperones, which work in synergy to stabilize proteins such as p53 and Fizzy-related homolog (Fzr) to synchronize the Anaphase Promoting Complex (APC/C). Dysfunction of this process activates the oncogenic pathway leading to the development of cancer. Additionally, an overview of current drugs targeting Hsp90 at various phases of clinical trials has been included.

[Association of genomic instability of CDH1 gene with clinicopathological characteristics of gastric cancer].

OBJECTIVE: To assess the association of genomic instability of epithelial cadherin 1 (CDH1) gene and clinicopathological characteristics of gastric cancer. METHODS: In total 120 paraffin-embedded gastric cancer tissue specimen were prepared, and genomic DNA was extracted. The genomic instability of the CDH1 gene was analyzed by immunohistochemistry and silver staining PCR-single-strand conformation polymorphism. RESULTS: The number of information individuals (heterozygotes) was 98 for the D16S752 locus. The detection rates for microsatellite instability (MSI) and loss of heterozygosity (LOH) at the D16S752 locus and the positive rate of CDH1 protein were 19.39%, 16.33% and 51.02%, respectively. The detection rate of MSI in TNM stages I or II was significantly higher than that in stages III or IV (P<0.05) while the detection rate of LOH was significantly lower than that in stages III or IV (P<0.05). The positive rate of CDH1 protein in TNM stages III or IV was significantly lower than that in stages I or II (P<0.05). The detection rate of MSI of cases with lymph node metastasis was significantly lower than that of without lymph node metastasis (P<0.05) while the detection rate of LOH was significantly higher than that without lymph node metastasis (P<0.05). The positive rate of CDH1 protein in patients with lymph node metastasis was significantly lower than that in patients without lymph node metastasis (P<0.05). The positive rate of CDH1 protein in MSI-positive group was significantly higher than that in MSI-negative group (P<0.05), and the positive rate of CDH1 protein in the LOH-positive group was significantly lower than that the LOH-negative group (P<0.05). CONCLUSION: The genomic instability of the CDH1 gene is associated with the progression of gastric cancer. MSI at the D16S752 locus may be used as a molecular marker for early gastric cancer, while LOH at this locus mostly occurs in advanced gastric cancer and can be regarded as an effective indicators for malignancy evaluation and prognosis.

Insufficiency of FZR1 disturbs HSC quiescence by inhibiting ubiquitin-dependent degradation of RUNX1 in aplastic anemia.

FZR1 has been implicated as a master regulator of the cell cycle and quiescence, but its roles and molecular mechanisms in the pathogenesis of severe aplastic anemia (SAA) are unclear. Here, we report that FZR1 is downregulated in SAA HSCs compared with healthy control and is associated with decreased quiescence of HSC. Haploinsufficiency of Fzr1 shows impaired quiescence and self-renewal ability of HSC in two Fzr1 heterozygous knockout mouse models. Mechanistically, FZR1 insufficiency inhibits the ubiquitination of RUNX1 protein at lysine 125, leading to the accumulation of RUNX1 protein, which disturbs the quiescence of HSCs in SAA patients. Moreover, downregulation of Runx1 reversed the loss of quiescence and impaired long-term self-renew ability in Fzr1+/- HSCs in vivo and impaired repopulation capacity in BM from SAA patients in vitro. Our findings, therefore, raise the possibility of a decisive role of the FZR1-RUNX1 pathway in the pathogenesis of SAA via deregulation of HSC quiescence.

RNA methyltransferase NSUN2 promotes growth of hepatocellular carcinoma cells by regulating fizzy-related-1 in vitro and in vivo.

The aim of the study was to investigate the role of NSUN2 (NOP2/Sun RNA Methyltransferase Family Member 2) in hepatocellular carcinoma (HCC). The expressions of NSUN2 and FZR1 were measured. Cell viability, proliferation, and apoptosis were assessed. HCC xenograft in nude mouse model was established. Tumor weight and volume were examined. Tumor tissues were collected for immunohistochemistry (IHC). TCGA database analysis and clinical sample testing suggested that the transcript levels of NSUN2 and FZR1 were increased in HCC tissues. NSUN2 knockdown inhibited HCC cell viability and proliferation, and promoted cell apoptosis. Moreover, the effects of NSUN2 could be countered by overexpressing FZR1. In animal experiment, NSUN2 silencing suppressed tumor growth in nude mice by downregulating FZR1. In conclusion, NSUN2 has a regulatory effect on HCC cell proliferation and apoptosis. NSUN2 knockout could inhibit cellular processes in HCC and tumor growth, likely via FZR1 inhibition. This finding has not only revealed the role of NSUN2 in HCC growth, but also suggests a promising target for HCC treatment.

E3 Ubiquitin Ligase APC/CCdh1 Negatively Regulates FAH Protein Stability by Promoting Its Polyubiquitination.

Fumarylacetoacetate hydrolase (FAH) is the last enzyme in the degradation pathway of the amino acids tyrosine and phenylalanine in mammals that catalyzes the hydrolysis of 4-fumarylacetoacetate into acetoacetate and fumarate. Mutations of the FAH gene are associated with hereditary tyrosinemia type I (HT1), resulting in reduced protein stability, misfolding, accelerated degradation and deficiency in functional proteins. Identifying E3 ligases, which are necessary for FAH protein stability and degradation, is essential. In this study, we demonstrated that the FAH protein level is elevated in liver cancer tissues compared to that in normal tissues. Further, we showed that the FAH protein undergoes 26S proteasomal degradation and its protein turnover is regulated by the anaphase-promoting complex/cyclosome-Cdh1 (APC/C)Cdh1 E3 ubiquitin ligase complex. APC/CCdh1 acts as a negative stabilizer of FAH protein by promoting FAH polyubiquitination and decreases the half-life of FAH protein. Thus, we envision that Cdh1 might be a key factor in the maintenance of FAH protein level to regulate FAH-mediated physiological functions.

FZR1 as a novel biomarker for breast cancer neoadjuvant chemotherapy prediction.

The concept of breast-conserving surgery is a remarkable achievement of breast cancer therapy. Neoadjuvant chemotherapy is being used increasingly to shrink the tumor prior to surgery. Neoadjuvant chemotherapy is reducing the tumor size to make the surgery with less damaging to surrounding tissue and downstage locally inoperable disease to operable. However, non-effective neoadjuvant chemotherapy could increase the risks of delaying surgery, develop unresectable disease and metastatic tumor spread. The biomarkers for predicting the neoadjuvant chemotherapy effect are scarce in breast cancer treatment. In this study, we identified that FZR1 can be a novel biomarker for breast cancer neoadjuvant chemotherapy according to clinical patient cohort evaluation and molecular mechanism investigation. Transcriptomic data analysis indicated that the expression of FZR1 is correlated with the effect of neoadjuvant chemotherapy. Mechanistically, we demonstrate that FZR1 is pivotal to the chemotherapy drugs induced apoptosis and cell cycle arrest. FZR1 is involved in the stability of p53 by impairing the phosphorylation at ser15 site. We demonstrate that the expression of FZR1 detected by quantification of IHC can be an effective predictor of neoadjuvant chemotherapy in animal experiment and clinical patient cohort. To obtain more benefit for breast cancer patient, we propose that the FZR1 IHC score using at the clinical to predict the effect of neoadjuvant chemotherapy.

[Hereditary breast carcinomas pathologist's perspective]. / Les cancers héréditaires du sein vus par le pathologiste.

Breast cancers occurring in the context of a hereditary mutation of a predisposition gene represent 5 to 10% of all breast cancers, 20 to 25% of which being due to a mutation in the BRCA1 or BRCA2 genes. Authorization to market PARP inhibitors for breast cancer patients with hereditary BRCA1 and 2 mutations has recently been obtained. Given the annual frequency of breast cancer, morphological identification could facilitate the patient care process to limit the search for BRCA1 and 2 mutations to patients whose tumors have very specific characteristics. However, only a few morphological features have been recognized and differ depending on the mutated genes. Breast cancer occurring as part of a mutation in the BRCA1 gene is in 85% of cases of high-grade non-specific type invasive carcinomas with very limited contours, contain numerous lymphocytes in the stroma and are of triple-negative phenotype. Carcinomas associated with mutations in the BRCA2 genes and genes more recently recognized as associated with a risk of development of breast cancer (CHECK2, BMPR1A, BRIP1, PALB2, MUTYH) are most often non-specific invasive carcinomas, although other histological types are possible, grade III, luminal B phenotype. Breast cancer occurring in the context of a constitutional mutation of TP53 occurs in women under 35 years old are of non-specific histological type and with an amplification of HER2 in two thirds of the cases. Those associated with a PTEN mutation are readily of the apocrine type. Finally, very rarely, certain lobular-type breast cancers can occur in the context of a constitutional mutation of the CDH1 gene, which codes for the protein E-cadherin. The morphological and phenotypic characteristics may suggest to the pathologist a carcinoma of the breast occurring in a context of hereditary mutation. However, at the present time the only situations where a morphological sorting makes it possible to accelerate the genetic analysis are those of an invasive carcinoma of non-specific type of triple-negative phenotype in a woman of less than 50 years or that of a diagnosis of HER2 breast cancer amplified in a woman under 31 years of age (Chompret criteria). Family background and personal history are of great importance in the genetic counseling indication decision trees. Unfortunately, to date, no quality antibody has been developed against BRCA1 and 2 to help the pathologist identify hereditary cases. The immunohistochemical analysis of RAD51 could facilitate the identification of tumors possibly sensitive to PARP inhibitors. Progress to identify hereditary cancers is expected thanks to the development of artificial intelligence algorithms from digitized histological slides.

APC/C ubiquitin ligase: Functions and mechanisms in tumorigenesis.

The anaphase promoting complex/ cyclosome (APC/C), is an evolutionarily conserved protein complex essential for cellular division due to its role in regulating the mitotic transition from metaphase to anaphase. In this review, we highlight recent work that has shed light on our understanding of the role of APC/C coactivators, Cdh1 and Cdc20, in cancer initiation and development. We summarize the current state of knowledge regarding APC/C structure and function, as well as the distinct ways Cdh1 and Cdc20 are dysregulated in human cancer. We also discuss APC/C inhibitors, novel approaches for targeting the APC/C as a cancer therapy, and areas for future work.

APC/Cdh1 targets PECAM-1 for ubiquitination and degradation in endothelial cells.

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a member of the immunoglobulin superfamily and is expressed by hematopoietic and endothelial cells (ECs). Recent studies have shown that PECAM-1 plays a crucial role in promoting the development of the EC inflammatory response in the context of disturbed flow. However, the mechanistic pathways that control PECAM-1 protein stability remain largely unclear. Here, we identified PECAM-1 as a novel substrate of the APC/Cdh1 E3 ubiquitin ligase. Specifically, lentivirus-mediated Cdh1 depletion stabilized PECAM-1 in ECs. Conversely, overexpression of Cdh1 destabilized PECAM-1. The proteasome inhibitor MG132 blocked Cdh1-mediated PECAM-1 degradation. In addition, Cdh1 promoted K48-linked polyubiquitination of PECAM-1 in a destruction box-dependent manner. Furthermore, we demonstrated that compared with pulsatile shear stress (PS), oscillatory shear stress decreased the expression of Cdh1 and the ubiquitination of PECAM-1, therefore stabilizing PECAM-1 to promote inflammation in ECs. Hence, our study revealed a novel mechanism by which fluid flow patterns regulate EC homeostasis via Cdh1-dependent ubiquitination and subsequent degradation of PECAM-1.

Differential properties of mitosis-associated events following CHK1 and WEE1 inhibitor treatments in human tongue carcinoma cells.

CHK1 and WEE1 play pivotal roles in G2/M checkpoint following exogenous DNA damage and regulation of DNA replication under normal cellular conditions. Here, we monitored and compared the cell cycle kinetics of mitosis-associated events after CHK1 and WEE1 inhibitor treatments in a human tongue cancer cell line (SAS). A fluorescent ubiquitination-based cell cycle indicator (Fucci) that reflects SCFSKP2 and APCCDH1 E3 ligase activities was used to monitor cell cycle progression. Numerous γH2AX-positive cells were observed within the S phase population of cells following CHK1 inhibitor treatment, and polyploid cells exhibiting DNA damage emerged via abortive mitosis (endomitosis) at 24 h post treatment. While WEE1 inhibitor-treated cells exhibited similar polyploidy via endomitosis at later time points, they possessed fewer γH2AX foci during S phase, and polyploid cells exhibiting DNA damage were scarce. Instead, mitosis duration greatly extended and was accompanied by an abnormal emission of Fucci red fluorescence. Kinetic analysis of Fucci fluorescence revealed that abnormal emission occurred at early M phase in a manner independent of green fluorescence degradation as a marker of APCCDH1 activation. When an inhibitor of the essential spindle checkpoint factor MPS1 was co-treated with a WEE1 inhibitor, the elongated mitosis duration and abnormal red fluorescence were abrogated, and WEE1-induced reduction of clonogenic survival was offset. We demonstrate novel differential effects on mitosis-associated events following CHK1 and WEE1 inhibitor treatments.

Interplay between c-Src and the APC/C co-activator Cdh1 regulates mammary tumorigenesis.

The Anaphase Promoting Complex (APC) coactivator Cdh1 drives proper cell cycle progression and is implicated in the suppression of tumorigenesis. However, it remains elusive how Cdh1 restrains cancer progression and how tumor cells escape the inhibition of Cdh1. Here we report that Cdh1 suppresses the kinase activity of c-Src in an APC-independent manner. Depleting Cdh1 accelerates breast cancer cell proliferation and cooperates with PTEN loss to promote breast tumor progression in mice. Hyperactive c-Src, on the other hand, reciprocally inhibits the ubiquitin E3 ligase activity of APCCdh1 through direct phosphorylation of Cdh1 at its N-terminus, which disrupts the interaction between Cdh1 and the APC core complex. Furthermore, pharmacological inhibition of c-Src restores APCCdh1 tumor suppressor function to repress a panel of APCCdh1 oncogenic substrates. Our findings reveal a reciprocal feedback circuit of Cdh1 and c-Src in the crosstalk between the cell cycle machinery and the c-Src signaling pathway.

Plk4 Regulates Centriole Asymmetry and Spindle Orientation in Neural Stem Cells.

Defects in mitotic spindle orientation (MSO) disrupt the organization of stem cell niches impacting tissue morphogenesis and homeostasis. Mutations in centrosome genes reduce MSO fidelity, leading to tissue dysplasia and causing several diseases such as microcephaly, dwarfism, and cancer. Whether these mutations perturb spindle orientation solely by affecting astral microtubule nucleation or whether centrosome proteins have more direct functions in regulating MSO is unknown. To investigate this question, we analyzed the consequences of deregulating Plk4 (the master centriole duplication kinase) activity in Drosophila asymmetrically dividing neural stem cells. We found that Plk4 functions upstream of MSO control, orchestrating centriole symmetry breaking and consequently centrosome positioning. Mechanistically, we show that Plk4 acts through Spd2 phosphorylation, which induces centriole release from the apical cortex. Overall, this work not only reveals a role for Plk4 in regulating centrosome function but also links the centrosome biogenesis machinery with the MSO apparatus.

Hippo signaling is intrinsically regulated during cell cycle progression by APC/CCdh1.

The Hippo-YAP/TAZ signaling pathway plays a pivotal role in growth control during development and regeneration and its dysregulation is widely implicated in various cancers. To further understand the cellular and molecular mechanisms underlying Hippo signaling regulation, we have found that activities of core Hippo signaling components, large tumor suppressor (LATS) kinases and YAP/TAZ transcription factors, oscillate during mitotic cell cycle. We further identified that the anaphase-promoting complex/cyclosome (APC/C)Cdh1 E3 ubiquitin ligase complex, which plays a key role governing eukaryotic cell cycle progression, intrinsically regulates Hippo signaling activities. CDH1 recognizes LATS kinases to promote their degradation and, hence, YAP/TAZ regulation by LATS phosphorylation is under cell cycle control. As a result, YAP/TAZ activities peak in G1 phase. Furthermore, we show in Drosophila eye and wing development that Cdh1 is required in vivo to regulate the LATS homolog Warts with a conserved mechanism. Cdh1 reduction increased Warts levels, which resulted in reduction of the eye and wing sizes in a Yorkie dependent manner. Therefore, LATS degradation by APC/CCdh1 represents a previously unappreciated and evolutionarily conserved layer of Hippo signaling regulation.

Injury induces endothelial to mesenchymal transition in the mouse corneal endothelium in vivo via FGF2.

Purpose: To determine whether the mouse corneal endothelium enters endothelial to mesenchymal transition (EndoMT) following surgical injury in vivo. Methods: The corneal endothelium in anesthetized mice was surgically injured in vivo under direct visualization. The secretion of interleukin-1 beta (IL-1ß) and fibroblast growth factor 2 (FGF2) into the aqueous humor was analyzed with western blotting. The expression of FGF2, Snai1, Zeb1, Col1a1, Col1a2, Fn1, Vim, Cdk2, Ccne1, and Cdh1 was analyzed with semiquantitative RT-PCR in the mouse corneal endothelium ex vivo and in vivo. Knockdown of FGF2 was done using siRNA. Col8a2 was used as a corneal endothelial marker, and Keratocan (Ktcn) was used as a stromal marker. ß-actin was used as a loading control. Results: Sequential expression of IL-1ß and FGF2 was detected in the aqueous humor after surgical injury. FGF2 treatment induced expression of endothelial to mesenchymal transition-related genes including Snai1, and Zeb1 in the mouse ex vivo corneal endothelium. This led to increased expression of Col1a1, Col1a2, Fn1, and Vim and suppression of Cdh1 in a time-dependent manner. Expression of FGF2, Snai1, Zeb1, Col1a1, Col1a2, Fn1, Vim, Cdk2, and Ccne1 was completely abolished by FGF2 siRNA knockdown in the mouse corneal endothelium ex vivo. Surgical injury induced FGF2 expression in the in vivo mouse corneal endothelium. The injury-dependent expression of FGF2, Snai1, Zeb1, Col1a1, Col1a2, Fn1, Vim, Cdk2, and Ccne1 and the suppression of Cdh1 were inhibited by siRNA knockdown of FGF in the mouse corneal endothelium in vivo. Moreover, siRNA knockdown of FGF2 inhibited the formation of the injury-dependent retrocorneal membrane in the in vivo mouse corneal endothelium. Conclusions: These findings suggest that after surgical injury, FGF2 induces the expression of EndoMT-related genes Snai1, Zeb1, Col1a1, Col1a2, Fn1, Vim, Cdk2, and Ccne1 in the mouse corneal endothelium in vivo, similar to the human corneal endothelium ex vivo.

Human gastric cancer modelling using organoids.

OBJECTIVE: Gastric cancer is the second leading cause of cancer-related deaths and the fifth most common malignancy worldwide. In this study, human and mouse gastric cancer organoids were generated to model the disease and perform drug testing to delineate treatment strategies. DESIGN: Human gastric cancer organoid cultures were established, samples classified according to their molecular profile and their response to conventional chemotherapeutics tested. Targeted treatment was performed according to specific druggable mutations. Mouse gastric cancer organoid cultures were generated carrying molecular subtype-specific alterations. RESULTS: Twenty human gastric cancer organoid cultures were established and four selected for a comprehensive in-depth analysis. Organoids demonstrated divergent growth characteristics and morphologies. Immunohistochemistry showed similar characteristics to the corresponding primary tissue. A divergent response to 5-fluoruracil, oxaliplatin, irinotecan, epirubicin and docetaxel treatment was observed. Whole genome sequencing revealed a mutational spectrum that corresponded to the previously identified microsatellite instable, genomic stable and chromosomal instable subtypes of gastric cancer. The mutational landscape allowed targeted therapy with trastuzumab for ERBB2 alterations and palbociclib for CDKN2A loss. Mouse cancer organoids carrying Kras and Tp53 or Apc and Cdh1 mutations were characterised and serve as model system to study the signalling of induced pathways. CONCLUSION: We generated human and mouse gastric cancer organoids modelling typical characteristics and altered pathways of human gastric cancer. Successful interference with activated pathways demonstrates their potential usefulness as living biomarkers for therapy response testing.

APC/CCdh1 regulates the balance between maintenance and differentiation of hematopoietic stem and progenitor cells.

Hematopoietic stem and progenitor cells (HSPCs) represent the lifelong source of all blood cells and continuously regenerate the hematopoietic system through differentiation and self-renewal. The process of differentiation is initiated in the G1 phase of the cell cycle, when stem cells leave their quiescent state. During G1, the anaphase-promoting complex or cyclosome associated with the coactivator Cdh1 is highly active and marks proteins for proteasomal degradation to regulate cell proliferation. Following Cdh1 knockdown in HSPCs, we analyzed human and mouse hematopoiesis in vitro and in vivo in competitive transplantation assays. We found that Cdh1 is highly expressed in human CD34+ HSPCs and downregulated in differentiated subsets; whereas, loss of Cdh1 restricts myeloid differentiation, supports B cell development and preserves immature short-term HSPCs without affecting proliferation or viability. Our data highlight a role of Cdh1 as a regulator of balancing the maintenance of HSPCs and differentiation into mature blood cells.

Identification of selective inhibitors for diffuse-type gastric cancer cells by screening of annotated compounds in preclinical models.

BACKGROUND: Diffuse-type gastric cancer (DGC) exhibits rapid disease progression and poor patient prognosis. We have previously established an E-cadherin/p53 double conditional knockout (DCKO) mouse line as the first genetically engineered one, which morphologically and molecularly recapitulates human DGC. In this study, we explored low-molecular-weight drugs selectively eliminating mouse and human DGC cells. METHODS: We derived mouse gastric cancer (GC) cell lines from DGC of the DCKO mice demonstrating enhanced tumourigenic activity in immunodeficient mice and acquired tolerance to cytotoxic anti-cancer agents. RESULTS: We performed a synthetic lethal screening of 1535 annotated chemical compounds, and identified 27 candidates selectively killing the GC cell lines. The most potent drug mestranol, an oestrogen derivative, and other oestrogen receptor modulators specifically attenuated cell viability of the GC cell lines by inducing apoptosis preceded by DNA damage. Moreover, mestranol could significantly suppress tumour growth of the GC cells subcutaneously transplanted into nude mice, consistent with longer survival time in the female DCKO mice than in the male. Expectedly, human E-cadherin-mutant and -low gastric cancer cells showed higher susceptibility to oestrogen drugs in contrast to E-cadherin-intact ones in vitro and in vivo. CONCLUSIONS: These findings may lead to the development of novel therapeutic strategies targeting DGC.

O-GlcNAcylation affects ß-catenin and E-cadherin expression, cell motility and tumorigenicity of colorectal cancer.

O-GlcNAcylation, the addition of ß-N-acetylglucosamine (O-GlcNAc) moiety to Ser/Thr residues, is a sensor of the cell metabolic state. Cancer diseases such as colon, lung and breast cancer, possess deregulated O-GlcNAcylation. Studies during the last decade revealed that O-GlcNAcylation is implicated in cancer tumorigenesis and proliferation. The Wnt/ß-catenin signaling pathway and cadherin-mediated adhesion are also implicated in epithelial-mesenchymal transition (EMT), a key cellular process in invasion and cancer metastasis. Often, deregulation of the Wnt pathway is caused by altered phosphorylation of its components. Specifically, phosphorylation of Ser or Thr residues of ß-catenin affects its location and interaction with E-cadherin, thus facilitating cell-cell adhesion. Consistent with previous studies, the current study indicates that ß-catenin is O-GlcNAcylated. To test the effect of O-GlcNAcylation on cell motility and how O-GlcNAcylation might affect ß-catenin and E-cadherin functions, the enzyme machinery of O-GlcNAcylation was modulated either with chemical inhibitors or by gene silencing. When O-GlcNAcase (OGA) was inhibited, a global elevation of protein O-GlcNAcylation and increase in the expression of E-cadherin and ß-catenin were noted. Concomitantly with enhanced O-GlcNAcylation, ß-catenin transcriptional activity were elevated. Additionally, fibroblast cell motility was enhanced. Stable silenced cell lines with adenoviral OGA or adenoviral O-GlcNAc transferase (OGT) were established. Consistent with the results obtained by OGA chemical inhibition by TMG, OGT-silencing led to a significant reduction in ß-catenin level. In vivo, murine orthotropic colorectal cancer model indicates that elevated O-GlcNAcylation leads to increased mortality rate, tumor and metastasis development. However, reduction in O-GlcNAcylation promoted survival that could be attributed to attenuated tumor and metastasis development. The results described herein provide circumstantial clues that O-GlcNAcylation deregulates ß-catenin and E-cadherin expression and activity in fibroblast cell lines and this might influence EMT and cell motility, which may further influence tumor development and metastasis.

Combination therapy of PKCζ and COX-2 inhibitors synergistically suppress melanoma metastasis.

BACKGROUND: Metastatic malignant melanoma is one of the most aggressive malignancies and its treatment remains challenging. Recent studies demonstrate that the melanoma metastasis has correlations with the heightened activations of protein kinase C ζ (PKCζ) and cyclooxygenase-2 (COX-2) signaling pathways. Targeted inhibitions for PKCζ and COX-2 have been considered as the promising strategies for the treatment of melanoma metastasis. Thus, the PKCζ inhibitor J-4 and COX-2 inhibitor Celecoxib were combined to treat melanoma metastasis in this study. METHODS: The Transwell assay, Wound-healing assay and Adhesion assay were used to evaluate the inhibition of combined therapy of J-4 and Celecoxib on melanoma cells invasion, migration and adhesion in vitro, respectively. The impaired actin polymerization was observed by confocal microscope and inactivated signal pathways about PKCζ and COX-2 were confirmed by the Western blotting assay. The B16-F10/C57BL mouse melanoma model was used to test the inhibition of combined therapy of J-4 and Celecoxib on melanoma metastasis in vivo. RESULTS: The in vitro results showed that the combination of J-4 and Celecoxib exerted synergistic inhibitory effects on the migration, invasion and adhesion of melanoma B16-F10 and A375 cells with combination index less than 1. The actin polymerization and phosphorylation of Cofilin required in cell migration were severely impaired, which is due to the inactivation of PKCζ related signal pathways and the decrease of COX-2. The combined inhibition of PKCζ and COX-2 induced Mesenchymal-Epithelial Transition (MET) in melanoma cells with the expression of E-Cadherin increasing and Vimentin decreasing. The secretion of MMP-2/MMP-9 also significantly decreased after the combination treatment. In C57BL/6 mice intravenously injected with B16-F10 cells (5 × 104 cells/mouse), co-treatment of J-4 and Celecoxib also severely suppressed melanoma lung metastasis. The body weight monitoring and HE staining results indicated the low toxicity of the combination therapy. CONCLUSIONS: This study demonstrates that the combination therapy of PKCζ and COX-2 inhibitors can significantly inhibit melanoma metastasis in vitro and in vivo, which will be an efficient strategy for treatment of melanoma metastasis in clinics.

Lack of epithelial PPARγ causes cystic adenomatoid malformations in mouse fetal lung.

Peroxisome proliferator-activated receptor-γ (PPARγ) plays an important role in lipid and glucose metabolism. In this study, the function of PPARγ on lung development was investigated. Lung-specific Pparg conditional knockout mice (PpargΔLuEpC) were developed using Cre-Lox system. PpargΔLuEpC mice showed abnormal lung development with enlarged airspaces and followed by increase of apoptotic cells at E14.5 to E18.5. Gene analysis revealed that expression of Pmaip1, a gene related to apoptosis, was significantly increased while expression of Retnla, a gene related to anti-apoptosis, was dramatically decreased in the fetal lung (E14.5) of PpargΔLuEpC mice. In addition, expression of Pthlh, a gene phenotypically expressed in the congenital cystic adenomatoid malformation (CCAM), was increased at E14.5 to E18.5 in the lung of PpargΔLuEpC mice. Cell culture studies revealed that PPARγ could bind to promoter region of Pthlh gene as a repressor in the immortalized mouse lung epithelial cell line MLE-15. Surprisingly, phenotypic changes in MLE-15-shPparg cells, stably transfected with shPparg plasmid, were similar to the PpargΔLuEpC mice model. In addition, MLE-15-shPparg cells were easily detached from the cultured plate when cold phosphate buffered saline was applied. Furthermore, expression of Cdh1, a gene related to cell adhesion, was significantly reduced in the MLE-15-shPparg cells. Taken together, PPARγ may play an important role in fetal lung development via alveolar cell-to-cell adhesion system.