Role of tissue transglutaminase 2 in the acquisition of a mesenchymal-like phenotype in highly invasive A431 tumor cells.

BACKGROUND: Cancer progression is closely linked to the epithelial-mesenchymal transition (EMT) process. Studies have shown that there is increased expression of tissue tranglutaminase (TG2) in advanced invasive cancer cells. TG2 catalyzes the covalent cross-linking of proteins, exhibits G protein activity, and has been implicated in the modulation of cell adhesion, migration, invasion and cancer metastasis. This study explores the molecular mechanisms associated with TG2's involvement in the acquisition of the mesenchymal phenotype using the highly invasive A431-III subline and its parental A431-P cells. RESULTS: The A431-III tumor subline displays increased expression of TG2. This is accompanied by enhanced expression of the mesenchymal phenotype, and this expression is reversed by knockdown of endogenous TG2. Consistent with this, overexpression of TG2 in A431-P cells advanced the EMT process. Furthermore, TG2 induced the PI3K/Akt activation and GSK3ß inactivation in A431 tumor cells and this increased Snail and MMP-9 expression resulting in higher cell motility. TG2 also upregulated NF-κB activity, which also enhanced Snail and MMP-9 expression resulting in greater cell motility; interestingly, this was associated with the formation of a TG2/NF-κB complex. TG2 facilitated acquisition of a mesenchymal phenotype, which was reversed by inhibitors of PI3K, GSK3 and NF-κB. CONCLUSIONS: This study reveals that TG2 acts, at least in part, through activation of the PI3K/Akt and NF-κB signaling systems, which then induce the key mediators Snail and MMP-9 that facilitate the attainment of a mesenchymal phenotype. These findings support the possibility that TG2 is a promising target for cancer therapy.

Matrix metalloproteinase-9 cooperates with transcription factor Snail to induce epithelial-mesenchymal transition.

One of the most fundamental biological processes in tumor metastasis is the process of epithelial-mesenchymal transition (EMT). During EMT, zinc-finger-family of transcription factors such as Snail, Slug and Twist, and matrix metalloproteinases (MMPs) are upregulated, and this correlates with increased tumor cell invasion and motility. We previously obtained a highly invasive A431-III tumor subline, which is a rich source of MMP-9 and observed a plausible link between MMP levels and the promotion of EMT. To gain further understanding of EMT, we investigated the contribution of distinct MMPs to the induction of EMT. Exposing A431, cervical carcinoma parental cells, to MMP-9 stimulated a phenotypic alteration and cells became spindle-like as shown for A431-III cells. In the present communication, we document changes in gene expression profiles of A431-P and A431-III cells, including those of genes involved in cell adhesion, cytoskeleton reorganization, polarity, migration and transcription. Treatment of both A431-P and A431-III cells with GM6001, a broad spectrum MMP inhibitor, resulted in the diminution of vimentin and fibronectin, indicating a role for MMP-9 in the induction of EMT. Abrogation of MMP-9-mediated cell-cell contact in both A431-P and A431-III cells using MMP-9 siRNA resulted in decreased cell invasion, motility and altered cytoskeleton arrangement together with a reduction in Snail expression. Knockdown of Snail resulted in similar changes along with diminished MMP-9 expression. These data suggest a higher capacity of MMP-9 than that of Snail in eliciting the development of EMT in A431 cells. Based on these findings, we speculate that the overexpression of MMP-9 in A431-III cells might directly induce (or stimulate) EMT and that the transcriptional factor, Snail, could cooperatively engage in this phenomenon.

Effects of dietary flavonoids, luteolin, and quercetin on the reversal of epithelial-mesenchymal transition in A431 epidermal cancer cells.

Highly invasive A431-III cells, which are derived from parental A431-P cells, were originally isolated by three successive passages through a Boyden chamber using a Matrigel-coated membrane support. The greater invasion potential shown by A431-III cells was due to their increased ability to spread/migrate, which was associated with enhanced MMP activity. The tumor progression events evoked by A431-P cells compared to A431-III cells may help identify useful strategies for evaluating the epithelial-mesenchymal transition (EMT) and these cell lines could be a reliable model for evaluating tumor metastasis events. Using this approach, we evaluated the effects of luteolin and quercetin using the A431-P/A431-III EMT model. These flavonoids reversed cadherin switching, downregulated EMT markers, and nullified the invasion ability of A431-III cells. Overexpression of MMP-9 resulted in induction of the EMT in A431-P cells and this could be reversed by treating with luteolin or quercetin. Cotreatment of A431-P and A431-III cells with epidermal growth factor (EGF) plus luteolin or quercetin resulted in a more epithelial-like morphology, led to reduced levels of EGF-induced markers of EMT, and caused the restoration of cell-cell junctions. E-cadherin was decreased by EGF, but increased by luteolin and quercetin. Our results suggest that luteolin and quercetin are potentially beneficial agents that target and prevent the occurrence of EMT in epidermal carcinoma cells. These chemicals also have the ability to attenuate tumor progression in A431-III cells. Luteolin and quercetin show inherent potential as chemopreventive/antineoplastic agents and do this by abating tumor progression through a reversal of EMT.

Flavonoids Luteolin and Quercetin Inhibit RPS19 and contributes to metastasis of cancer cells through c-Myc reduction.

Flavonoids luteolin and quercetin can inhibit growth and metastasis of cancer cells. In our previous report, luteolin and quercetin was shown to block Akt/mTOR/c-Myc signaling. Here, we found luteolin and quercetin reduced protein level and transactivation activity of RPS19 in A431-III cells, which is isolated from parental A431 (A431-P) cell line. Further investigation the inhibitory mechanism of luteolin and quercetin on RPS19, we found c-Myc binding sites on RPS19 promoter. The Akt inhibitor LY294002, mTOR inhibitor rapamycin and c-Myc inhibitor 10058-F4 significantly suppressed RPS19 expression and transactivation activities. Overexpression and knockdown of c-Myc in cancer cells show RPS19 expression was regulated by c-Myc. Furthermore, Knockdown and overexpression of RPS19 was used to analyze of the function of RPS19 in cancer cells. The epithelial-mesenchymal transition (EMT) markers and metastasis abilities of cancer cells were also regulated by RPS19. These data suggest that luteolin and quercetin might inhibit metastasis of cancer cells by blocking Akt/mTOR/c-Myc signaling pathway to suppress RPS19-activated EMT signaling.

Dietary flavonoids, luteolin and quercetin, inhibit invasion of cervical cancer by reduction of UBE2S through epithelial-mesenchymal transition signaling.

We previously reported that the dietary flavonoids, luteolin and quercetin, might inhibit the invasiveness of cervical cancer by reversing epithelial-mesenchymal transition (EMT) signaling. However, the regulatory mechanism exerted by luteolin and quercetin is still unclear. This study analyzed the invasiveness activation by ubiquitin E2S ligase (UBE2S) through EMT signaling and inhibition by luteolin and quercetin. We found that UBE2S expression was significantly higher in highly invasive A431 subgroup III (A431-III) than A431-parental (A431-P) cells. UBE2S small interfering (si)RNA knockdown and overexpression experiments showed that UBE2S increased the migratory and invasive abilities of cancer cells through EMT signaling. Luteolin and quercetin significantly inhibited UBE2S expression. UBE2S showed a negative correlation with von Hippel-Lindau (VHL) and a positive correlation with hypoxia-induced factor (Hif)-1α. Our findings suggest that high UBE2S in malignant cancers contributes to cell motility through EMT signaling and is reversed by luteolin and quercetin. UBE2S might contribute to Hif-1α signaling in cervical cancer. These results show the metastatic inhibition of cervical cancer by luteolin and quercetin through reducing UBE2S expression, and provide a functional role for UBE2S in the motility of cervical cancer. UBE2S could be a potential therapeutic target in cervical cancer.

Dietary Flavonoids Luteolin and Quercetin Suppressed Cancer Stem Cell Properties and Metastatic Potential of Isolated Prostate Cancer Cells.

A highly invasive Du145-III subline was isolated by three successive passages of the parental Du145 prostate tumor cell line (Du145-P) through a Boyden chamber with matrigel-coated membrane support. Du145-III cells showed great invasion potential based on their increased ability to spread/migrate and enhanced expression/secretion of the matrix metalloproteinase 9 (MMP9). Du145-III cells exerted vasculogenic mimicry (VM) properties, reminiscent of endothelial cell characteristics and expressed elevated levels of cancer stem cell (CSC) markers, including Nanog, Sox2, CD44 and ABCG2 and ability to self-renew. Of prominence, MMP9 was required for the induction of VM and for increased stemness in Du145-III cells. Using Du145-III as a model, the effects of dietary flavonoids, luteolin and quercetin, were evaluated on stemness and invasion capacity of Du145-III cells in relation to JNK signaling pathway activation. These flavonoids depressed the malignancy of highly invasive Du145-III cells, VM, anchorage-independent spheroid formation and expression of certain CSC markers. Since luteolin and quercetin were able to target CSC cells and prevent cancer cell invasiveness, may serve as potential anti-angiogenesis and anti-metastasis agents.

Impact of flavonoids on matrix metalloproteinase secretion and invadopodia formation in highly invasive A431-III cancer cells.

Metastasis is a major cause of mortality in cancer patients. Invadopodia are considered to be crucial structures that allow cancer cells to penetrate across the extracellular matrix (ECM) by using matrix metalloproteinases (MMPs). Previously, we isolated a highly invasive A431-III subline from parental A431 cells by Boyden chamber assay. The A431-III cells possess higher invasive and migratory abilities, elevated levels of MMP-9 and an enhanced epithelial-mesenchymal transition (EMT) phenotype. In this study, we discovered that A431-III cells had an increased potential to form invadopodia and an improved capacity to degrade ECM compared with the original A431 cells. We also observed enhanced phosphorylation levels of cortactin and Src in A431-III cells; these phosphorylated proteins have been reported to be the main regulators of invadopodia formation. Flavonoids, almost ubiquitously distributed in food plants and plant food products, have been documented to exhibit anti-tumor properties. Therefore, it was of much interest to explore the effects of flavonoid antioxidants on the metastatic activity of A431-III cells. Exposure of A431-III cells to two potent dietary flavonoids, namely luteolin (Lu) and quercetin (Qu), caused inhibition of invadopodia formation and decrement in ECM degradation. We conclude that Lu and Qu attenuate the phosphorylation of cortactin and Src in A431-III cells. As a consequence, there ensues a disruption of invadopodia generation and the suppression of MMP secretion. These changes, in concert, bring about a reduction in metastasis.