Neurotensin-induced Erk1/2 phosphorylation and growth of human colonic cancer cells are independent from growth factors receptors activation

Neurotensin (NT) promotes the proliferation of human colonic cancer cells by undefined mechanisms. We already demonstrated that, in the human colon adenocarcinoma cell line HT29, the effects of NT were mediated by a complex formed between the NT receptor-1 (NTSR1) and-3 (NTSR3). Here we examined cellular mechanisms that led to NT-induced MAP kinase phosphorylation and growth factors receptors transactivation in colonic cancer cells and proliferation in HT29 cells. With the aim to identify upstream signaling involved in NT-elicited MAP kinase activation, we found that the stimulatory effects of the peptide were totally independent from the activation of the epidermal growth factor receptor (EGFR) both in the HT29 and the HCT116 cells. NT was unable to promote phosphorylation of EGFR and to compete with EGF for its binding to the receptor. Pharmacological approaches allowed us to differentiate EGF and NT signaling in HT29 cells since only NT activation of Erk1/2 was shown to be sensitive to PKC inhibitors and since only NT increased the intracellular level of calcium. We also observed that NT was not able to transactivate Insulin-like growth factor receptor.Our findings indicate that, in the HT29 and HCT116 cell lines, NT stimulates MAP kinase phosphorylation and cell growth by a pathway which does not involve EGF system but rather NT receptors which transduce their own intracellular effectors. These results indicate that depending on the cell line used, blocking EGFR is not the general rule to inhibit NT-induced cancer cell proliferation.     

Ultraviolet irradiation can induce evasion of colon cancer cells from stimulation of epidermal growth factor

Receptor down-regulation is the most prominent regulatory system of EGF receptor (EGFR) signal attenuation and a critical target for therapy against colon cancer, which is highly dependent on the function of the EGFR. In this study, we investigated the effect of ultraviolet-C (UV-C) on down-regulation of EGFR in human colon cancer cells (SW480, HT29, and DLD-1). UV-C caused inhibition of cell survival and proliferation, concurrently inducing the decrease in cell surface EGFR and subsequently its degradation. UV-C, as well as EGFR kinase inhibitors, decreased the expression level of cyclin D1 and the phosphorylated level of retinoblastoma, indicating that EGFR down-regulation is correlated to cell cycle arrest. Although UV-C caused a marked phosphorylation of EGFR at Ser-1046/1047, UV-C also induced activation of p38 MAPK, a stress-inducible kinase believed to negatively regulate tumorigenesis, and the inhibition of p38 MAPK canceled EGFR phosphorylation at Ser-1046/1047, as well as subsequent internalization and degradation, suggesting that p38 MAPK mediates EGFR down-regulation by UV-C. In addition, phosphorylation of p38 MAPK induced by UV-C was mediated through transforming growth factor-β-activated kinase-1. Moreover, pretreatment of the cells with UV-C suppressed EGF-induced phosphorylation of EGFR at tyrosine residues in addition to cell survival signal, Akt. Together, these results suggest that UV-C irradiation induces the removal of EGFRs from the cell surface that can protect colon cancer cells from oncogenic stimulation of EGF, resulting in cell cycle arrest. Hence, UV-C might be applied for clinical strategy against human colon cancers.     

Retinoids arrest breast cancer cell proliferation: retinoic acid selectively reduces the duration of receptor tyrosine kinase signaling

Retinoic acid (RA) induces cell cycle arrest of hormone-dependent human breast cancer (HBC) cells. Previously, we demonstrated that RA-induced growth arrest of T-47D HBC cells required the activity of the RA-induced protein kinase, protein kinase Calpha (PKCalpha) [J. Cell Physiol. 172 (1997) 306]. Here, we demonstrate that RA treatment of T-47D cells interfered with growth factor signaling to downstream, cytoplasmic and nuclear targets. RA treatment did not inhibit epidermal growth factor (EGF) receptor activation but resulted in rapid inactivation. The lack of sustained EGFR activation was associated with transient rather than sustained association of the EGFR with the Shc adaptor proteins and activation of Erk 1/2 and with compromised induction of expression of immediate early response genes. Inhibiting the activity of PKCalpha, a retinoic acid-induced target gene, prevented the effects of RA on cell proliferation and EGF signaling. Constitutive expression of PKCalpha, in the absence of RA, decreased cell proliferation and decreased EGF signaling. RA treatment increased steady-state levels of the protein tyrosine phosphatase PTP-1C and all measured effects of RA on EGF receptor function were reversed by the tyrosine phosphate inhibitor orthovanadate. These results indicate that RA-induced target genes, particularly PKCalpha, prevent sustained growth factor signaling, uncoupling activated receptor tyrosine kinases and nuclear targets that are required for cell cycle progression.     

Histone deacetylase 1: a target of 9-hydroxystearic acid in the inhibition of cell growth in human colon cancer

Recent studies have shown that an endogenous lipoperoxidation product, 9-hydroxystearic acid (9-HSA), acts in colon carcinoma cells (HT29) as a growth inhibitor by inducing p21(WAF1) in an immediate-early, p53-independent manner and that p21(WAF1) is required for 9-HSA-mediated growth arrest in HT29 cells. It is conceivable, therefore, to hypothesize that the cytostatic effect induced by this agent is at least partially associated with a molecular mechanism that involves histone deacetylase 1 (HDAC1) inhibition, as demonstrated for sodium butyrate and other specific inhibitors, such as trichostatin A and hydroxamic acids. Here, we show that, after administration, 9-HSA causes an accumulation of hyperacetylated histones and strongly inhibits the activity of HDAC1. The interaction of 9-HSA with the catalytic site of the enzyme has been highlighted by computational modeling of the human HDAC1, using its homolog from the hyperthermophilic Aquifex aeolicus as a template. Consistent with the experimental data, we find that 9-HSA can bind to the active site of the protein, showing that the inhibition of the enzyme can be explained at the molecular level by the ligand-protein interaction.     

A Switch Role of Src in the Biphasic EGF Signaling of ER-Negative Breast Cancer Cells

It is well established that epidermal growth factor (EGF) is a potent mitogen in cells expressing EGF receptor (EGFR). However, a body of evidence indicated that the effects of mitogenic EGF signaling exhibit a non-monotonic, or biphasic dose response curve; EGF at low concentrations elicits a mitogenic signaling pathway to stimulate cell proliferation while at high concentrations, EGF inhibits cell growth. However, the molecular mechanism underlying this paradoxical effect of EGF on cell proliferation remains largely unknown. Here, we investigated the molecular mechanisms underlying the biphasic EGF signaling in ER-negative breast cancer MDA-MB-231 and MDA-MB-436 cells, both of which express endogenous EGFR. We found that EGF at low concentrations induced the phosphorylation of the Src-Y416 residue, an event to activate Src, while at high concentrations allowed Src-Y527 phosphorylation that inactivates Src. EGF at 10 ng/ml also induced phosphorylation of the MAPK/ERK and activated cyclin D1 promoter activity through the Src/EGFR/STAT5 pathways but not at a higher concentration (500 ng/ml). Our results thus demonstrated that Src functions as a switch of EGF signaling depending on concentrations of EGF.     

Evidence of an EGF/TGE-alpha--independent pathway for estrogen-regulated cell proliferation

To elucidate the relationship between epidermal growth factor (EGF)/transforming growth factor (TGF-alpha) and estradiol-17 beta (E) in cell proliferation, we examined their effects on the breast cancer cell line, CAMA-1. While E was able to consistently induce cell proliferation under a variety of experimental conditions, EGF/TGF-alpha was without effect. Despite the presence of the receptor (EGFR) gene, mature EGFR protein and mRNA were not detected by radioreceptor assay, 35S Met-labelling, and the Intron Differential RNA/PCR method under conditions in which cells remain responsive to E. Furthermore, TGF-alpha is not an autocrine factor in CAMA-1 cells. We demonstrated unequivocally that EGF/TGF-alpha interaction with EGFR is not an obligatory event in mediating estrogen-stimulated cell proliferation.     

Activation of G‐protein coupled estrogen receptor inhibits the proliferation of cervical cancer cells via sustained activation of ERK1/2

Cervical cancer is one of the most common gynaecological women cancer and suggested to be modulated by estrogenic signals. G protein‐coupled receptor (GPER), a seven‐transmembrane G protein‐coupled receptor, has been reported to regulate the cell proliferation of various cancers. But there is no study investigating the effects of GPER on the progression of cervical cancer. In the present study, we revealed for the first time that GPER was also highly expressed in various human cervical cancer cells. Activation of GPER via its specific agonist G‐1 induced G2/M cell cycle arrest and down regulation of cyclin B via a time dependent manner. Furthermore, G‐1 treatment induced sustained activation of extracellular‐signal‐regulated kinases (ERK)1/2 via epidermal growth factor receptor (EGFR) signals. Both inhibitors of ERK1/2 and EGFR significantly abolished G‐1‐induced suppression of cell proliferation and down regulation of cyclin B. Generally, our study revealed that GPER is highly expressed in human cervical cancer cells and its activation inhibits cell proliferation via EGFR/ERK1/2 signals. It suggested that G‐1 can be considered as a potential new pharmacological tool to reduce the growth of cervical cancer. Copyright © 2015 John Wiley & Sons, Ltd.     

Retinoids suppress epidermal growth factor-associated cell proliferation by inhibiting epidermal growth factor receptor-dependent ERK1/2 activation.

Human papillomavirus (HPV) is an important etiological agent in the genesis of cervical cancer. HPV-positive cervical tumors and human papillomavirus-positive cell lines display increased epidermal growth factor receptor (EGFR) expression, which is associated with increased cell proliferation. ECE16-1 cells are an HPV-immortalized human ectocervical epithelial cell line that is a model of HPV-associated cervical neoplasia and displays elevated EGFR levels. In the present study, we evaluated the effects of receptor-selective retinoid ligands on EGFR-associated signal transduction. We show that retinoic acid receptor (RAR)-selective ligands reduce EGFR level and the magnitude and duration of EGFR activation in EGF-stimulated cells. These effects are reversed by cotreatment with an RAR antagonist. To identify the mechanism, we examined the effects of retinoid treatments on EGF-dependent signaling. Stimulation with EGF causes a biphasic activation of the ERK1/2 MAPK. The first peak of activation is present at 20 min, and the second is present at 36 h. This activation subsequently leads to an increase in the cyclin D1 level and increased cell proliferation. Simultaneous treatment with EGF and a RAR-selective retinoid inhibits both phases of ERK1/2 activation, completely eliminates the cyclin D1 induction, and suppresses EGF-dependent cell proliferation. This effect is specific as retinoid treatment does not alter the level or activity of other EGFR-regulated kinases, including AKT and the MAPKs p38 and JNK. Retinoid X receptor-selective ligands, in contrast, did not regulate these responses. These results suggest that RAR ligand-associated down-regulation of EGFR activity reduces cell proliferation by reducing the magnitude and duration of EGF-dependent ERK1/2 activation.     

Heregulin beta1 promotes breast cancer cell proliferation through Rac/ERK-dependent induction of cyclin D1 and p21Cip1

Accumulating evidence indicates that heregulins, EGF (epidermal growth factor)-like ligands, promote breast cancer cell proliferation and are involved in the progression of breast cancer towards an aggressive and invasive phenotype. However, there is limited information regarding the molecular mechanisms that mediate these effects. We have recently established that HRG (heregulin beta1) promotes breast cancer cell proliferation and migration via cross-talk with EGFR (EGF receptor) that involves the activation of the small GTPase Rac1. In the present paper we report that Rac1 is an essential player for mediating the induction of cyclin D1 and p21(Cip1) by HRG in breast cancer cells. Inhibition of Rac function by expressing either the Rac-GAP (GTPase-activating protein) beta2-chimaerin or the dominant-negative Rac mutant N17Rac1, or Rac1 depletion using RNAi (RNA interference), abolished the cyclin D1 and p21(Cip1) induction by HRG. Interestingly, the proliferative effect of HRG was impaired not only when the expression of Rac1 or cyclin D1 was inhibited, but also when cells were depleted of p21(Cip1) using RNAi. Inhibition of EGFR, PI3K (phosphoinositide 3-kinase; kinases required for Rac activation by HRG) or MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] also blocked the up-regulation of cyclin D1 and p21(Cip1) by HRG. In addition, we found that HRG activates NF-kappaB (nuclear factor kappaB) in a Rac1- and MEK-dependent fashion, and inhibition of NF-kappaB abrogates cyclin D1/p21(Cip1) induction and proliferation by HRG. Taken together, these findings establish a central role for Rac1 in the control of HRG-induced breast cancer cell-cycle progression and proliferation through up-regulating the expression of cyclin D1 and p21(Cip1).     

Additive and inhibitory effects of simultaneous treatment with growth factors on DNA synthesis through MAPK pathway and G1 cyclins in rat hepatocytes

Several growth factors play an important role in liver regeneration. Once hepatic injury occurs, liver regeneration is stimulated by hepatocyte growth factor (HGF), transforming growth factor (TGF)-alpha, and heparin-binding epidermal growth factor-like growth factor (HB-EGF), whereas TGF-beta1 terminates liver regeneration. In this study, we analyzed the effect of a combination of HGF and epidermal growth factor (EGF) on mitogen-activated protein kinase (MAPK) activity and G1 cyclin expression in primary cultured rat hepatocytes. Treatment with a combination of HGF and EGF, in comparison with that of either HGF or EGF, induced tyrosine phosphorylation of both c-Met and EGF receptor (EGFR) independently and additively stimulated MAPK activity and cyclin D1 expression, resulting in additive stimulation of DNA synthesis. On the other hand, although TGF-beta1 treatment did not affect tyrosine phosphorylation of c-Met and EGFR, MAPK activity, and cyclin D1 expression, which were stimulated by HGF and EGF, DNA synthesis was completely inhibited through a marked decrease in cyclin E expression. These results indicate that potent mitogens, such as HGF, TGF-alpha, and HB-EGF, could induce the additive enhancement of liver regeneration cooperatively through an increase in Ras/MAPK activity followed by cyclin D1 expression, and that TGF-beta1 suppresses the growth factor-induced signals between cyclin D1 and cyclin E, resulting in the inhibition of DNA synthesis.     

Targeting Androgen Receptor/Src Complex Impairs the Aggressive Phenotype of Human Fibrosarcoma Cells

Background

Hormones and growth factors influence the proliferation and invasiveness of human mesenchymal tumors. The highly aggressive human fibrosarcoma HT1080 cell line harbors classical androgen receptor (AR) that responds to androgens triggering cell migration in the absence of significant mitogenesis. As occurs in many human cancer cells, HT1080 cells also express epidermal growth factor receptor (EGFR).

Experimental

Findings: We report that the pure anti-androgen Casodex inhibits the growth of HT1080 cell xenografts in immune-depressed mice, revealing a novel role of AR in fibrosarcoma progression. In HT1080 cultured cells EGF, but not androgens, robustly increases DNA synthesis. Casodex abolishes the EGF mitogenic effect, implying a crosstalk between EGFR and AR. The mechanism underlying this crosstalk has been analyzed using an AR-derived small peptide, S1, which prevents AR/Src tyrosine kinase association and androgen-dependent Src activation. Present findings show that in HT1080 cells EGF induces AR/Src Association, and the S1 peptide abolishes both the assembly of this complex and Src activation. The S1 peptide inhibits EGF-stimulated DNA synthesis, cell matrix metalloproteinase-9 (MMP-9) secretion and invasiveness of HT1080 cells. Both Casodex and S1 peptide also prevent DNA synthesis and migration triggered by EGF in various human cancer-derived cells (prostate, breast, colon and pancreas) that express AR.

Conclusion

This study shows that targeting the AR domain involved in AR/Src association impairs EGF signaling in human fibrosarcoma HT1080 cells. The EGF-elicited processes inhibited by the peptide (DNA synthesis, MMP-9 secretion and invasiveness) cooperate in increasing the aggressive phenotype of HT1080 cells. Therefore, AR represents a new potential therapeutic target in human fibrosarcoma, as supported by Casodex inhibition of HT1080 cell xenografts. The extension of these findings in various human cancer-derived cell lines highlights the conservation of this process across divergent cancer cells and identifies new potential targets in the therapeutic approach to human cancers.     

A cell-based high-throughput screen for epidermal growth factor receptor pathway inhibitors

Epidermal growth factor receptor (EGFR) is a valid drug target for development of target-based therapeutics against non-small-cell lung cancer. In this study, we established a high-throughput cell-based assay to screen for compounds that may inhibit EGFR activation and/or EGFR-mediated downstream signaling pathway. This drug screening platform is based on the characterization of an EGFR-transfected 32D cell line (32D-EGFR). The expression of EGFR in 32D cells allowed cell proliferation in the presence of either epidermal growth factor (EGF) or interleukin 3 (IL-3) and provided a system for both screening and counterscreening of EGFR pathway-inhibitory compounds. After the completion of primary and secondary screenings in which 32D-EGFR cells were grown under the stimulation of either EGF or IL-3, 9 of 20,000 compounds were found to selectively inhibit the EGF-dependent proliferation, but not the IL-3-dependent proliferation, of 32D-EGFR cells. Subsequent analysis showed that 3 compounds of the 9 initial hits directly inhibited the kinase activity of recombinant EGFR in vitro and the phosphorylation of EGFR in H1299 cells transfected with EGFR. Thus, this 32D-EGFR assay system provides a promising approach for identifying novel EGFR and EGFR signaling pathway inhibitors with potential antitumor activity.     

Activation of PPAR{gamma} by curcumin inhibits Moser cell growth and mediates suppression of gene expression of cyclin D1 and EGFR

Colorectal cancer is a leading cause of cancer-related morbidity and mortality in the United States. Curcumin, the yellow pigment in turmeric, possesses inhibitory effects on growth of a variety of tumor cells by reducing cell proliferation and inducing apoptosis. Effects of the peroxisome proliferator-activated receptor-gamma (PPARgamma) on stimulating cell differentiation and on inducing cell cycle arrest have attracted attention from the perspective of treatment and prevention of cancer. The aim of this study was to elucidate the mechanisms by which curcumin inhibits colon cancer cell growth. In the present report, we observed that curcumin, in a dose-dependent manner, inhibited the growth of Moser cells, a human colon cancer-derived cell line, and stimulated the trans-activating activity of PPARgamma. Further studies demonstrated that activation of PPARgamma was required for curcumin to inhibit Moser cell growth. Activation of PPARgamma mediated curcumin suppression of the expression of cyclin D1, a critical protein in the cell cycle, in Moser cells. In addition, curcumin blocked EGF signaling by inhibiting EGF receptor (EGFR) tyrosine phosphorylation and suppressing the gene expression of EGFR mediated by activation of PPARgamma. In addition to curcumin reduction of the level of phosphorylated PPARgamma, inhibition of cyclin D1 expression played a major and significant role in curcumin stimulation of PPARgamma activity in Moser cells. Taken together, our results demonstrated for the first time that curcumin activation of PPARgamma inhibited Moser cell growth and mediated the suppression of the gene expression of cyclin D1 and EGFR. These results provided a novel insight into the roles and mechanisms of curcumin in inhibition of colon cancer cell growth and potential therapeutic strategies for treatment of colon cancer.     

Epidermal growth factor (EGF) induces apoptosis in a transfected cell line expressing EGF receptor on its membrane

Interaction between epidermal growth factor (EGF) and EGF receptor (EGFR) promotes cell growth in most cell lines, but in a number of cell lines, EGF paradoxically inhibits proliferation. In the present study, we established a cell line expressing full-length human EGFR on membrane with a GFP fluorescence reporter at the C-terminal and studied the effects of EGF on cell proliferation in the transfected cell line. Our results suggested that low concentrations of EGF promoted proliferation, while high concentrations of EGF induced loss of adhesion, cell cycle arrest, apoptosis, and inhibition of proliferation. The effects of EGF on cell proliferation correlated well with the expression levels of EGFR. High concentrations of EGF induced both EGFR expression and apoptosis in a dose-dependent manner. Our study reported, for the first time, a relationship between the effects of EGF on cell proliferation and levels of EGFR expression in one cell line expressing different levels of EGFR caused by different concentrations of EGF treatment. The study should provide considerable insight into the effects of EGF on cell proliferation and tumor cell metastasis.     

Down-regulation of epidermal growth factor receptor-signaling pathway by binding of GRP78/BiP to the receptor under glucose-starved stress conditions

GRP78/BiP, a molecular chaperone in the endoplasmic reticulum, is induced under such adverse conditions for cell survival as glucose starvation. Induction of GRP78 has been shown to coincide with G1 cell cycle arrest, which is an important cellular defense system. In this study, we investigated involvement of GRP78 in the mechanism of growth arrest by using human epidermoid carcinoma A431 cells. Under a chemical stress condition with 2-deoxyglucose, GRP78 was induced 3–4-fold. In the stressed cells, an underglycosylated form of epidermal growth factor receptor (EGFR) was produced and the mature form was decreased. We found that the molecular chaperone GRP78 in the endoplasmic reticulum formed a stable complex with the underglycosylated EGFR but did not with the mature form. This complex formation occurred specifically under the stress conditions, and the complex was dissociated upon removal of the stress. Treatment of the GRP78-underglycosylated EGFR complex with ATP resulted in a release of the underglycosylated EGFR from GRP78, indicating that the complex could be formed through the chaperone function of GRP78. In accordance with the complex formation with endoplasmic reticulum-resident GRP78, the underglycosylated EGFR could not be translocated to the cell surface. As a result, EGF could not induce expression of cyclin D3, a G1 cyclin, in the stressed cells, whereas it did in non-stressed cells. These results indicated that, in the stressed cells, GRP78 participated in down-regulation of EGF-signaling pathway by forming a stable complex with EGFR and inhibiting EGFR translocation to the cell surface. J. Cell. Physiol. 177:282–288, 1998. © 1998 Wiley-Liss, Inc.