Combined treatment with cetuximab and STA9090 has synergistic anticancer effects on human non-small cell lung cancer.

Cetuximab (CET), a human murine chimeric IgG monoclonal antibody and an inhibitor of epidermal growth factor receptor (EGFR), has been shown to be effective in treating various types of cancer. However, its use is hindered by limitations such as resistance development, variability in patient response, side effects, and challenges in biomarker identification. Therefore, CET is often combined with other targeted therapies or chemotherapies to enhance its effectiveness. In this study, we investigate the anticancer effects and underlying mechanisms of the combination of CET, an EGFR inhibitor, and STA9090, an inhibitor of heat shock protein 90 (Hsp90), in both in vitro and in vivo models of non-small cell lung cancer (NSCLC). The results demonstrate significantly stronger effects on NSCLC cells in response to combination therapy than to treatment with either agent alone, indicating that the combination of CET and STA9090 has potential synergistic effects. Additionally, the combination therapy inhibits tumor growth in a xenograft nude mouse model more effectively than treatment with either agent alone, suggesting improved efficacy when used together. Furthermore, the synergistic effects of the combination therapy are likely due to inactivation of the receptor tyrosine kinase (RTK) pathway, which is overly activated in cancer and contributes to tumor growth, angiogenesis, and metastasis. Consequently, our findings suggest that STA9090 has potent direct antitumor activity and synergizes with CET against NSCLC tumors. It is highly likely that these synergistic effects are mediated through RTK pathway inactivation caused by the combination. Therefore, our findings strongly and consistently support the potential synergistic effect of STA9090, an RTK inhibitor, in combination with EGFR-targeting agents.

Hedgehog ligand and receptor cooperatively regulate EGFR stability and activity in non-small cell lung cancer.

PURPOSE: The hyperactivation of epidermal growth factor receptor (EGFR) plays a crucial role in non-small cell lung cancer (NSCLC). Hedgehog (Hh) signaling has been implicated in the tumorigenesis and progression of various cancers, however, its function in NSCLC cells remains controversial. Herein, we present a novel finding that challenges the current understanding of Hh signaling in tumor growth. METHODS: Expression of Hh ligands and receptor were assessed using TCGA datasets, immunoblotting and immunohistochemical. Biological function of Hh ligands and receptor in NSCLC were tested using colony formation, cell count kit-8 (CCK-8) and xenograft assays. Biochemical effect of Hh ligands and receptor on regulating EGFR stability and activity were checked via immunoblotting. RESULTS: Expression of Hh ligands and receptor was suppressed in NSCLC tissues, and the lower expression levels of these genes were associated with poor prognosis. Ptch1 binds to EGFR and facilitates its poly-ubiquitylation and degradation independent of downstream transcriptional signaling. Moreover, Hh ligands cooperate with Ptch1 to regulate the protein stability and activity of EGFR. This unique mechanism leads to a suppressive effect on NSCLC tumor growth. CONCLUSION: Non-canonical Hh signaling pathway, involving cooperation between Hh ligands and their receptor Ptch1, facilitates the degradation of EGFR and attenuates its activity in NSCLC. These findings provide novel insights into the regulation of EGFR protein stability and activity, offer new diagnostic indicators for molecular typing of NSCLC and identify potential targets for targeted therapy of this challenging disease.

Phosphorylated SHMT2 Regulates Oncogenesis Through m6A Modification in Lung Adenocarcinoma.

Targeting cancer-specific metabolic processes is a promising therapeutic strategy. Here, this work uses a compound library that directly inhibits metabolic enzymes to screen the potential metabolic targets in lung adenocarcinoma (LUAD). SHIN1, the specific inhibitor of serine hydroxymethyltransferase 1/2 (SHMT1/2), has a highly specific inhibitory effect on LUAD cells, and this effect depends mainly on the overexpression of SHMT2. This work clarifies that mitogen-activated protein kinase 1 (MAPK1)-mediated phosphorylation at Ser90 is the key mechanism underlying SHMT2 upregulation in LUAD and that this phosphorylation stabilizes SHMT2 by reducing STIP1 homology and U-box containing protein 1 (STUB1)-mediated ubiquitination and degradation. SHMT2-Ser90 dephosphorylation decreases S-adenosylmethionine levels in LUAD cells, resulting in reduced N6-methyladenosine (m6A) levels in global RNAs without affecting total protein or DNA methylation. Methylated RNA immunoprecipitation sequencing (MeRIP-Seq) and RNA sequencing (RNA-Seq) analyses further demonstrate that SHMT2-Ser90 dephosphorylation accelerates the RNA degradation of oncogenic genes by reducing m6A modification, leading to the inhibition of tumorigenesis. Overall, this study elucidates a new regulatory mechanism of SHMT2 during oncogenesis and provides a theoretical basis for targeting SHMT2 as a therapeutic target in LUAD.

Identification of genes and pathways associated with menopausal status in breast cancer patients using two algorithms.

BACKGROUND: Menopausal status has a known relationship with the levels of estrogen, progesterone, and other sex hormones, potentially influencing the activity of ER, PR, and many other signaling pathways involved in the initiation and progression of breast cancer. However, the differences between premenopausal and postmenopausal breast cancer patients at the molecular level are unclear. METHODS: We retrieved eight datasets from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) associated with menopausal status in breast cancer patients were identified using the MAMA and LIMMA methods. Based on these validated DEGs, we performed Gene Ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Protein-protein interaction (PPI) networks were constructed. We used DrugBank data to investigate which of these validated DEGs are targetable. Survival analysis was performed to explore the influence of these genes on breast cancer patient prognosis. RESULTS: We identified 762 DEGs associated with menopausal status in breast cancer patients. PPI network analysis indicated that these genes are primarily involved in pathways such as the cell cycle, oocyte meiosis and progesterone-mediated oocyte maturation pathways. Notably, several genes played roles in multiple signaling pathways and were associated with patient survival. These genes were also observed to be targetable according to the DrugBank database. CONCLUSION: We identified DEGs associated with menopausal status in breast cancer patients. The association of these genes with several key pathways may promote understanding of the complex characterizations of breast cancer. Our findings offer valuable insights for developing new therapeutic strategies tailored to the menopausal status of breast cancer patients.

Multi-Lineage Human Endometrial Organoids on Acellular Amniotic Membrane for Endometrium Regeneration.

Asherman's syndrome is an endometrial regeneration disorder resulting from injury to the endometrial basal layer, causing the formation of scar tissue in the uterus and cervix. This usually leads to uterine infertility, menstrual disorders, and placental abnormalities. While stem cell therapy has shown extensive progress in repairing the damaged endometrium and preventing intrauterine adhesion, issues of low engraftment rates, rapid senescence, and the risk of tumorigenesis remain to be resolved for efficient and effective application of this technology in endometrial repair. This study addressed these challenges by developing a co-culture system to generate multi-lineage endometrial organoids (MLEOs) comprising endometrial epithelium organoids (EEOs) and endometrial mesenchymal stem cells (eMSCs). The efficacy of these MLEOs was investigated by seeding them on a biocompatible scaffold, the human acellular amniotic membrane (HAAM), to create a biological graft patch, which was subsequently transplanted into an injury model of the endometrium in rats. The results indicated that the MLEOs on the HAAM patch facilitated endometrial angiogenesis, regeneration, and improved pregnancy outcomes. The MLEOs on the HAAM patch could serve as a promising strategy for treating endometrial injury and preventing Asherman's syndrome.

ATF1 promotes the malignancy of lung adenocarcinoma cells by transcriptionally regulating ZNF143 expression.

The clinical oncogenic functions and mechanisms of activating transcription factor 1 (ATF1) in the progression of lung adenocarcinoma have not been completely elucidated. In this study, by employing human lung adenocarcinoma tissues and cells, we detect the correlation of ATF1 expression with the clinicopathological features and prognosis of patients with lung adenocarcinoma and find that ATF1 promotes lung adenocarcinoma cell proliferation and migration by transcriptionally enhancing zinc finger protein 143 (ZNF143) expression. ATF1 and ZNF143 are strongly expressed in lung adenocarcinoma tissues compared with those in the adjacent normal tissues, and high ATF1 and ZNF143 expressions are related to poor disease-free survival of lung adenocarcinoma patients. ATF1 overexpression results in increased proliferation and migration of lung adenocarcinoma cells, whereas knockdown of ATF1 inhibits cell proliferation and migration. Furthermore, ATF1 transcriptionally regulates the expression of ZNF143, and ATF1 and ZNF143 expressions are positively correlated in lung adenocarcinoma tissues. ZNF143 knockdown blocks lung adenocarcinoma cell migration, which is mediated by ATF1 upregulation. Hence, this study provides a potential therapeutic candidate for the treatment of lung adenocarcinoma.

Tea domain transcription factor TEAD4 mitigates TGF-ß signaling and hepatocellular carcinoma progression independently of YAP.

Tea domain transcription factor 4 (TEAD4) plays a pivotal role in tissue development and homeostasis by interacting with Yes-associated protein (YAP) in response to Hippo signaling inactivation. TEAD4 and YAP can also cooperate with transforming growth factor-ß (TGF-ß)-activated Smad proteins to regulate gene transcription. Yet, it remains unclear whether TEAD4 plays a YAP-independent role in TGF-ß signaling. Here, we unveil a novel tumor suppressive function of TEAD4 in liver cancer via mitigating TGF-ß signaling. Ectopic TEAD4 inhibited TGF-ß-induced signal transduction, Smad transcriptional activity, and target gene transcription, consequently suppressing hepatocellular carcinoma cell proliferation and migration in vitro and xenograft tumor growth in mice. Consistently, depletion of endogenous TEAD4 by siRNAs enhanced TGF-ß signaling in cancer cells. Mechanistically, TEAD4 associates with receptor-regulated Smads (Smad2/3) and Smad4 in the nucleus, thereby impairing the binding of Smad2/3 to the histone acetyltransferase p300. Intriguingly, these negative effects of TEAD4 on TGF-ß/Smad signaling are independent of YAP, as impairing the TEAD4-YAP interaction through point mutagenesis or depletion of YAP and/or its paralog TAZ has little effect. Together, these results unravel a novel function of TEAD4 in fine tuning TGF-ß signaling and liver cancer progression in a YAP-independent manner.

Identification of genes and pathways associated with sex in Non-smoking lung cancer population.

INTRODUCTION: Women represent a higher proportion than men among those with lung cancer in nonsmokers compared to smokers. The reason for this abnormally higher proportion is not yet clear, but sex differences suggest there may be a genetic component at play. MATERIALS AND METHODS: The gene expression determined by Illumina RNA Sequencing and the relevant clinical information of lung cancer patients was download from TCGA. The differentially expressed genes (DEGs) were screened between males and females in both nonsmoking and smoking populations. The top 50 validated DEGs are represented with heatmaps. Based on the DEGs, GO functional and KEGG pathway enrichment analyses were performed. PPI networks were constructed to further illustrate the direct and indirect associations among the DEGs. Survival analysis was performed to explore whether these genes can affect lung cancer patient prognosis. RESULTS: In non-smoking patients, there were significantly more females than males (female 73.0% vs male 27.0%, P < 0.001). Such difference was not found in smoking patients (female 50.7% vs male 49.3%, P = 0.770). A total of 898 DEGs were identified in the non-smoking population, while a total of 992 DEGs were identified in the smoking population. Of these, only 122 genes were shared by both populations. Some pathways were enriched specifical in non-smoking population, such as cAMP signaling pathway and ovarian steroidogenesis. Several proteins related to estrogen function and MAPK/PI3K signaling, such as KRT16, ERBB4 and NTF4, showed differential effects on the lung adenocarcinoma progression in non-smoking males or females. CONCLUSIONS: Some genetic differences between male and female in non-smoking lung adenocarcinoma patients have been identified. Potentially, ER signaling and MAPK/PI3K signaling partially participated in this discrepancy.

Aberrant expression of the extracellular matrix component Biglycan regulated by Hedgehog signalling promotes colorectal cancer cell proliferation.

Hedgehog (Hh) signalling plays essential roles in regulating embryonic development and contributes to tumour initiation, growth and progression in multiple cancers. The detailed mechanism by which Hh signalling participates in tumour growth warrants thorough study, although several downstream target genes have been identified. Herein, a set of novel targets of Hh signalling was identified in multiple types of tumour cells via RNA-Seq analysis. Among these targets, the expression regulation and oncogenic function of the extracellular matrix component biglycan (BGN) were investigated. Further investigation verified that Hh signalling activates the expression of BGN via the transcription factor Gli2, which directly binds to the promoter region of BGN. Functional assays revealed that BGN facilitates tumour cell growth and proliferation in colorectal cancer (CRC) cells, and xenograft assays confirmed that BGN also promotes tumour growth . Moreover, analysis of clinical CRC samples showed that both the protein and mRNA levels of BGN are increased in CRC tissues compared to those in adjacent tissues, and higher expression of BGN is correlated with poorer prognosis of CRC patients, further confirming the function of BGN in CRC. Taken together, aberrantly activated Hh signalling increases the expression of BGN, possibly regulates the extracellular matrix, and thereby promotes tumour growth in CRC.

DNAJC5 promotes hepatocellular carcinoma cells proliferation though regulating SKP2 mediated p27 degradation.

DNAJC5 (DnaJ heat shock protein family (Hsp40) member C5), also known as cysteine tandem protein (CSPα), is important for maintaining the normal function of nerve tissues, but its oncogenic function remains unknown. Here, we report a unique mechanism underlying the oncogenic function of DNAJC5. DNAJC5 protein expression is highly detectable in human hepatocellular carcinoma (HCC) tissues and is strongly related to a poor prognosis among HCC patients. DNAJC5 overexpression promotes HCC cell proliferation and reduced the ratio of cells in G1 phase of the cell cycle. Furthermore, DNAJC5 interacts with SKP2 and enhances the degradation of p27 (a cyclin-dependent kinase inhibitor1B) by promoting formation of the SKP2-p27 complex. In contrast, DNAJC5 knockdown rescues the SKP2-mediated decrease in p27 protein levels. These results reveal that the DNAJC5-SKP2-p27 pathway is a novel mechanism for the oncogenic function of DNAJC5 in HCC.

Positive feedback of SuFu negating protein 1 on Hedgehog signaling promotes colorectal tumor growth.

Hedgehog (Hh) signaling plays a critical role in embryogenesis and tissue homeostasis, and its deregulation has been associated with tumor growth. The tumor suppressor SuFu inhibits Hh signaling by preventing the nuclear translocation of Gli and suppressing cell proliferation. Regulation of SuFu activity and stability is key to controlling Hh signaling. Here, we unveil SuFu Negating Protein 1 (SNEP1) as a novel Hh target, that enhances the ubiquitination and proteasomal degradation of SuFu and thus promotes Hh signaling. We further show that the E3 ubiquitin ligase LNX1 plays a critical role in the SNEP1-mediated degradation of SuFu. Accordingly, SNEP1 promotes colorectal cancer (CRC) cell proliferation and tumor growth. High levels of SNEP1 are detected in CRC tissues and are well correlated with poor prognosis in CRC patients. Moreover, SNEP1 overexpression reduces sensitivity to anti-Hh inhibitor in CRC cells. Altogether, our findings demonstrate that SNEP1 acts as a novel feedback regulator of Hh signaling by destabilizing SuFu and promoting tumor growth and anti-Hh resistance.

Identification of genes and pathways related to breast cancer metastasis in an integrated cohort.

BACKGROUND: Breast cancer is the most common malignant disease in women. Metastasis is the most common cause of death from this cancer. Screening genes related to breast cancer metastasis may help elucidate the mechanisms governing metastasis and identify molecular targets for antimetastatic therapy. The development of advanced algorithms enables us to perform cross-study analysis to improve the robustness of the results. MATERIALS AND METHODS: Ten data sets meeting our criteria for differential expression analyses were obtained from the Gene Expression Omnibus (GEO) database. Among these data sets, five based on the same platform were formed into a large cohort using the XPN algorithm. Differentially expressed genes (DEGs) associated with breast cancer metastasis were identified using the differential expression via distance synthesis (DEDS) algorithm. A cross-platform method was employed to verify these DEGs in all ten selected data sets. The top 50 validated DEGs are represented with heat maps. Based on the validated DEGs, Gene Ontology (GO) functional and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed. Protein interaction (PPI) networks were constructed to further illustrate the direct and indirect associations among the DEGs. Survival analysis was performed to explore whether these genes can affect breast cancer patient prognosis. RESULTS: A total of 817 DEGs were identified using the DEDS algorithm. Of these DEGs, 450 genes were validated by the second algorithm. Enriched KEGG pathway terms demonstrated that these 450 DEGs may be involved in the cell cycle and oocyte meiosis in addition to their functions in ECM-receptor interaction and protein digestion and absorption. PPI network analysis for the proteins encoded by the DEGs indicated that these genes may be primarily involved in the cell cycle and extracellular matrix. In particular, several genes played roles in multiple signalling pathways and were related to patient survival. These genes were also observed to be targetable in the CTD2 database. CONCLUSIONS: Our study analysed multiple cross-platform data sets using two different algorithms, helping elucidate the molecular mechanisms and identify several potential therapeutic targets of metastatic breast cancer. In addition, several genes exhibited promise for applications in targeted therapy against metastasis in future research.

The Hedgehog signaling pathway promotes chemotherapy resistance via multidrug resistance protein 1 in ovarian cancer.

Various studies have revealed that the Hedgehog (Hh) signaling pathway promotes ovarian cancer invasion, migration and drug resistance. Previous studies by our group have identified a set of genes, including multidrug resistance gene 1 (MDR1), that are regulated by Hh signaling in ovarian cancer. However, the association between Hh signaling activation and MDR1 expression requires further validation. In the present study, reverse transcription­quantitative PCR or western blot assays were used to evaluate the mRNA and protein expression levels of MDR1, Sonic Hh (Shh), glioma­associated oncogene 2 (Gli2), Gli1 and γ­phosphorylated H2A.X variant histone (γ­H2AX). MTT and colony­formation assays were performed to determine the effect of cisplatin (DDP) after inhibiting the Hh pathway in ovarian cancer cells. The results indicated that MDR1, Gli2 and Shh levels were much higher in SK­OV­3 cells with acquired DDP resistance than in native SK­OV­3 cells. ES­2 cells with overexpression of Gli2 were capable of efficiently forming colonies in the presence of low DDP concentrations. By contrast, Gli2 knockdown in SK­OV­3 cells decreased the colony­forming ability under the same concentration of DDP. As determined by MTT assays, knockdown of Gli2 or targeting of the Hh signaling pathway with either Gli­antagonist 61 (GANT61) or cyclopamine, in combination with DDP treatment, diminished the viability of ES­2 and SK­OV­3 cells, whereas Gli2 overexpression increased the viability of ES­2 cells in the presence of DDP. Knockdown of Gli2 or targeting the Hh signaling pathway with GANT61 also increased γ­H2AX levels but decreased the expression of MDR1 in the presence of DDP. MDR1 expression is regulated by the Hh signaling pathway and is likely a downstream transcription factor of Gli2. In conclusion, targeting the Hh signaling pathway increases the sensitivity of ovarian cancer to DDP. MDR1 is a target gene of the Hh signaling pathway and this pathway may affect chemoresistance of ovarian cancer to DDP via MDR1.

The critical role of dysregulated Hh-FOXM1-TPX2 signaling in human hepatocellular carcinoma cell proliferation.

BACKGROUND: Aberrant activation of the Hedgehog (Hh) signaling pathway is frequently observed in hepatocellular carcinoma (HCC), nevertheless, the precise molecular mechanism remains unclear. Forkhead box M1 (FOXM1), a target of the Hh pathway, is a key oncofetal transcription factor and a master cell cycle regulator. Targeting protein for Xenopus kinesin-like protein 2 (TPX2) is an oncogene critical for mitosis. However, how these molecular events affect HCC progression remains unclear. METHODS: Realtime PCR, immunohistochemistry, western blotting, and analyses of datasets TCGA and Gene Expression Omnibus (GEO) were conducted to assess the expression of TPX2 and FOXM1 at the mRNA and protein levels in HCC samples or HCC cells. Expression and knockdown of TPX2 and FOXM1 were performed to assess their role in regulating HCC cell proliferation in vitro and in vivo. Dual luciferase report assay and chromosome immunoprecipitation (ChIP) were investigated to seek the FOXM1 binding sites in the promoter of TPX2. RESULTS: Specific antagonists (cyclopamine and GANT61) of the Hh pathway down-regulated TPX2, whereas activation of Hh signaling stimulated TPX2 expression. Furthermore, TPX2 over-expression accelerated HCC cell proliferation when upstream events of Hh signaling were inhibited, and TPX2 knockdown significantly alleviated Sonic Hh ligand (Shh)-induced HCC cell proliferation. Reporter assays and ChIP showed that FOXM1 bound to the TPX2 promoter, confirming that TPX2 is a direct downstream target of FOXM1. Xenograft model further verified the cell function and expression regulation of TPX2 and FOXM1 in vivo. Furthermore, FOXM1 regulated TPX2 activity to drive HCC proliferation. Immunohistochemical (IHC) analysis indicated that FOXM1 and TPX2 were highly-expressed in HCC samples and cohort study revealed that FOXM1 and TPX2 may act as negative predictors for the prognosis of patients with HCC. CONCLUSIONS: TPX2 acts as a novel downstream target and effector of the Hh pathway, and Hh signaling contributes to HCC proliferation via regulating the FOXM1-TPX2 cascade, suggesting that this signaling axis may be a novel therapeutic target for HCC.

FOXM1 promotes hepatocellular carcinoma progression by regulating KIF4A expression.

BACKGROUND: Forkhead box M1 (FOXM1) is a proliferation-associated transcription factor of the forkhead box proteins superfamily, which includes four isoforms FOXM1a, b, c, and d. FOXM1 has been implicated in hepatocellular carcinoma (HCC) progression, but the underlying molecular mechanism remains elusive. In this study, we aim to clarify the molecular basis for FOXM1-mediated HCC progression. METHODS: Bioinformatic analysis was used to explore the differentially expressed genes predicting HCC proliferation. The expression of FOXM1 and kinesin family member (KIF)4A was confirmed by western blotting and immunohistochemistry in HCC tissues. Kaplan-Meier survival analysis was conducted to analyze the clinical impact of FOXM1 and KIF4A on HCC. The effect of FOXM1 on the regulation of KIF4A expression was studied in cell biology experiments. The interaction between KIF4A and FOXM1 was analyzed by chromatin immunoprecipitation and luciferase experiments. A series of experiments was performed to explore the functions of FOXM1/KIF4A in HCC progression, such as cell proliferation, cell growth, cell viability, and cell cycle. A xenograft mouse model was used to explore the regulatory effect of FOXM1-KIF4A axis on HCC tumor growth. RESULTS: FOXM1 and KIF4A were overexpressed in human primary HCC tissues compared to that in matched adjacent normal liver tissue and are significant risk factors for HCC recurrence and shorter survival. We found that KIF4A was dominantly regulated by FOXM1c among the four isoforms, and further identified KIF4A as a direct downstream target of FOXM1c. Inhibiting FOXM1 decreased KIF4A expression in HCC cells, whereas its overexpression had the opposite effect. FOXM1-induced HCC cell proliferation was dependent on elevated KIF4A expression as KIF4A knockdown abolished FOXM1-induced proliferation of HCC cells both in vitro and in vivo. CONCLUSION: The FOXM1-KIF4A axis mediates human HCC progression and is a potential therapeutic target for HCC treatment.

Aberrant Activation Of Hedgehog Signalling Promotes Cell Migration And Invasion Via Matrix Metalloproteinase-7 In Ovarian Cancer Cells.

Background: Accumulating evidence indicates that aberrant activation of the Hedgehog (Hh) signalling pathway by Glioma-associated oncogene (Gli) transcription factors is involved in the aggressive progression of cancers, including ovarian cancer. Whereas the molecular mechanism underlying this phenomenon remains unelucidated. Matrix metalloproteinase-7 (MMP-7) facilitates degradation of the extracellular matrix, promoting the invasion of tumour cells, and is associated with cancer progression and metastasis. In previous reports, we identified a set of genes regulated by Hh signalling in ovarian tumour cells among which MMP-7 was identified as a potential Hh target gene candidate. However, establishing an association between Hh signalling activation and MMP-7 expression requires further validation, and the function of this regulation remains unknown. Methods: A cDNA microarray was utilized to identify potential downstream targets of Hh signalling. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to evaluate mRNA expression, and immunoblotting (IB) was conducted to evaluate protein expression. The invasive and migratory capabilities of tumour cells were tested with the transwell and wound healing assays, respectively. The mRNA levels of Gli2 and MMP-7 in normal ovarian tissues and cancerous tissues in various stages together with the corresponding clinical information were acquired from the indicated GEO datasets to elucidate associations between MMP-7 expression and cancer progression and prognosis. Additionally, immunohistochemistry (IHC) was performed in multiple ovarian cancers, benign tumours and normal tissues to evaluate Gli2 and MMP-7 protein expression. Results: MMP-7 expression was regulated by the Hh ligand, antagonist and downstream transcript factor Gli2, demonstrating this gene as an Hh target. MMP-7 facilitated the invasion and migration of ovarian tumour cells, indicating its key function in ovarian cancer progression. IHC analysis demonstrated abnormally increased Gli2 and MMP-7 expression levels in benign tumours and ovarian cancer tissues. Moreover, high MMP-7 levels were significantly associated with poor overall survival (OS) and poor progression-free survival (PFS) in ovarian cancer patients. Conclusion: Aberrant activation of the Hh-Gli-MMP-7 signalling axis is essential for acceleration of the progression and metastasis of human ovarian cancer, implicating its use as a novel therapeutic target of ovarian cancer. In addition, MMP-7 can potentially serve as a prognostic marker of ovarian cancer.

GLI2 promotes cell proliferation and migration through transcriptional activation of ARHGEF16 in human glioma cells.

BACKGROUND: The Hedgehog (Hh) signaling pathway plays critical roles in modulating embryogenesis and maintaining tissue homeostasis, with glioma-associated oncogene (GLI) transcription factors being the main mediators. Aberrant activation of this pathway is associated with various human malignancies including glioblastoma, although the mechanistic details are not well understood. METHODS: We performed a microarray analysis of genes that are differentially expressed in glioblastoma U87 cells overexpressing GLI2A, the active form of GLI2, relative to the control cells. Chromatin immunoprecipitation and dual-luciferase assays were used to determine whether Rho guanine nucleotide exchange factor 16 (ARHGEF16) is a downstream target of GLI2. Then, transwell migration, EdU and soft-agar colony formation assays were employed to test effects of ARHGEF16 on glioma cancer cell migration and proliferation, and the effects of GLI2/ARHGEF16 signaling on tumor growth were examined in vivo. Finally, we performed yeast two-hybrid assay, Co-IP and GST-pull down to identify factors that mediate effects of ARHGEF16. RESULTS: We found that ARHGEF16 mRNA level was upregulated in U87 cells overexpressing GLI2A relative to control cells. GLI2 binds to the ARHGEF16 promoter and activates gene transcription. Glioma cells U87 and U118 overexpressing ARHGEF16 showed enhanced migration and proliferation relative to the control cells, while knockdown of ARHGEF16 in H4 cells led to decreased cell proliferation compared to the control H4 cells. In contrast to the promoting effect of GLI2A overexpression on glioma xenograft growth, both GLI2 inhibition and ARHGEF16 knockdown retarded tumor growth. Cytoskeleton-associated protein 5 (CKAP5) was identified as an interaction protein of ARHGEF16, which is important for the stimulatory effects of ARHGEF16 on glioma cell migration and proliferation. CONCLUSIONS: These results suggest that therapeutic strategies targeting the GLI2/ARHGEF16/CKAP5 signaling axis could inhibit glioma progression and recurrence.

Receptor for Activated C Kinase 1 (RACK1) Promotes Dishevelled Protein Degradation via Autophagy and Antagonizes Wnt Signaling.

Wnt signaling plays a critical role in embryonic development, tissue homeostasis, and cancer development. Dishevelled (Dvl) is an essential and central component in Wnt signaling, and its stability and activity is tightly regulated. It has been shown that Dvl can be degraded via both the proteasome and autophagy-lysosome pathways. Here we report that receptor for activated C kinase 1 (RACK1) negatively regulates Dishevelled stability and Wnt signaling. RACK1 interacts with Dvl proteins and promotes their lysosomal degradation, and this effect is enhanced by autophagy induction. RACK1 also interacts with LC3 and enhances the association of LC3 with Dvl2, thereby leading to degradation of Dvl proteins through autophagy. These findings reveal a novel regulatory function of RACK1 in Wnt signaling by modulating Dvl stability.

Smad7 Protein Interacts with Receptor-regulated Smads (R-Smads) to Inhibit Transforming Growth Factor-ß (TGF-ß)/Smad Signaling.

TGF-ß is a pleiotropic cytokine that regulates a wide range of cellular actions and pathophysiological processes. TGF-ß signaling is spatiotemporally fine-tuned. As a key negative regulator of TGF-ß signaling, Smad7 exerts its inhibitory effects by blocking receptor activity, inducing receptor degradation or interfering with Smad-DNA binding. However, the functions and the molecular mechanisms underlying the actions of Smad7 in TGF-ß signaling are still not fully understood. In this study we report a novel mechanism whereby Smad7 antagonizes TGF-ß signaling at the Smad level. Smad7 oligomerized with R-Smad proteins upon TGF-ß signaling and directly inhibited R-Smad activity, as assessed by Gal4-luciferase reporter assays. Mechanistically, Smad7 competes with Smad4 to associate with R-Smads and recruits the E3 ubiquitin ligase NEDD4L to activated R-Smads, leading to their polyubiquitination and proteasomal degradation. Similar to the R-Smad-Smad4 oligomerization, the interaction between R-Smads and Smad7 is mediated by their mad homology 2 (MH2) domains. A positive-charged basic region including the L3/ß8 loop-strand module and adjacent amino acids in the MH2 domain of Smad7 is essential for the interaction. These results shed new light on the regulation of TGF-ß signaling by Smad7.

The AP-1 transcription factor homolog Pf-AP-1 activates transcription of multiple biomineral proteins and potentially participates in Pinctada fucata biomineralization.

Activator protein-1 (AP-1) is an important bZIP transcription factor that regulates a series of physiological processes by specifically activating transcription of several genes, and one of its well-chartered functions in mammals is participating in bone mineralization. We isolated and cloned the complete cDNA of a Jun/AP-1 homolog from Pinctada fucata and called it Pf-AP-1. Pf-AP-1 had a highly conserved bZIP region and phosphorylation sites compared with those from mammals. A tissue distribution analysis showed that Pf-AP-1 was ubiquitously expressed in P. fucata and the mRNA level of Pf-AP-1 is extremely high in mantle. Pf-AP-1 expression was positively associated with multiple biomineral proteins in the mantle. The luciferase reporter assay in a mammalian cell line showed that Pf-AP-1 significantly up-regulates the transcriptional activity of the promoters of KRMP, Pearlin, and Prisilkin39. Inhibiting the activity of Pf-AP-1 depressed the expression of multiple matrix proteins. Pf-AP-1 showed a unique expression pattern during shell regeneration and pearl sac development, which was similar to the pattern observed for biomineral proteins. These results suggest that the Pf-AP-1 AP-1 homolog is an important transcription factor that regulates transcription of several biomineral proteins simultaneously and plays a role in P. fucata biomineralization, particularly during pearl and shell formation.