Induction of integrin ß3 by sustained ERK activity promotes the invasiveness of TGFß-induced mesenchymal tumor cells.

The emerging roles of integrin ß3 in the epithelial-mesenchymal transition (EMT) and drug resistance underline its significance in cancer metastasis and recurrence. However, the molecular mechanism underlying the distinctive expression of integrin ß3 is less understood. In the present report, we demonstrated that repetitive exposure to transforming growth factor ß (TGFß), a potent inducer of the EMT, significantly increased the expression of integrin ß3 in A549 lung cancer cells with distinct mesenchymal properties, such as actin filament reorganization and invasiveness. Notably, integrin ß3 expression was associated to cancer cell invasion and migration, and was determined not by Smad4-dependent pathways but by sustained ERK1/2 activity in the mesenchymal cancer cells. These data suggest that ERK1/2 plays an important role in mediating non-canonical TGFß signal pathways for integrin ß3 expression. Therefore, the targeting of the MEK/ERK activity seems to be a promising therapeutic approach to suppressing EMT-associated cancer progression that potentially occurs in TGFß-enriched microenvironments, which would lead to the suppression of the metastatic potential of integrin ß3-positive cancer cells.

Chronic TGFß stimulation promotes the metastatic potential of lung cancer cells by Snail protein stabilization through integrin ß3-Akt-GSK3ß signaling.

Chronic exposure to TGFß, a frequent occurrence for tumor cells in the tumor microenvironment, confers more aggressive phenotypes on cancer cells by promoting their invasion and migration while at the same time increasing their resistance to the growth-inhibitory effect of TGFß. In this study, a transdifferentiated (TD) A549 cell model, established by chronically exposing A549 cells to TGFß, showed highly invasive phenotypes in conjunction with attenuation of Smad-dependent signaling. We show that Snail protein, the mRNA expression of which strongly correlates with a poor prognosis in lung cancer patients, was highly stable in TD cells after TGFß stimulation. The increased protein stability of Snail in TD cells correlated with elevated inhibitory phosphorylation of GSK3ß, resulting from the high Akt activity. Notably, integrin ß3, whose expression was markedly increased upon sustained exposure to TGFß, was responsible for the high Akt activity as well as the increased Snail protein stability in TD cells. Consistently, clinical database analysis on lung cancer patients revealed a negative correlation between overall survival and integrin ß3 mRNA levels. Therefore, we suggest that the integrin ß3-Akt-GSK3ß signaling axis plays an important role in non-canonical TGFß signaling, determining the invasive properties of tumor cells chronically exposed to TGFß.

GalNAc-T14 promotes metastasis through Wnt dependent HOXB9 expression in lung adenocarcinoma.

While metastasis, the main cause of lung cancer-related death, has been extensively studied, the underlying molecular mechanism remains unclear. A previous clinicogenomic study revealed that expression of N-acetylgalactosaminyltransferase (GalNAc-T14), is highly inversely correlated with recurrence-free survival in those with non-small cell lung cancer (NSCLC). However, the underlying molecular mechanism(s) has not been determined. Here, we showed that GalNAc-T14 expression was positively associated with the invasive phenotype. Microarray and biochemical analyses revealed that HOXB9, the expression of which was increased in a GalNAc-T14-dependent manner, played an important role in metastasis. GalNAc-T14 increased the sensitivity of the WNT response and increased the stability of the ß-catenin protein, leading to induced expression of HOXB9 and acquisition of an invasive phenotype. Pharmacological inhibition of ß-catenin in GalNAc-T14-expressing cancer cells suppressed HOXB9 expression and invasion. A meta-analysis of clinical genomics data revealed that expression of GalNAc-T14 or HOXB9 was strongly correlated with reduced recurrence-free survival and increased hazard risk, suggesting that targeting ß-catenin within the GalNAc-T14/WNT/HOXB9 axis may be a novel therapeutic approach to inhibit metastasis in NSCLC.

Loss of E-cadherin activates EGFR-MEK/ERK signaling, which promotes invasion via the ZEB1/MMP2 axis in non-small cell lung cancer.

Loss of E-cadherin, a hallmark of epithelial-mesenchymal transition (EMT), can significantly affect metastatic dissemination. However, the molecular mechanism of EMT-associated metastatic dissemination by loss of E-cadherin still remains unclear in non-small cell lung cancers (NSCLCs). In the present study, we show that the knockdown of E-cadherin was sufficient to convert A549 NSCLC cells into mesenchymal type with the concurrent up-regulation of typical EMT inducers such as ZEB1 and TWIST1. Interestingly, the EMT-induced cells by E-cadherin depletion facilitate invasion in a matrix metalloproteinase-2 (MMP2)-dependent manner with aberrant activation of EGFR signaling. We demonstrated that the elevated invasiveness was a result of the activated EGFR-MEK/ERK signaling, which in turn leads to ZEB1 dependent MMP2 induction. These results suggest that the EGFR-MEK/ERK/ZEB1/MMP2 axis is responsible for promoted invasion in EMT-induced NSCLCs. Consistently, ERK activation and loss of E-cadherin were both observed in the disseminating cancer cells at the invasive tumor fronts in NSCLS cancer tissues. Thereby, these data suggest that the EGFR-MEK/ERK signaling would be a promising molecular target to control aberrant MMP2 expression and consequent invasion in the EMT-induced NSCSLs.

Human lung cancer-associated fibroblasts enhance motility of non-small cell lung cancer cells in co-culture.

The metastatic potential of non-small cell lung cancer (NSCLC) cells has been shown to be associated with the tumor microenvironment. Cancer-associated fibroblasts (CAFs) are a major component of the tumor microenvironment, regulating tumor cell function by secreting growth factors, chemokines, and extracellular matrix (ECM). In this study, we examined the role of CAFs in the tumor progression of NSCLC. Firstly, we established primary cultures of CAFs and matched normal fibroblasts (NFs) from patients with resected NSCLC. CAFs exhibited greater expression of the pan-mesenchymal marker α-smooth muscle actin (α-SMA) than did NFs, although they displayed similar morphology. Furthermore, we employed a direct co-culture assay with human NSCLC A549 and H358 cells, and found that CAFs were more potent in inducing the epithelial-to-mesenchymal transition (EMT) phenotype than NFs, as indicated by an elongated and disseminated appearance. CAF-induced EMT led to an increase in motility and a decrease in proliferation of NSCLC cells through SMAD family number-3 (SMAD3)-dependent up-regulation of the growth inhibitory gene p21(CIP1) [cyclin-dependent kinase inhibitor-1A (CDKN1A)] and α-SMA. Taken together, these findings provide evidence that lung CAFs have tumor-promoting capacity distinct from NFs and might play a significant role in the metastatic potential of NSCLC.

Extract of Aneilema keisak inhibits transforming growth factor-ß-dependent signalling by inducing Smad2 downregulation in keloid fibroblasts.

Keloids are characterised by the excessive accumulation of extracellular matrix (ECM), especially overabundant collagen formation. In keloid fibroblasts (KFs), transforming growth factor (TGF)-ß-dependent signalling is closely associated with a variety of keloid pathologic responses such as ECM production and fibroblast overgrowth. Thus, inhibition of TGF-ß signalling would be a potential therapeutic approach to prevent keloid scar formation. Thereby, we aimed to identify a novel TGF-ß signalling blocker among natural products using a simplified screening approach. We discovered that the extract of Aneilema keisak (A.K-Ex) lowered TGF-ß-dependent signalling by reducing Smad2 protein levels. Given that KFs showed altered dependency on TGF-ß for survival and proliferation, A.K-Ex-mediated reduction in Smad2 protein levels significantly inhibited the major characteristics of KFs such as cell growth, migration and collagen synthesis, suggesting that A.K-Ex exhibits possible therapeutic activity on keloids.

Oncogenic challenges in stem cells and the link to cancer initiation.

Adult stem cells, which are characterized by self-renewal and multi-potency, are classified as specialized cell types, responsible for the regeneration of damaged tissues. There is growing evidence that senescence of stem cells (or stem cell aging) is closely associated with a variety of aging-related diseases such as tissue atrophy, degenerative diseases and onset of cancers. Alterations in the systemic environment during aging may trigger stress signaling in stem cells and reduce stem cell characteristics, resulting in loss of differentiation potential and defective self-renewal (referred to as mal-differentiation). Thus, it has been suggested that aging-related disorders such as retarded regeneration of damaged tissue and onset of cancer may result from the mal-differentiation of stem cells. In particular, many types of cancers such as leukemia, intestinal cancer, skin cancer and sarcoma have been shown to originate from adult stem cells after a variety of oncogenic challenges. This review summarizes recent studies on cancers originating from stem cells, demonstrating possible molecular mechanisms that govern the susceptibility of stem cells to oncogenic challenges.

A new hepatocytic isoform of PLZF lacking the BTB domain interacts with ATP7B, the Wilson disease protein, and positively regulates ERK signal transduction.

The promyelocytic leukemia zinc finger (PLZF) protein has been described as a transcriptional repressor of the BTB-domain/zinc-finger family, and shown to regulate the expression of Hox genes during embryogenesis and the expression of cyclin A in the cell cycle progression. Here, a 45-kDa isoform of PLZF without a BTB domain was identified via yeast two-hybrid screening using the C-terminal region of ATP7B as bait in our determination of the biological roles of the Wilson disease protein outside of its copper-binding domain. Our immunoprecipitation experiments showed that the hepatocytic isoform of PLZF could specifically interact with the C-terminal region of ATP7B. The immunostaining of HepG2 cells revealed that the ATP7B and PLZF proteins were apparently colocalized into the trans-Golgi complexes. It was also determined that disruption of PLZF expression in the HepG2 cells affected an attenuation of ERK activity in a dose-dependent manner. The hepatocytic activities of ERK kinase were found to be enhanced as the result of PLZF or ATP7B expression, but this enhancement was abrogated by the deletion of the C-terminal region of ATP7B. Furthermore, a transgenic Drosophila strain that ectopically expressed the hepatocytic deltaBTB-PLZF exhibited phenotypic changes in eye and wing development, and these alterations were fully recovered as the result of ATP7B expression, indicating the obvious in vivo interaction between the two proteins. Those PLZF-induced abnormalities were attributed to the enhancement of ERK signaling, as was shown by phenotypic reversions with loss-of-function mutations in ERK signal transduction in Drosophila. These data suggest the existence of a mechanism that regulates ERK signaling via the C-terminus of ATP7B and the ATP7B-interacting hepatocytic PLZF.

Stimulation of Oct-4 activity by Ewing's sarcoma protein.

The Oct-4 gene encodes a transcription factor that is expressed in embryonic stem (ES) cells and germ cells. Oct-4 is known to function as a transcriptional activator of genes involved in maintaining an undifferentiated totipotent state and possibly in preventing expression of genes activated during differentiation. In addition, it is a putative proto-oncogene and a critical player in the genesis of human testicular germ cell tumors. Although much effort has gone toward characterizing Oct-4, there is still little known about the molecular mechanisms and the proteins that regulate Oct-4 function. To identify cofactors that control Oct-4 function in vivo, we used a recently developed bacterial two-hybrid screening system and isolated a novel ES cell-derived cDNA encoding Ewing's sarcoma protein (EWS). EWS is a proto-oncogene and putative RNA-binding protein involved in human cancers. By using glutathione-S-transferase (GST) pull-down assays, we were able to confirm the interaction between Oct-4 and EWS in vitro, and moreover, coimmunoprecipitation and colocalization studies have shown that these proteins also associate in vivo. We have mapped the EWS-interacting region to the POU domain of Oct-4. In addition, three independent sites on EWS are involved in binding to Oct-4. In this study, we report that Oct-4 and EWS are coexpressed in the pluripotent mouse and human ES cells. Consistent with its ability to bind to and colocalize with Oct-4, ectopic expression of EWS enhances the transactivation ability of Oct-4. Moreover, a chimeric protein generated by fusion of EWS (1-295) to the GAL4 DNA-binding domain significantly increases promoter activity of a reporter containing GAL4 DNA-binding sites, suggesting the presence of a strong activation domain within EWS. Taken together, our results suggest that Oct-4-mediated transactivation is stimulated by EWS.