Na+/K+-ATPase β2-subunit (AMOG) expression abrogates invasion of glioblastoma-derived brain tumor-initiating cells

Background

Mechanisms of glioma invasion remain to be fully elucidated. Glioma cells within glioblastoma multiforme (GBM) range from well-differentiated tumor cells to less-differentiated brain tumor-initiating cells (BTICs). The β2-subunit of Na⁺/K⁺-ATPase, called the adhesion molecule on glia (AMOG), is highly expressed in normal glia but is thought to be universally downregulated in GBM. To test our hypothesis that expression of AMOG is heterogeneous in GBM and confers a less invasive phenotype, we compared it between BTICs and differentiated cells from patient-matched GBM and then tested GBM invasion in vitro after AMOG overexpression.

Methods

Immunohistochemistry, immunoblotting, and real-time PCR were used to characterize AMOG protein and mRNA expression in tumor samples, BTICs, and differentiated cells. Matrigel invasion assay, scratch assay, and direct cell counting were used for testing in vitro invasion, migration, and proliferation, respectively.

Results

While AMOG expression is heterogeneous in astrocytomas of grades II–IV, it is lost in most GBM. BTICs express higher levels of AMOG mRNA and protein compared with patient-matched differentiated tumor cells. Overexpression of AMOG decreased GBM cell and BTIC invasion without affecting migration or proliferation. Knockdown of AMOG expression in normal human astrocytes increased invasion.

Conclusions

AMOG expression inhibits GBM invasion. Its downregulation increases invasion in glial cells and may also represent an important step in BTIC differentiation. These data provide compelling evidence implicating the role of AMOG in glioma invasion and provide impetus for further investigation.     

Gefitinib selectively inhibits tumor cell migration in EGFR-amplified human glioblastoma

Background

Tissue invasion is a hallmark of most human cancers and remains a major source of treatment failure in patients with glioblastoma (GBM). Although EGFR amplification has been previously associated with more invasive tumor behavior, existing experimental models have not supported quantitative evaluation of interpatient differences in tumor cell migration or testing of patient-specific responses to therapies targeting invasion. To explore these questions, we optimized an ex vivo organotypic slice culture system allowing for labeling and tracking of tumor cells in human GBM slice cultures.

Methods

With use of time-lapse confocal microscopy of retrovirally labeled tumor cells in slices, baseline differences in migration speed and efficiency were determined and correlated with EGFR amplification in a cohort of patients with GBM. Slices were treated with gefitinib to evaluate anti-invasive effects associated with targeting EGFR.

Results

Migration analysis identified significant patient-to-patient variation at baseline. EGFR amplification was correlated with increased migration speed and efficiency compared with nonamplified tumors. Critically, gefitinib resulted in a selective and significant reduction of tumor cell migration in EGFR-amplified tumors.

Conclusions

These data provide the first identification of patient-to-patient variation in tumor cell migration in living human tumor tissue. We found that EGFR-amplified GBM are inherently more efficient in their migration and can be effectively targeted by gefitinib treatment. These data suggest that stratified clinical trails are needed to evaluate gefitinib as an anti-invasive adjuvant for patients with EGFR-amplified GBM. In addition, these results provide proof of principle that primary slice cultures may be useful for patient-specific screening of agents designed to inhibit tumor invasion.     

Inhibition of HSP27 alone or in combination with pAKT inhibition as therapeutic approaches to target SPARC-induced glioma cell survival

Background

The current treatment regimen for glioma patients is surgery, followed by radiation therapy plus temozolomide (TMZ), followed by 6 months of adjuvant TMZ. Despite this aggressive treatment regimen, the overall survival of all surgically treated GBM patients remains dismal, and additional or different therapies are required. Depending on the cancer type, SPARC has been proposed both as a therapeutic target and as a therapeutic agent. In glioma, SPARC promotes invasion via upregulation of the p38 MAPK/MAPKAPK2/HSP27 signaling pathway, and promotes tumor cell survival by upregulating pAKT. As HSP27 and AKT interact to regulate the activity of each other, we determined whether inhibition of HSP27 was better than targeting SPARC as a therapeutic approach to inhibit both SPARC-induced glioma cell invasion and survival.

Results

Our studies found the following. 1) SPARC increases the expression of tumor cell pro-survival and pro-death protein signaling in balance, and, as a net result, tumor cell survival remains unchanged. 2) Suppressing SPARC increases tumor cell survival, indicating it is not a good therapeutic target. 3) Suppressing HSP27 decreases tumor cell survival in all gliomas, but is more effective in SPARC-expressing tumor cells due to the removal of HSP27 inhibition of SPARC-induced pro-apoptotic signaling. 4) Suppressing total AKT1/2 paradoxically enhanced tumor cell survival, indicating that AKT1 or 2 are poor therapeutic targets. 5) However, inhibiting pAKT suppresses tumor cell survival. 6) Inhibiting both HSP27 and pAKT synergistically decreases tumor cell survival. 7) There appears to be a complex feedback system between SPARC, HSP27, and AKT. 8) This interaction is likely influenced by PTEN status. With respect to chemosensitization, we found the following. 1) SPARC enhances pro-apoptotic signaling in cells exposed to TMZ. 2) Despite this enhanced signaling, SPARC protects cells against TMZ. 3) This protection can be reduced by inhibiting pAKT. 4) Combined inhibition of HSP27 and pAKT is more effective than TMZ treatment alone.

Conclusions

We conclude that inhibition of HSP27 alone, or in combination with pAKT inhibitor IV, may be an effective therapeutic approach to inhibit SPARC-induced glioma cell invasion and survival in SPARC-positive/PTEN-wildtype and SPARC-positive/PTEN-null tumors, respectively.     

CMTM3 is frequently reduced in clear cell renal cell carcinoma and exhibits tumor suppressor activities

Purpose

CKLF-like MARVEL transmembrane domain containing member 3 (CMTM3) is silenced in many kinds of cancers and inhibits tumor cells growth. We investigated the expression and role of CMTM3 in clear cell renal cell carcinoma (ccRCC).

Methods

The expression of CMTM3 was detected in ccRCC tissue microarray, specimens, and cell lines by immunohistochemistry, quantitative real-time polymerase chain reaction (qRT-PCR) and western blot, respectively. After transfected with CMTM3 plasmid or vector, the proliferation and migration of ccRCC 786-0 cells were determined by MTT assay and transwell assay, respectively. Furthermore, the anchorage-independent growth of transfected cells was assessed using soft agar colony formation assay.

Results

CMTM3 was down-regulated in 84 % (63/75) of ccRCC tissues and its expression had no correlation with the gender, age, clinical staging and histologic grade. CMTM3 protein was undetectable by western blot in most detected ccRCC specimens and two RCC cell lines (786-0 and ACHN). qRT-PCR analysis showed that CMTM3 mRNA was dramatically down-regulated in 40 ccRCC cancer tissues as compared with the paired adjacent normal ones. Restoration of CMTM3 significantly suppressed the anchorage-independent growth, proliferation and migration of 786-0 cells.

Conclusion

These results indicate that CMTM3 is significantly down-regulated in ccRCC and exerts remarkable tumor-suppressive functions in 786-0 cells. Reduction of CMTM3 expression may contribute to the pathogenesis of ccRCC and CMTM3 may be a potentially target for therapeutic strategy.     

CD44 enhances invasion of basal-like breast cancer cells by upregulating serine protease and collagen-degrading enzymatic expression and activity

Introduction

Basal-like breast cancers (BL-BCa) have the worst prognosis of all subgroups of this disease. Hyaluronan (HA) and the HA receptor CD44 have a long-standing association with cell invasion and metastasis of breast cancer. The purpose of this study was to establish the relation of CD44 to BL-BCa and to characterize how HA/CD44 signaling promotes a protease-dependent invasion of breast cancer (BrCa) cells.

Methods

CD44 expression was determined with immunohistochemistry (IHC) analysis of a breast cancer tissue microarray (TMA). In vitro experiments were performed on a panel of invasive BL-BCa cell lines, by using quantitative polymerase chain reaction (PCR), immunoblotting, protease activity assays, and invasion assays to characterize the basis of HA-induced, CD44-mediated invasion.

Results

Expression of the hyaluronan (HA) receptor CD44 associated with the basal-like subgroup in a cohort of 141 breast tumor specimens (P = 0.018). Highly invasive cells of the representative BL-BCa cell line, MDA-MB-231 (MDA-MB-231Hi) exhibited increased invasion through a basement membrane matrix (Matrigel) and collagen. In further experiments, HA-induced promotion of CD44 signaling potentiated expression of urokinase plasminogen activator (uPA) and its receptor uPAR, and underpinned an increased cell-associated activity of this serine protease in MDA-MB-231Hi and a further BL-BCa cell line, Hs578T cells. Knockdown of CD44 attenuated both basal and HA-stimulated uPA and uPAR gene expression and uPA activity. Inhibition of uPA activity by using (a) a gene-targeted RNAi or (b) a small-molecule inhibitor of uPA attenuated HA-induced invasion of MDA-MB-231Hi cells through Matrigel. HA/CD44 signaling also was shown to increase invasion of MDA-MB-231 cells through collagen and to potentiate the collagen-degrading activity of MDA-MB-231Hi cells. CD44 signaling was subsequently shown to upregulate expression of two potent collagen-degrading enzymes, the cysteine protease cathepsin K and the matrix metalloprotease MT1-MMP. RNAi- or shRNA-mediated depletion of CD44 in MDA-MB-231Hi cells decreased basal and HA-induced cathepsin K and MT1-MMP expression, reduced the collagen-degrading activity of the cell, and attenuated cell invasion through collagen. Pharmacologic inhibition of cathepsin K or RNAi-mediated depletion of MT1-MMP also attenuated MDA-MB-231Hi cell invasion through collagen.

Conclusion

HA-induced CD44 signaling increases a diverse spectrum of protease activity to facilitate the invasion associated with BL-BCa cells, providing new insights into the molecular basis of CD44-promoted invasion.     

MicroRNA-301a promotes migration and invasion by targeting TGFBR2 in human colorectal cancer

Background

MicroRNAs (miRNAs) have been reported to play crucial roles in regulating a variety of genes pivotal for tumor metastasis. MicroRNA-301a (miR-301a) is overexpressed and displays oncogenic activity in many cancers. However, little is known about the potential roles of miR-301a in colorectal cancer (CRC).

Methods

Taqman probe stem-loop real-time PCR was used to quantitatively measure the expression level of miR-301a in 48 cases of CRC tissues and the matched adjacent non-tumor mucosa as well as in CRC cell lines. miR-301a mimics and inhibitors were used to up-regulate and down-regulate miR-301a in CRC cells, respectively; lentivirus was used to construct miR-301a stably up- and down-regulated CRC cell lines. Metastasis ability was evaluated by transwell and wound healing assays while invasion was measured by transwell coated with matrix gel in vitro; in vivo metastasis was performed on nude mice model. The target of miR-301a was predicted by TargetScan software and validated by qRT-PCR, immunohistochemistry, western blot and luciferase reporter gene assay.

Results

The expression of miR-301a was significantly higher in lymph node metastasis positive CRC samples compared with negative ones. Downregulation of miR-301a significantly inhibited the migration and invasion both in vitro and in vivo while forced up-regulation of miR-301a promoted migration and invasion. TGFBR2 was identified to be the downstream target of miR-301a. Knockdown of TGFBR2 in cells treated by miR-301a inhibitor elevated the previously abrogated migration and invasion.

Conclusions

Our data indicated that miR-301a correlated with the metastatic and invasive ability in human colorectal cancers and miR-301a exerted its role as oncogene by targeting TGFBR2.

     

Silencing of GM3 synthase suppresses lung metastasis of murine breast cancer cells

Background

Gangliosides are sialic acid containing glycosphingolipids that are ubiquitously distributed on vertebrate plasma membranes. GM3, a precursor for most of the more complex ganglioside species, is synthesized by GM3 synthase. Although total ganglioside levels are significantly higher in breast tumor tissue than in normal mammary tissue, the roles played by gangliosides in breast cancer formation and metastasis are not clear.

Methods

To investigate the roles of gangliosides in breast tumor development, GM3 synthase was silenced in the highly metastatic 4T1 cells and over-expressed in the non-metastatic 67NR cells. The behavior of breast cancer cells was examined in vitro using migration assay, invasion assay, and soft agar assay. Tumor formation and metastasis in vivo were examined using a well established mouse mammary tumor model.

Results

GM3 synthase silencing in 4T1 cells significantly inhibited cell migration, invasion and anchorage-independent growth in vitro, and lung metastasis in vivo. In addition, over-expression of GM3 synthase in nonmetastatic 67NR cells significantly induced cell migration and anchorage-independent growth. Further studies indicated that activation of the phosphoinositide-3 kinase/Akt pathway, and consequently inhibition of nuclear factor of activated T cell (NFAT)1 expression, could be the mechanism underlying the suppression of breast cancer migration/invasion induced by GM3 synthase silencing.

Conclusion

Our findings indicate that GM3 synthase silencing suppressed lung metastasis in murine breast cancer cells. The molecular mechanism that underlies GM3 synthase mediated migration and invasion was inhibition of the phosphoinositide-3 kinase/Akt pathway. The findings suggest that GM3 synthase may be of value as a therapeutic target in breast cancer.     

Role of LOXs and COX-2 on FAK activation and cell migration induced by linoleic acid in MDA-MB-231 breast cancer cells

Background

Epidemiological studies and animal models suggest a link between high levels of dietary fat intake and an increased risk of developing breast cancer. Particularly, free fatty acids (FFAs) are involved in several processes, including proliferation, migration and invasion, in breast cancer cells. Linoleic acid (LA) is a dietary n-6 polyunsaturated fatty acid that is known to induce proliferation and invasion in breast cancer cells. So far, however, the contribution of LA to focal adhesion kinase (FAK) activation and cell migration in breast cancer cells has not been studied.

Results

Here, we show that LA promotes FAK and Src activation, as well as cell migration, in MDA-MB-231 breast cancer cells. FAK activation and cell migration require Src, Gi/Go, COX-2 and LOXs activities, whereas both are independent of Δ6 desaturase activity. In addition, we show that cell migration requires FAK activity, whereas FAK activation requires Src activity, thus suggesting a reciprocal catalytic activation mechanism of FAK and Src.

Conclusions

In summary, our findings show that LA induces FAK activation and cell migration in MDA-MB-231 breast cancer cells.     

Integrin α3 is overexpressed in glioma stem-like cells and promotes invasion

Background:

Glioma stem-like cell (GSC) properties are responsible for gliomagenesis and recurrence. GSCs are invasive but its mechanism remains to be elucidated. Here, we attempted to identify the molecules that promote invasion in GSCs.

Methods:

Neurospheres and CD133⁺ cells were collected from glioblastoma (GBM) specimens and glioma cell lines by sphere-formation method and magnetic affinity cell sorting, respectively. Differential expression of gene candidates, its role in invasion and its signaling pathway were evaluated in glioma cell lines.

Results:

Neurospheres from surgical specimens attached to fibronectin and laminin, the receptors of which belong to the integrin family. Integrin α3 was overexpressed in CD133⁺ cells compared with CD133⁻ cells in all the glioma cell lines (4 out of 4). Immunohistochemistry demonstrated the localisation of integrin α3 in GBM cells, including invading cells, and in the tumour cells around the vessels, which is believed to be a stem cell niche. The expression of integrin α3 was correlated with migration and invasion. The invasion activity of glioma cells was linked to the phosphorylation of extracellular signal–regulated kinase (ERK) 1/2.

Conclusion:

Our results suggest that integrin α3 contributes to the invasive nature of GSCs via ERK1/2, which renders integrin α3 a prime candidate for anti-invasion therapy for GBM.     

Reversibility of regorafenib effects in hepatocellular carcinoma cells

Purpose

Multikinase growth inhibitors inhibit their target kinases with varying potency. Patients often require lower doses or therapy breaks due to drug toxicities. To evaluate the effects of drug withdrawal on hepatocellular carcinoma cells after incubation with growth-inhibitory concentrations of regorafenib, cell growth, migration and invasion, and signaling were examined.

Methods

Cell proliferation, motility, and invasion were analyzed by MTT, wound healing, and invasion assays, respectively, and MAPK pathway protein markers were analyzed by Western blot.

Results

After regorafenib removal, cell growth, migration, and invasion recovered. Repeated drug exposure resulted in changes in cell growth patterns. Recovery could be blocked by sub-growth-inhibitory concentrations of either doxorubicin or vitamin K1. Recovery of growth was associated with increased phospho-JNK, phospho-p38, and phospho-STAT3 levels. The recovery of growth, migration, and signaling were blocked by a JNK inhibitor.

Conclusions

Removal of regorafenib from growth-inhibited cells resulted in a JNK-dependent recovery of growth and migration.     

Inhibition of phosphatidylcholine-specific phospholipase C results in loss of mesenchymal traits in metastatic breast cancer cells

Introduction

Acquisition of mesenchymal characteristics confers to breast cancer (BC) cells the capability of invading tissues different from primary tumor site, allowing cell migration and metastasis. Regulators of the mesenchymal-epithelial transition (MET) may represent targets for anticancer agents. Accruing evidence supports functional implications of choline phospholipid metabolism in oncogene-activated cell signaling and differentiation. We investigated the effects of D609, a xanthate inhibiting phosphatidylcholine-specific phospholipase C (PC-PLC) and sphingomyelin synthase (SMS), as a candidate regulator of cell differentiation and MET in the highly metastatic BC cell line MDA-MB-231.

Methods

PC-PLC expression and activity were investigated using confocal laser scanning microscopy (CLSM), immunoblotting and enzymatic assay on human MDA-MB-231 compared with MCF-7 and SKBr3 BC cells and a nontumoral immortalized counterpart (MCF-10A). The effects of D609 on PC-PLC and SMS activity, loss of mesenchymal markers and changes in migration and invasion potential were monitored in MDA-MB-231 cells by enzymatic assays, CLSM, immunoblotting and transwell chamber invasion combined with scanning electron microscopy examinations. Cell proliferation, formation and composition of lipid bodies and cell morphology were investigated in D609-treated BC cells by cell count, CLSM, flow-cytometry of BODIPY-stained cells, nuclear magnetic resonance and thin-layer chromatography.

Results

PC-PLC (but not phospholipase D) showed 2- to 6-fold activation in BC compared with nontumoral cells, the highest activity (up to 0.4 pmol/??g protein/min) being detected in the poorly-differentiated MDA-MB-231 cells. Exposure of the latter cells to D609 (50 ??g/mL, 24-72 h) resulted into 60-80% PC-PLC inhibition, while SMS was transiently inhibited by a maximum of 21%. These features were associated with progressive decreases of mesenchymal traits such as vimentin and N-cadherin expression, reduced galectin-3 and milk fat globule EGF-factor 8 levels, ??-casein formation and decreased in vitro cell migration and invasion. Moreover, proliferation arrest, changes in cell morphology and formation of cytosolic lipid bodies typical of cell differentiation were induced by D609 in all investigated BC cells.

Conclusions

These results support a critical involvement of PC-PLC in controlling molecular pathways responsible for maintaining a mesenchymal-like phenotype in metastatic BC cells and suggests PC-PLC deactivation as a means to promote BC cell differentiation and possibly enhance the effectiveness of antitumor treatments.     

Downregulation of CD147 expression alters cytoskeleton architecture and inhibits gelatinase production and SAPK pathway in human hepatocellular carcinoma cells

Background

CD147 plays a critical role in the invasive and metastatic activity of hepatocellular carcinoma (HCC) cells by stimulating the surrounding fibroblasts to express matrix metalloproteinases (MMPs). Tumor cells adhesion to extracellular matrix (ECM) proteins is the first step to the tumor metastasis. MMPs degrade the ECM to promote tumor metastasis. The aim of this study is to investigate the effects of small interfering RNA (siRNA) against CD147 (si-CD147) on hepatocellular carcinoma cells' (SMMC-7721) architecture and functions.

Methods

Flow cytometry and western blot assays were employed to detect the transfection efficiency of si-CD147. Confocal microscopy was used to determine the effects of si-CD147 on SMMC-7721 cells' cytoskeleton. Invasion assay, gelatin zymography and cell adhesion assay were employed to investigate the effects of si-CD147 on SMMC-7721 cells' invasion, gelatinase production and cell adhesive abilities. Western blot assay was utilized to detect the effects of si-CD147 on focal adhesion kinase (FAK), vinculiln and mitogen-activated protein kinase (MAPK) expression in SMMC-7721 cells.

Results

Downregulation of CD147 gene induced the alteration of SMMC-7721 cell cytoskeleton including actin, microtubule and vimentin filaments, and inhibited gelatinase production and expression, cells invasion, FAK and vinculin expression. si-CD147 also blocked SMMC-7721 cells adhesion to collagen IV and phosphorylation level of SAPK/JNKs. SAPK/JNKs inhibitor SP600125 inhibited gelatinase production and expression.

Conclusion

CD147 is required for normal tumor cell architecture and cell invasion. Downregulation of CD147 affects HCC cell structure and function. Moreover, the alteration of cell behavior may be related to SAPK/JNK Pathway. siRNA against CD147 may be a possible new approach for HCC gene therapy.