Wnt/beta-catenin signaling inhibits death receptor-mediated apoptosis and promotes invasive growth of HNSCC

The Wnt/beta-catenin signaling pathway plays a critical role in cell proliferation and oncogenesis. It has been found to be chronically activated in a variety of human cancers, including head and neck squamous cell carcinoma (HNSCC). Previously, we have found that the activation of the Wnt/beta-catenin signaling pathway inhibits mitochondria-mediated apoptosis. In this study, we extended our studies to determine whether the Wnt/beta-catenin signaling pathway inhibited death receptor-mediated apoptosis in HNSCC cells. We found that Wnt/beta-catenin inhibited not only tumor necrosis factor (TNF)/c-Myc-mediated apoptosis, but also cell detachment-mediated apoptosis (anoikis) which is dependent on the death receptor signaling pathway. Interestingly, we also observed that the Wnt/beta-catenin signaling pathway induced HNSCC cell scattering and promoted cell invasion in the Matrigel, both of which are hallmarks for the invasive growth of HNSCC. Consistently, the over-expression of beta-catenin promoted HNSCC tumor growth in nude mice. Taken together, our results suggest that the Wnt/beta-catenin signaling pathway plays dual functions in HNSCC development: promoting both cell survival and invasive growth of HNSCC cells.     

ras Oncogene triggers up-regulation of cIAP2 and XIAP in intestinal epithelial cells: epidermal growth factor receptor-dependent and -independent mechanisms of ras-induced transformation

Detachment of normal epithelial cells from the extracellular matrix (ECM) triggers apoptosis, a phenomenon called anoikis. Conversely, carcinomas (cancers of epithelial origin) represent three-dimensional disorganized multicellular masses in which cells are deprived of adhesion to the ECM but remain viable. Resistance of cancer cells to anoikis is thought to be critical for tumor progression. However, the knowledge about molecular mechanisms of this type of resistance remains limited. Herein we report that ras oncogene, an established inhibitor of anoikis, triggers a significant upregulation of anti-apoptotic proteins cIAP2 and XIAP in intestinal epithelial cells. We also observed that the effect of ras on cIAP2 requires ras-induced autocrine production of transforming growth factor alpha (TGF-alpha), a ligand for epidermal growth factor receptor, whereas ras-triggered up-regulation of XIAP is TGF-alpha-independent. Moreover, overexpression of either cIAP2 or XIAP in nonmalignant intestinal epithelial cell was found to block anoikis. In addition, an established IAP antagonist Smac or Smac-derived cell-permeable peptide suppressed ras-induced anoikis resistance and subsequent anchorage-independent growth of ras-transformed cells. We conclude that ras-induced overexpression of cIAP2 and XIAP significantly contributes to the ability of ras-transformed intestinal epithelial cells to survive in the absence of adhesion to the ECM and grow in a three-dimensional manner.     

BMP4 enhances anoikis resistance and chemoresistance of breast cancer cells through canonical BMP signaling

Bone morphogenetic proteins (BMPs) regulate cell fate during development and mediate cancer progression. In this study, we investigated the role of BMP4 in proliferation, anoikis resistance, metastatic migration, and drug resistance of breast cancer cells. We utilized breast cancer cell lines and clinical samples representing different subtypes to understand the functional effect of BMP4 on breast cancer. The BMP pathway was inhibited with the small molecule inhibitor LDN193189 hydrochloride (LDN). BMP4 signaling enhanced the expression of stem cell genes CD44, ALDH1A3, anti-apoptotic gene BCL2 and promoted anoikis resistance in MDA-MB-231 breast cancer cells. BMP4 enhanced self-renewal and chemoresistance in MDA-MB-231 by upregulating Notch signaling while LDN treatment abrogated anoikis resistance and proliferation of anoikis resistant breast cancer cells in the osteogenic microenvironment. Conversely, BMP4 downregulated proliferation, colony-forming ability, and suppressed anoikis resistance in MCF7 and SkBR3 cells, while LDN treatment promoted tumor spheroid formation and growth. These findings indicate that BMP4 has a context-dependent role in breast cancer. Further, our data with MDA-MB-231 cells representing triple-negative breast cancer suggest that BMP inhibition might impair its metastatic spread and colonization.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12079-021-00649-9.     

Curcumin sensitizes non-small cell lung cancer cell anoikis through reactive oxygen species-mediated Bcl-2 downregulation

Anoikis, an apoptosis triggered by loss of cell anchorage, has been shown to be a principal mechanism of inhibition of tumor metastasis. Recently, anti-apoptotic Bcl-2 and Cav-1 proteins have been demonstrated to be highly associated with tumor metastasis and apoptosis resistance. Curcumin, a major active component of turmeric, Curcuma longa, has been shown to inhibit neoplastic evolution and tumor progression; however, the underlying mechanisms are unclear. In this study, we investigated the effect of curcumin on cell anoikis as a possible mechanism of anti-tumorigenic action of curcumin, and evaluated the potential role of Bcl-2 and Cav-1 in this process. Our results showed that ectopic expression of either Bcl-2 or Cav-1 induced anoikis resistance of lung carcinoma H460 cells. Curcumin downregulated Bcl-2 protein during anoikis and sensitized the cells to detachment-induced apoptosis, whereas it had no significant effect on Cav-1 protein expression. Bcl-2 down-regulation as well as anoikis enhancement by curcumin were inhibited by superoxide anion scavenger, Mn(III)tetrakis(4-benzoic acid) porphyrin chloride, but were unaffected by other ROS scavengers including catalase and deferoxamine, suggesting that superoxide anion is a key player in the downregulation of Bcl-2 by curcumin. Furthermore, we provided evidence that curcumin decreased Bcl-2 level through ubiquitin-proteasomal degradation which sensitized cells to detachment-induced apoptosis. These findings indicate a novel pathway for curcumin regulation of Bcl-2 and provide a key mechanism of anoikis regulation that may be exploited for metastatic cancer treatment.     

Potential involvement of the cyclooxygenase-2 pathway in hepatocellular carcinoma-associated angiogenesis

Angiogenesis plays a crucial role in tumor development and growth. The present study was carried out to investigate the potential involvement of the cyclooxygenase-2 (Cox-2) pathway in the regulation of angiogenesis in hepatocellular carcinoma (HCC). We inhibited Cox-2 expression in HCC cell line HuH-7 by selective Cox-2 inhibitor (SC-58635) or Cox-2 siRNA. Conditioned media (CMs) from HuH-7 cells were used in angiogenic assays in vitro and in vivo. Compared with CMs from untreated and negative siRNA treated HuH-7 cells, CMs from SC-58635 and Cox-2 siRNA treated HuH-7 dramatically suppressed the proliferation, migration, and differentiation of human umbilical vein endothelial cells (HUVECs) in vitro and neovascularization in vivo. These inhibitory effects could be partially reversed by the addition of exogenous PGE2 to CMs. Furthermore, Cox-2 inhibition by SC-58635 resulted in PGE2 reduction accompanied by the down-regulation of four PGE2 receptor (EP receptor) subtypes. Treatment with SC-58635 led to the down-expression of proangiogenic factors such as VEGF, HGF, FGF2, ANGPT1 and ANGPT2 in HCC. An approximately 78% reduction of VEGF level has been found in the CM from SC-58635 treated HuH-7. Our results suggest an involvement of Cox-2 in the control of HCC-associated angiogenesis. PGE2 as a vital angiogenic factor may act directly on endothelial cells to promote HuH-7-stimulated angiogenic process. Moreover, Cox-2/PGE2/EP/VEGF pathway possibly also contributes to tumor angiogenesis in HCC. This study provides the rationale for clinical studies of Cox-2 inhibitors on the treatment or chemoprevention of HCC.     

Osteopontin contributes to hepatocyte growth factor-induced tumor growth and metastasis formation

The cytokine hepatocyte growth factor (HGF)/scatter factor-1 and its cognate receptor, Met, are involved in the etiology and progression of many types of cancer. Despite recent advances in understanding the signal transduction pathways activated by HGF, the mechanism by which HGF exerts its tumorigenic effect is not well understood. To identify proteins that may be involved in mediating HGF-induced cell motility, invasiveness, and tumorigenesis, we used two separate differential display screening methods to identify changes in gene expression that are initiated by HGF in an epithelial cell culture system. Among several known and unknown genes whose expression was modified, osteopontin (OPN), a protein previously associated with tumorigenesis, was found to be upregulated within 6 h following HGF stimulation. OPN expression was dependent on activation of the PI-3 kinase pathway. Autocrine secretion of HGF resulted in sustained expression of OPN. Downregulation of opn expression by stable antisense transfection attenuated OPN expression and repressed HGF-induced invasiveness in vitro and decreased HGF-mediated tumor growth and metastasis formation in vivo. Constitutive expression of OPN in itself exerted partial invasiveness in vitro, but its expression itself was not sufficient to initiate tumor growth or metastasis formation in vivo. Thus, together with other molecules, OPN activity contributes to HGF-induced tumor growth and invasiveness.     

Cell detachment triggers p38 mitogen-activated protein kinase-dependent overexpression of Fas ligand. A novel mechanism of Anoikis of intestinal epithelial cells

Many cell types undergo apoptosis when they are detached from the extracellular matrix (ECM). This phenomenon has been termed anoikis. Most epithelial cells, which are normally attached to a type of ECM called basement membrane, are particularly sensitive to anoikis. Conversely, carcinoma cells tend to be resistant to anoikis, and this resistance plays a critical role in tumor invasion and metastasis. We reported previously that detachment-induced down-regulation of the anti-apoptotic molecule Bcl-X(L) makes a significant contribution to anoikis of intestinal epithelial cells. Here we demonstrate that exogenous Bcl-X(L), no matter how highly expressed in these cells, can significantly attenuate anoikis but cannot completely prevent it, suggesting that at least another pro-apoptotic event is activated by the loss of cell-ECM contacts. Indeed, in this study we identified a novel mechanism of anoikis in intestinal epithelial cells that involves detachment-induced overexpression of Fas ligand. We also demonstrated that this elevation in Fas ligand expression requires a detachment-induced increase of p38 mitogen-activated protein kinase activity. We conclude that the activation of at least two different pro-apoptotic events is required for anoikis of intestinal epithelial cells.     

Hepatocyte growth factor induces GATA-4 phosphorylation and cell survival in cardiac muscle cells

Hepatocyte growth factor (HGF) is released in response to myocardial infarction and may play a role in regulating cardiac remodeling. Recently, HGF was found to inhibit the apoptosis of cardiac muscle cells. Because GATA-4 can induce cell survival, the effects of HGF on GATA-4 activity were investigated. Treatment of HL-1 cells or primary adult rat cardiac myocytes with HGF, at concentrations that can be detected in the human serum after myocardial infarction, rapidly enhances GATA-4 DNA-binding activity. The enhanced DNA-binding activity is associated with the phosphorylation of GATA-4. HGF-induced phosphorylation and activation of GATA-4 is abolished by MEK inhibitors or the mutation of the ERK phosphorylation site (S105A), suggesting that HGF activates GATA-4 via MEK-ERK pathway-dependent phosphorylation. HGF enhances the expression of anti-apoptotic Bcl-x(L), and this is blocked by dominant negative mutants of MEK or GATA-4. Forced expression of wild-type GATA-4, but not the GATA-4 mutant (S105A) increases the expression of Bcl-x(L). Furthermore, expression of the GATA-4 mutant (S105A) suppresses HGF-mediated protection of cells against daunorubicin-induced apoptosis. These results demonstrate that HGF protects cardiac muscle cells against apoptosis via a signaling pathway involving MEK/ERK-dependent phosphorylation of GATA-4.     

Transforming growth factor-alpha prevents detachment-induced inhibition of c-Src kinase activity, Bcl-XL down-regulation, and apoptosis of intestinal epithelial cells

Detachment of epithelial cells from the extracellular matrix (ECM) results in apoptosis, a phenomenon often referred to as anoikis. Acquisition of anoikis resistance is now thought to be a prerequisite for the progression of carcinomas. Colorectal cancer cells frequently secrete epidermal growth factor receptor (EGFR) ligands, which are known to have anti-apoptotic activity. However, whether these ligands have the ability to inhibit anoikis of intestinal epithelial cells is unclear, since at least in some cell types efficient EGFR signaling requires cell-ECM adhesion. Here we report that transforming growth factor-alpha (TGF-alpha), an EGFR ligand that is frequently secreted by colorectal cancer cells, strongly inhibits anoikis of the non-malignant rat intestinal epithelial cell lines, IEC-18 and RIE-1. TGF-alpha exerts its anti-anoikis effect by preventing detachment-induced inhibition of c-Src kinase activity. We also show that Fas activation, a molecular event known to play a critical role in anoikis, is not suppressed by TGF-alpha. On the other hand, this growth factor strongly inhibits the detachment-induced down-regulation of Bcl-X(L), another change that is involved in the induction of anoikis. We further demonstrate that this inhibition occurs in a c-Src-dependent manner. We conclude that TGF-alpha has the ability to suppress anoikis of intestinal epithelial cells, at least in part, by reverting the loss of c-Src activity and Bcl-X(L) expression induced by detachment from the ECM.     

Hepatocyte growth factor is a novel stimulator of glucose uptake and metabolism in skeletal muscle cells

Skeletal muscle plays a major role in glucose and lipid metabolism. Active hepatocyte growth factor (HGF) is present in the extracellular matrix in skeletal muscle. However, the effects of HGF on glucose and lipid metabolism in skeletal muscle are completely unknown. We therefore examined the effects of HGF on deoxyglucose uptake (DOGU), glucose utilization, and fatty acid oxidation (FAO) in skeletal muscle cells. HGF significantly enhanced DOGU in mouse soleus muscles in vitro. Furthermore, HGF significantly increased: (i) DOGU in a time- and dose-dependent manner; (ii) glucose utilization; and (iii) plasma membrane expression of Glut-1 and Glut-4 in the rat skeletal muscle model of L6 myotubes. HGF-mediated effect on DOGU was dependent on the activation of phosphatidylinositol 3-kinase signaling pathway. On the other hand, HGF markedly and significantly decreased FAO in L6 myotubes without affecting the activities of carnitine palmitoyltransferase I and II. Collectively, these results indicate that HGF is a potent activator of glucose transport and metabolism and also a strong inhibitor of FAO in rodent myotubes. HGF, through its ability to stimulate glucose transport and metabolism and to impair FAO, may participate in the regulation of glucose disposal in skeletal muscle.     

EphB6 promotes anoikis by modulating EphA2 signaling

Anoikis is a specific type of apoptosis induced by detachment of epithelial cells from extracellular matrix, and acquiring resistance to anoikis is an important step that enables cancer cells to metastasize. EphA2, which is overexpressed in a variety of human cancers, is phosphorylated by Akt on serine 897 and mediates ligand ephrin-independent promotion of anoikis resistance through the RhoG activator Ephexin4. EphB6 is frequently silenced in invasive and metastatic cancers; however, its role in cancer progression is poorly understood. Here we show that EphB6 interacts with EphA2 and suppresses EphA2-mediated promotion of anoikis resistance in MCF7 breast cancer cells. On the other hand, knockdown of EphB6 promotes anoikis resistance. We further show that expression of EphB6 decreases serine 897 phosphorylation of EphA2 and suppresses EphA2–Ephexin4 interaction and the RhoG activation. These findings implicate EphB6 as a negative regulator of EphA2 oncogenic signaling.     

Hepatic stellate cell promoted hepatoma cell invasion via the HGF/c-Met signaling pathway regulated by p53

The biological behaviors of hepatocellular carcinoma (HCC) are complex mainly due to heterogeneity of progressive genetic and epigenetic mutations as well as tumor environment. Hepatocyte growth factor (HGF)/c-Met signaling pathway is regarded to be a prototypical example for stromal-epithelial interactions during developmental morphogenesis, wound healing, organ regeneration and cancer progression. And p53 plays as an important regulator of Met-dependent cell motility and invasion. Present study showed that 2 HCC cell lines, Hep3B and HepG2, displayed different invasive capacity when treated with HGF which was secreted by hepatic stellate cells (HSCs). We found that HGF promoted Hep3B cells invasion and migration as well as epithelial-mesenchymal transition (EMT) occurrence because Hep3B was p53 deficient, which leaded to the c-Met over-expression. Then we found that HGF/c-Met promoted Hep3B cells invasion and migration by upregulating Snail expression. In conclusion, HGF/c-Met signaling is enhanced by loss of p53 expression, resulting in increased ability of invasion and migration by upregulating the expression of Snail.     

Control of adhesion-dependent cell survival by focal adhesion kinase

The interactions of integrins with extracellular matrix proteins can activate focal adhesion kinase (FAK) and suppress apoptosis in normal epithelial and endothelial cells; this subset of apoptosis has been termed "anoikis." Here, we demonstrate that FAK plays a role in the suppression of anoikis. Constitutively activated forms of FAK rescued two established epithelial cell lines from anoikis. Both the major autophosphorylation site (Y397) and a site critical to the kinase activity (K454) of FAK were required for this effect. Activated FAK also transformed MDCK cells, by the criteria of anchorage-independent growth and tumor formation in nude mice. We provide evidence that this transformation resulted primarily from the cells' resistance to anoikis rather than from the activation of growth factor response pathways. These results indicate that FAK can regulate anoikis and that the conferral of anoikis resistance may suffice to transform certain epithelial cells.     

HAb18G/CD147 cell-cell contacts confer resistance of a HEK293 subpopulation to anoikis in an E-cadherin-dependent manner

Background  

Acquisition of resistance to "anoikis" facilitates the survival of cells under independent matrix-deficient conditions, such as cells in tumor progression and the production of suspension culture cells for biomedical engineering. There is evidence suggesting that CD147, an adhesion molecule associated with survival of cells in tumor metastasis and cell-cell contacts, plays an important role in resistance to anoikis. However, information regarding the functions of CD147 in mediating cell-cell contacts and anoikis-resistance remains limited and even self-contradictory.