Yongjie Ma

Reduction of Akt2 inhibits migration and invasion of glioma cells

Malignant gliomas have a tendency to invade diffusely into surrounding healthy brain tissues, thereby precluding their successful surgical removal. The serine/threonine kinase Akt2 is well known as an important regulator of cell survival and growth. In this study, we show that siRNA‐mediated depletion of Akt2 inhibited migration and invasion of glioma cells. In addition, we demonstrate the mechanisms by which Akt2 functions to promote cell migration and invasion. Phosphorylation of cofilin, a critical step of actin polymerization, and phosphorylation of Girdin, essential for the integrity of the actin cytoskeleton and cell migration, were impaired. Furthermore, epidermal growth factor‐induced ACAP1 phosphorylation and integrin β1 phosphorylation were also blocked, consistent with defects in adhesion. Thus, Akt2 regulates both cell adhesion and cytoskeleton rearrangement during migration. Decreased MMP‐9 expression in Akt2 knocked‐down glioma cells was subsequently confirmed by Western blotting, consistent with the decreased invasion in vitro and in vivo. These results suggest that Akt2 contributes to glioma cells migration and invasion by regulating the formation of cytoskeleton, influencing adhesion and increasing expression of MMP‐9. Our immunohistochemistry results by using human gliomas tissue sections also indicated that Akt2 expression was closely related with the malignancy of gliomas. This is coincident with our in vivo and in vitro results from cell lines. All of these results indicate that Akt2 is a critical factor in gliomas invasion. This study identifies that Akt2 is a potentially antiinvasion target for therapeutic intervention in gliomas.

Reduction of protein kinase C ζ inhibits migration and invasion of human glioblastoma cells

Glioblastomas are the most aggressive forms of primary brain tumors with their tendency to invade surrounding healthy brain tissues, rendering them largely incurable. In this report, we used small‐interference RNA technology to knock down the expression of protein kinase C (PKC) ζ, which resulted in specific and massive impairment of glioblastoma cell migration and invasion. We also explained the fundamental molecular processes of glioblastoma migration and invasion in which PKCζ is a participant. The silence of PKCζ expression likewise impaired the phosphorylation of LIN‐11, Isl1 and MEC‐3 protein domain kinase (LIMK) and cofilin, which is a critical step in cofilin recycling and actin polymerization. Consistent with the defects in cell adhesion, phosphorylation of integrin β1 was also dampened. Therefore, PKCζ regulated both cytoskeleton rearrangement and cell adhesion, which contributed to cell migration. Additionally, there was down‐regulation of matrix metalloprotease‐9 expression in siPKCζ/LN‐229 cells, which coincided with decreased invasion both in vitro and in vivo. These results indicate that PKCζ is involved in the control of glioblastoma cell migration and invasion by regulating the cytoskeleton rearrangement, cell adhesion, and matrix metalloprotease‐9 expression. Collectively, these findings suggest that PKCζ is a potential therapeutic target for glioblastoma infiltration.

Role of aquaporin-4 in the regulation of migration and invasion of human glioma cells

Glioblastoma is the most aggressive form of primary brain tumor with a tendency to invade surrounding healthy brain tissues, rendering tumors of this type largely incurable. Aquaporin-4 (AQP4) is a key molecule involved in maintaining water and ion homeostasis in the central nervous system and has been recently reported to play a role in cell migration in addition to its well-known function in brain edema. Increased AQP4 expression has been demonstrated in glioblastoma multiforme (GBM), suggesting that it is also involved in malignant brain tumors. Here, we identify a novel role for aquaporin-4 in glioblastoma cell migration and invasion. In the present study, we used small-interference RNA technology and a pharmacological inhibitor to knock down the expression of AQP4, which resulted in specific and massive impairment of glioblastoma cell migration and invasion in vitro and in vivo. In addition, we demonstrated the possible mechanisms by which AQP4 functions in the process of glioblastoma cell invasion. The downregulation of matrix metalloprotease-2 (MMP-2) expression in LN229 cells with AQP4 reduction coincided with decreased cell invasive ability. Furthermore, our study showed that AQP4 may also be involved in the regulation of glioblastoma cell adhesion. The expression of β-catenin and connexin 43 were increased in AQP4-downregulated LN229 cells consistent with their enhanced cell-cell adhesion ability. In summary, our results indicate that AQP4 is involved in the control of glioblastoma cell migration and invasion and may be a potential therapeutic target for glioblastoma cell infiltration.

Akt2 is required for macrophage chemotaxis

Tumor‐associated macrophages play an important role in tumorigenesis and metastasis. Trafficking of macrophages to the proximity of tumors is mediated by CSF‐1, a growth factor. In this study, we investigated the role of PKB/Akt in CSF‐1‐induced macrophage migration. Disruption of Akt2 expression by small interference RNA impaired chemotaxis of both THP‐1 cells and mouse peritoneal macrophages. Phosphorylation of PKCζ, an essential component in chemotaxis signaling pathway, was reduced. LIMK/Cofilin, downstream of PKCζ, regulated cytoskeleton rearrangement during cell migration. Disruption of Akt2 expression inhibited CSF‐1‐induced LIMK/Cofilin phosphorylation, which contributed to defects in actin polymerization and chemotaxis. Furthermore, MCP‐1, a chemokine, ‐induced macrophage chemotaxis was also impaired. Taken together, our results demonstrated that Akt2 plays an essential role in both CSF‐1‐ and chemokine‐induced chemotaxis of macrophages.

Pivotal Advance: PKCζ is required for migration of macrophages

The crosstalk, mediated by chemoattractants, between cancer cells and tumor‐associated macrophages, plays an important role in tumor invasion and metastasis. Our previous study reported that atypical protein kinase C ζ (PKCζ) regulates epidermal growth factor‐induced chemotaxis of human breast cancer cells. In this study, we investigated the role of PKCζ in CSF‐1‐induced chemotaxis of macrophages. Knockdown of PKCζ by small interference RNA impaired CSF‐1‐induced chemotaxis of human acute monocytic leukemia cell line THP‐1, which was probably a result of a decrease in CSF‐1‐induced phosphorylation of LIN‐11, Is11, and MEC‐3 protein domain kinase (LIMK)/cofilin and actin polymerization. Furthermore, silencing PKCζ expression also impaired migration of mouse peritoneal macrophages. Scratch analysis indicated that PKCζ was required for macrophage migration. Therefore, PKCζ is required for CSF‐1‐induced chemotaxis of macrophages. Blocking activation of PKCζ will be a novel strategy to inhibit cancer metastasis by blocking migration of cancer cells and macrophages.

Intersectin1-s is involved in migration and invasion of human glioma cells

Malignant gliomas have a tendency to invade diffusely into surrounding healthy brain tissues, thereby precluding their successful surgical removal. Intersectin1 (ITSN1) as a molecular linker in the central nervous system is well known as an important regulator of endocytosis and exocytosis. ITSN1 has two isoforms: ITSN1‐l and ITSN1‐s. In this study, we show that siRNA‐mediated down regulation of ITSN1‐s inhibited migration and invasion of glioma cells. In addition, we demonstrate the possible mechanisms by which ITSN1‐s functions in migration and invasion. Several key proteins, including cofilin, LIMK, PAK, FAK, integrin β1, and MMP‐9, which are critical for cells migration and invasion, were probably involved in ITSN1‐s signaling pathways. These results suggest that ITSN1‐s contributes to glioma cells migration and invasion by regulating the formation of cytoskeleton, influencing adhesion and increasing expression of MMP‐9. Our results indicate that ITSN1‐s is a critical factor in glioms invasion and identify that ITSN1‐s is a new potentially antiinvasion target for therapeutic intervention in gliomas.

Reduction of intersectin1-s induced apoptosis of human glioblastoma cells

Malignant gliomas have a high proliferation ability and high tendency to invade diffusely into surrounding healthy brain tissues, thereby precluding their successful surgical removal. Intersectin1 (also called ITSN1) as a molecular linker in the central nervous system is well known as an important regulator of endocytosis and exocytosis. ITSN1 has two isoforms: ITSN1-l and ITSN1-s. In this study, we show that siRNA-mediated down regulation of ITSN1-s induced glioma cells apoptosis. In addition, we demonstrate the possible mechanisms by which ITSN1-s functions in glioma cells apoptosis. Our data demonstrate that several key proteins, including FAK, Akt, Bcl-2, BAD which are critical for cells apoptosis were probably involved in ITSN1-s signaling pathways. Our results indicate that ITSN1-s is an effecter in regulation of gliomas cells apoptosis, and identify that ITSN1-s may be a new potentially anti-apoptosis target for therapeutic of gliomas.

Expression of aquaporin1, a water channel protein, in cytoplasm is negatively correlated with prognosis of breast cancer patients.

Aquaporin1 (AQP1) belongs to a highly conserved family of aquaporin proteins which facilitate water flux across cell membranes. Although emerging evidences indicated the cytoplasm was important for AQP1 localization, the function of AQP1 corresponding to its cytoplasmic distribution has rarely been explored until present. In our clinical study, we reported for the first time that AQP1 was localized dominantly in the cytoplasm of cancer cells of invasive breast cancer patients and cytoplasmic AQP1 was an independent prognostic factor. High expression of AQP1 indicated a shorter survival, especially in luminal subtype. Moreover, in line with our findings in clinic, cytoplasmic expression of AQP1 was further validated in both primary cultured breast cancer cells and AQP1 over-expressing cell lines, in which the functional importance of cytoplasmic AQP1 was confirmed in vitro. In conclusion, our study provided the first evidence that cytoplasmic expression of AQP1 promoted breast cancer progression and it could be a potential prognostic biomarker for breast cancer.

Low Expression of Slit2 and Robo1 is Associated with Poor Prognosis and Brain-specific Metastasis of Breast Cancer Patients.

Brain metastasis is a significant unmet clinical problem in breast cancer treatment. It is always associated with poor prognosis and high morbidity. Recently, Slit2/Robo1 pathway has been demonstrated to be involved in the progression of breast carcinoma. However, until present, there are no convincing reports that suggest whether the Slit2/Robo1 axis has any role in brain metastasis of breast cancer. In this study, we investigated the correlation between Slit2/Robo1 signaling and breast cancer brain metastasis for the first time. Our results demonstrated that (1) Invasive ductal carcinoma patients with low expression of Slit2 or Robo1 exhibited worse prognosis and brain-specific metastasis, but not liver, bone or lung. (2) Lower expression of Slit2 and Robo1 were observed in patients with brain metastasis, especially in their brain metastasis tumors, compared with patients without brain metastasis. (3) The interval from diagnosis of breast cancer to brain metastasis and brain metastasis to death were both much shorter in patients with low expression of Slit2 or Robo1 compared with the high expression group. Overall, our findings indicated that Slit2/Robo1 axis possibly be regarded as a significant clinical parameter for predicting brain metastasis in breast cancer patients.

Girdin, an actin-binding protein, is critical for migration, adhesion, and invasion of human glioblastoma cells

Girdin, an actin‐binding protein, possesses versatile functions in a multitude of cellular processes. Although several studies have shown that Girdin is involved in the cell DNA synthesis, actin cytoskeleton rearrangement, and cell motility, the molecular mechanisms of Girdin in tumor development and progression remain elusive. In this study, through over‐expression and siRNA experiments, we found that Girdin increased migration of LN229 human glioblastoma cells. On the other hand, reducing Girdin impaired F‐actin polymerization, which is essential for cell morphogenesis and motility. Matrix metalloproteinase 2, critical in human glioma migration and invasion, was down‐regulated upon Girdin reduction and led to decreased invasion in vitro and in vivo. In addition, silencing Girdin expression impaired the phosphorylation of two important adhesion molecules, integrin β1 and focal adhesion kinase, resulting in cell adhesion defects. Our immunohistochemical study on human gliomas tissue sections indicated that Girdin expression was positively related with glioma malignancy, supporting the in vitro and in vivo results from cell lines. Collectively, our findings suggest a critical role for Girdin in glioma infiltration. We show that reduction of Girdin, an actin‐binding protein, leads to impaired F‐actin polymerization and down‐regulated expression of matrix metallopeptidase protein 2 (MMP‐2), phosphorylated integrin β1, and phosphorylated focal adhesion kinase (FAK), which resulted in decreased migration, adhesion, and invasion of glioblastoma cells. Girdin was positively correlated with glioma malignancy and negatively associated with clinical prognosis, suggesting Girdin as a critical regulator in glioma infiltration.