Quqin Lu

Down-Regulation of Gli Transcription Factor Leads to the Inhibition of Migration and Invasion of Ovarian Cancer Cells via Integrin β4-Mediated FAK Signaling

Background Recent evidence suggests that aberrant activation of Hedgehog (Hh) signaling by Gli transcription factors is characteristic of a variety of aggressive human carcinomas including ovarian cancer. Therefore, chemotherapeutic agents that inhibit activation of Gli transcription factors have emerged as promising novel therapeutic drugs for ovarian cancer. Results In this study, we show that activation of Hh signaling promoted cellular migration and invasion, whereas blockade of Hh signaling with GANT61 suppressed cellular migration and invasion in ovarian cancer cells. After treatment with GANT61, cDNA microarray analyses revealed changes in many genes such as Integrin β4 subunit (ITGB4), focal adhesion kinase (FAK), etc. Furthermore, ITGB4 expression was up-regulated by Sonic Hedgehog (Shh) ligand and down-regulated by Hh signaling inhibitor. The Shh-mediated ovarian cell migration and invasion was blocked by neutralizing antibodies to ITGB4. In addition, phosphorylations of FAK were increased by Shh and decreased by Hh signaling inhibitor. Inhibition of Gli1 expression using siRNA mimicked the effects of GANT61 treatment, supporting the specificity of GANT61. Further investigations showed that activation of FAK was required for Shh-mediated cell migration and invasion. Finally, we found that down-regulation of Gli reduced the expression of ITGB4 and the phosphorylated FAK, resulting in the inhibition of tumor growth in vivo. Conclusions The Hh signaling pathway induces cell migration and invasion through ITGB4-mediated activation of FAK in ovarian cancer. Our findings suggest that the diminishment of crosstalk between phosphorylated FAK and ITGB4 due to the down-regulation of Gli family transcription factors might play a pivotal role for inhibiting ovarian cancer progression.

Suppression of growth and migration by blocking the hedgehog signaling pathway in gastric cancer cells

PurposePrevious studies have indicated that Hedgehog signaling is essential for gastric cancer development, but its precise role is still unclear. The aim of this study was to clarify the role of Hedgehog signaling in gastric cancer development.MethodsThe expression of key Hedgehog signaling components in clinical samples of sequential gastric cancer stages was assessed by immunohistochemistry. The roles and regulatory mechanisms of Hedgehog signaling in human gastric cancer cells and normal gastric epithelial cells were investigated using multiple cell biological approaches and cDNA microarray analyses.ResultsHedgehog signaling was found to be abnormally activated in a ligand-independent manner during gastric cancer development. Gli1 over-expression and reduced SuFu expression were found to be typical events in gastric cancer tissues. Gli1 over-expression was found to correlate with a poorly differentiated histology, advanced clinical stage, membrane serosa infiltration and lymph node metastasis in patients with gastric cancer. Data obtained from multiple cell biological assays showed that human gastric cancer cells require active Hedgehog signaling for survival, proliferation, migration and colony formation. N-Shh treatment significantly enhanced the migration, invasion and colony formation of gastric cancer cells. Moreover, the results of cDNA microarray analyses indicated that after treatment with cyclopamine or GANT61 (inhibitors of Hedgehog signaling), differentially expressed genes in gastric cancer cells were enriched in the apoptosis and MAPK pathways. Inhibitors of the Hedgehog pathway were found to suppress gastric cancer cell growth via apoptosis induction.ConclusionsOur findings indicate a vital role of the activated Hedgehog signaling pathway in promoting gastric initiation and progression. The Hedgehog signaling pathway may serve as a target for gastric cancer therapy.

SPOP Suppresses Tumorigenesis by Regulating Hedgehog/Gli2 Signaling Pathway in Gastric Cancer

BackgroundRecent evidence suggests that aberrant activation of Hedgehog (Hh) signaling by Gli transcription factors is characteristic of a variety of aggressive human carcinomas including gastric cancer. Speckle-type POZ protein, SPOP, is an E3 ubiquitin ligase adaptor, and it is found to inhibit oncogenic signaling. However, the molecular mechanisms are largely unknown.MethodsIn this study, we characterized the expression of SPOP in 88 pairs of gastric cancer tissues and adjacent tissues by immunohistochemical staining and Western blotting. The relationship between SPOP expression and clinical pathologic factors was analyzed. Transfected gastric cancer cell lines were used in cell viability, wound healing and colony formation assays. The interaction of SPOP with Gli2 and other related apoptotic proteins was assessed by immunoprecipitation, Western blotting, real-time PCR and dual luciferase reporter assays. Intracellular interaction of SPOP and Gli2 was visualized by immunofluorescent staining in gastric cancer cells.ResultsImmunohistochemical staining of SPOP can be detected in gastric cancer tissues but much less than adjacent gastric tissues (P < 0.01). High SPOP expression is negatively correlated with lymph node metastasis, poor histological differentiation, and tumor malignancy according to TNM staging. In vitro experiments revealed that over-expression of SPOP prevented tumor cells from proliferation, migration and colony formation in gastric cancer cell lines. Likewise, repression of SPOP promoted cell viability, migration, proliferation, and attenuated apoptosis. Mechanistic studies revealed that increasing SPOP accelerated Gli2 degradation but regardless of Gli2 synthesis. Furthermore, cytoplasmic Gli2 decreased markedly along with the abundant expression of SPOP in MKN45 cells.ConclusionsOur findings indicate that SPOP plays critical roles in suppressing gastric tumorigenesis through inhibiting Hh/Gli2 signaling pathway. It may provide an alternative strategy for developing therapeutic agents of gastric cancer in future.

Aberrantly activated Gli2-KIF20A axis is crucial for growth of hepatocellular carcinoma and predicts poor prognosis.

Glioma-associated oncogene 2 (Gli2), a primary transcriptional regulator of Hedgehog (Hh) signaling, is essential for hepatocellular carcinoma (HCC) growth and survival. However, the underlying molecular mechanism and crucial downstream targets of Gli2 in human HCC are not fully understood. Here, we report the identification of kinesin family member 20A (KIF20A) as a novel downstream target of Gli2, which is important for HCC proliferation and tumor growth. Inhibition of Hh signaling leads to a remarkable decrease of KIF20A expression in HCC cells, whereas overexpression of Gli2 elevates KIF20A expression by activating Forkhead Box M1 (FoxM1)-MMB complex-mediated transcription of this kinesin gene. Gli2-induced HCC cell growth requires enhanced expression of KIF20A, and knockdown of Gli2 or KIF20A represses the proliferation of HCC cells in vitro and in vivo. Correlated with these results, analyses of clinical HCC samples show that Gli2, FoxM1 and KIF20A are highly elevated in primary HCC samples and represent significant risk factors for HCC recurrence and survival. Conclusion: KIF20A is an important downstream target gene of Hh signaling. And, the Gli2-KIF20A axis is essential for the proliferation and growth of human HCC cells. Our study also suggests Gli2-KIF20A axis as a potential target for future therapeutic intervention and as an independent prognostic biomarker for HCC.

Heat shock protein 90β stabilizes focal adhesion kinase and enhances cell migration and invasion in breast cancer cells.

Focal adhesion kinase (FAK) acts as a regulator of cellular signaling and may promote cell spreading, motility, invasion and survival in malignancy. Elevated expression and activity of FAK frequently correlate with tumor cell metastasis and poor prognosis in breast cancer. However, the mechanisms by which the turnover of FAK is regulated remain elusive. Here we report that heat shock protein 90β (HSP90β) interacts with FAK and the middle domain (amino acids 233-620) of HSP90β is mainly responsible for this interaction. Furthermore, we found that HSP90β regulates FAK stability since HSP90β inhibitor 17-AAG triggers FAK ubiquitylation and subsequent proteasome-dependent degradation. Moreover, disrupted FAK-HSP90β interaction induced by 17-AAG contributes to attenuation of tumor cell growth, migration, and invasion. Together, our results reveal how HSP90β regulates FAK stability and identifies a potential therapeutic strategy to breast cancer.

Aberrant activation of hedgehog signaling promotes cell proliferation via the transcriptional activation of forkhead Box M1 in colorectal cancer cells

BackgroundRecent evidence suggests that the aberrant activation of Hedgehog (Hh) signaling by Gli transcription factors is characteristic of a variety of aggressive human carcinomas, including colorectal cancer (CRC). Forkhead box M1 (FoxM1) controls the expression of a number of cell cycle regulatory proteins, and FoxM1 expression is elevated in a broad range of human malignancies, which suggests that it plays a crucial role in tumorigenesis. However, the mechanisms underlying FoxM1 expression are not fully understood. Here, we aim to further investigate the molecular mechanism by which Gli1 regulates FoxM1 in CRC.MethodsWestern blotting and immunohistochemistry (IHC) were used to evaluate FoxM1 and Gli1 protein expression, respectively, in CRC tissues and matched adjacent normal mucosa. BrdU (5-bromo-2′-deoxyuridine) and clone formation assays were used to clarify the influence of FoxM1 on CRC cell growth and proliferation. Chromatin immunoprecipitation (ChIP) and luciferase experiments were performed to explore the potential mechanisms by which Gli1 regulates FoxM1. Additionally, the protein and mRNA expression levels of Gli1 and FoxM1 in six CRC cell lines were measured using Western blotting and real-time PCR. Finally, the effect of Hh signaling on the expression of FoxM1 was studied in cell biology experiments, and the effects of Hh signaling activation and FoxM1 inhibition on the distribution of CRC cells among cell cycle phases was assessed by flow cytometry.ResultsGli1 and FoxM1 were abnormally elevated in human CRC tissues compared with matched adjacent normal mucosa samples, and FoxM1 is a downstream target gene of the transcription factor Gli1 in CRC and promoted CRC cell growth and proliferation. Moreover, the aberrant activation of Hh signaling promoted CRC cell proliferation by directly binding to the promoter of FoxM1 and transactivating the activity of FoxM1 in CRC cells.ConclusionThe dysregulation of the Hh-Gli1-FoxM1 axis is essential for the proliferation and growth of human CRC cells and offers a potent target for therapeutic intervention in CRC.

Inhibition of Hedgehog signaling pathway impedes cancer cell proliferation by promotion of autophagy.

Multiple lines of evidence implicate that aberrant activation of Hedgehog (Hh) signaling is involved in a variety of human cancers. However, the molecular mechanisms underlying how cancer cells respond to Hh inhibition remain to be elucidated. In this study, we found that blockade of Hh signaling suppresses cell proliferation in human cancer cells. Microarray analysis revealed that differentially expressed genes (DEGs) in human cancer cells are enriched in autophagy pathway in response to the inhibition of Hh signaling. Interestingly, inhibition of Hh signaling induced autophagy, whereas activation of Hh signaling by ligand treatments prevented the induction of autophagy. In addition, inhibition of autophagy by 3-methyladenine (3-MA) partially suppressed cytotoxicity induced by inhibition of Hh signaling. Finally, in autophagy deficient cells, cytotoxic effect triggered by inhibition of Hh signaling was partially reversed, indicating the modulation of autophagy by Hh signaling is autophagy-specific. These results suggest that inhibition of Hh signaling impedes cancer cell proliferation in part through induction of autophagy.

Arl13b promotes gastric tumorigenesis by regulating Smo trafficking and activation of the Hedgehog signaling pathway

Inhibitors of the Hedgehog (Hh) pathway transducer Smoothened (Smo) have been approved for cancer treatment, but Smo mutations often lead to tumor resistance and it remains unclear how Smo is regulated. In this study, we identified the small GTPase Arl13b as a novel partner and regulator of Smo. Arl13b regulated Smo stability, trafficking, and localization, which are each crucial for Hh signaling. In gastric cancer cells, Arl13b stimulated proliferation, migration, and invasion in vitro and in vivo In clinical specimens of gastric cancer, Arl13b expression correlated strongly with tumor size and depth of invasion; patients with high levels of Arl13b had a poor prognosis. Our results show how Arl13b participates in Hh pathway activation in gastric cancer. Cancer Res; 77(15); 4000-13. ©2017 AACR.

Aldehyde dehydrogenase 1A1 stabilizes transcription factor Gli2 and enhances the activity of Hedgehog signaling in hepatocellular cancer.

The Gli transcription factors are primary transcriptional regulators that mediate the activation of Hedgehog (Hh) signaling. Recent studies have revealed that Gli proteins are also regulated transcriptionally and post-translationally through noncanonical mechanisms, independent of Hh signaling. However, the precise mechanisms involved in the regulation of Gli proteins remain unclear. Using a differential mass-spectrometry approach, we found that aldehyde dehydrogenase 1A1 (ALDH1A1) is associated with transcription factor Gli2. Overexpression of ALDH1A1 increased Gli2 protein levels; in contrast, ALDH1A1 depletion facilitated Gli2 degradation. In addition, Gli2 mRNA expression was not affected by ectopic expression of ALDH1A1, indicating the role of ALDH1A1 in the stabilization of Gli2. Further investigation showed that ALDH1A1 prolonged the stability of Gli2 protein in a catalytic-independent manner. Finally, we showed that overexpression of ALDH1A1 activated the Hh signaling pathway and promoted cell growth, migration and invasion in hepatocellular cancer cells. Together, these results illustrate regulatory roles of ALDH1A1 in the activation of the Hh signaling pathway and highlight a novel mechanism for the aberrant activation of the Hh signaling pathway in hepatocellular cancer cells.

The Inhibition of Heat Shock Protein 90 Facilitates the Degradation of Poly-Alanine Expanded Poly (A) Binding Protein Nuclear 1 via the Carboxyl Terminus of Heat Shock Protein 70-Interacting Protein.

Background Since the identification of poly-alanine expanded poly(A) binding protein nuclear 1 (PABPN1) as the genetic cause of oculopharyngeal muscular dystrophy (OPMD), considerable progress has been made in our understanding of the pathogenesis of the disease. However, the molecular mechanisms that regulate the onset and progression of the disease remain unclear. Results In this study, we show that PABPN1 interacts with and is stabilized by heat shock protein 90 (HSP90). Treatment with the HSP90 inhibitor 17-AAG disrupted the interaction of mutant PABPN1 with HSP90 and reduced the formation of intranuclear inclusions (INIs). Furthermore, mutant PABPN1 was preferentially degraded in the presence of 17-AAG compared with wild-type PABPN1 in vitro and in vivo. The effect of 17-AAG was mediated through an increase in the interaction of PABPN1 with the carboxyl terminus of heat shock protein 70-interacting protein (CHIP). The overexpression of CHIP suppressed the aggregation of mutant PABPN1 in transfected cells. Conclusions Our results demonstrate that the HSP90 molecular chaperone system plays a crucial role in the selective elimination of abnormal PABPN1 proteins and also suggest a potential therapeutic application of the HSP90 inhibitor 17-AAG for the treatment of OPMD.