Correction to: ECM1 regulates cell proliferation and trastuzumab resistance through activation of EGF-signaling.

After the publication of this work [1] errors were found in Figs. 1a and 5d.

Titanium dioxide nanoparticles induce apoptosis by interfering with EGFR signaling in human breast cancer cells.

Titanium dioxide nanoparticles, due to their smaller size and increased surface area comparted to the bulk form, are known to be bioreactive and have unexpected toxicological outcomes. Previous studies have shown that nanoscale titanium dioxide induces reactive oxygen species (ROS)-mediated cytotoxicity and genotoxicity. Although many reports have discussed the ROS-mediated cytotoxic effects of titanium dioxide nanoparticles (TiO2-NPs), their effects on the receptor-ligand association are unknown. In this study, the possibility that TiO2-NPs can interfere with the receptor-ligand binding was assessed by monitoring alterations in the phosphorylation status of proteins downstream of the epidermal growth factor receptor (EGFR) signaling cascade. TiO2-NPs blocked ligand-induced EGFR autophosphorylation, leading to the deactivation of EGFR downstream effectors such as Akt and extracellular signal-regulated kinase signaling, inducing cell death.

Toxicological assessment of phthalates and their alternatives using human keratinocytes.

Phthalates are mainly used as binders and plasticizers in various industrial products including detergents, surfactants, waxes, paints, pharmaceuticals, food products, and cosmetics. However, they have been reported to be endocrine disruptors, which are chemicals that can mimic or disturb endocrines, causing interference to the endocrine system. Recently, there have been numerous reports showing that phthalates have negative health impacts such as asthma, breast cancer, obesity, type II diabetes, and male infertility. Due to these effects, there is an urgent need for phthalate alternatives. In this study, the potential cytotoxicity of phthalates and their substitutes were screened in HaCaT cells, a human keratinocyte cell line, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) thiazolyl blue assay, immunocytochemistry, flow cytometric analysis, and western blotting. We confirmed that common phthalates such as butyl benzyl phthalate (BBP), di-n-butyl phthalate (DBP), and di-2-ethylhexyl phthalate (DEHP) have genotoxic effects, leading to cell death. Among the known phthalate substitutes, tributyl O-acetylcitrate (ATBC), triethyl 2-acetylcitrate (ATEC), and trihexyl O-acetylcitrate (ATHC) were tested for cytotoxicity. As a result, ATEC showed similar levels of cytotoxicity with the phthalates whereas ATBC and ATHC did not show significant cytotoxicity even in high doses (5 mg/ml).

Comparative toxicological evaluation of nonylphenol and nonylphenol polyethoxylates using human keratinocytes.

Nonylphenol polyethoxylates (NPEOs) are a major group of nonionic surfactants widely used in various detergents, cleaners, plastics, papers, and agro-chemical products. Nonylphenol (NP), which is a final degraded metabolite derived from NPEOs, has been reported as an endocrine disrupter, known to mimic or disturb reproductive hormone functions. Concern about the hazards of NP and NPEOs has generated legal restrictions and action plans worldwide. Considering the fact that NP and NPEOs are majorly used in the production of products such as detergents, shampoos, and cosmetics which frequently come into contact with the skin, we investigated the effects of NP and NPEOs on a human keratinocyte cell line (HaCaT). In this study, the toxicity of NP and NPEOs was screened in HaCaT cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide thiazolyl blue assay and Western blotting. The potential cytotoxicity of substitutes was assessed by dose-response assays, relative cell viability, and genotoxicity caused by specific alterations in DNA damage response proteins (including ataxia-telangiectasia mutated, p53, Chk1, Chk2, and Histone H2A.X). We demonstrated that NP and NPEOs are toxic to HaCaT cells, as revealed by the decreased cell viability after 24 h treatment. NPs and NPEOs also induced apoptosis and DNA damage as shown by the activation of Poly(ADP-ribose) polymerase, Caspase-3, and Histone H2A.X.

Silencing of Glut1 induces chemoresistance via modulation of Akt/GSK-3ß/ß-catenin/survivin signaling pathway in breast cancer cells.

Cancer cells require increased aerobic glycolysis to support rapid cell proliferation. For their increased energy demands, cancer cells express glucose transporter (Glut) proteins at a high level. Glut1 is associated with basal-like breast cancer and is considered a potential therapeutic target. To investigate the possibility of Glut1 as a therapeutic target in breast cancer cells, we downregulated Glut1 in triple-negative breast cancer (TNBC) cell lines using a short hairpin system. We determined whether Glut1 silencing might enhance anti-proliferative effect of chemotherapeutic agents. Contrary to our hypothesis, ablation of Glut1 attenuated apoptosis and increased drug resistance via upregulation of p-Akt/p-GSK-3ß (Ser9)/ß-catenin/survivin. These results indicated that the potential of Glut1 as a therapeutic target should be carefully reevaluated.

Ablation of CD44 induces glycolysis-to-oxidative phosphorylation transition via modulation of the c-Src-Akt-LKB1-AMPKα pathway.

Cluster of differentiation 44 (CD44) is a transmembrane glycoprotein that has been identified as a cancer stem cell marker in various cancer cells. Although many studies have focused on CD44 as a cancer stem cell marker, its effect on cancer cell metabolism remains unclear. To investigate the role of CD44 on cancer cell metabolism, we established CD44 knock-down cells via retroviral delivery of shRNA against CD44 in human breast cancer cells. Silencing of CD44 decreased the glycolytic phenotype of cancer cells, affecting glucose uptake, ATP production, and lactate production. We also found that ablation of the CD44-induced lactate dehydrogenase (LDH) isoenzyme results in a shift to LDH1 due to LDHA down-regulation and LDHB up-regulation, implying the importance of LDH isoenzyme modulation on cancer metabolism. The expression of glycolysis-related proteins including hypoxia inducible factor-1α (HIF-1α) and LDHA was decreased by CD44 silencing. These effects were due to the up-regulation of liver kinase B1 (LKB1)/AMP-activated protein kinase (AMPK)α activity by reduction in c-Src and Akt activity in CD44 knock-down cells. Finally, induction of LKB1/AMPKα activity blocked the expression of HIF-1α and its target gene, LDHA. Inversely, LDHB expression was repressed by HIF-1α. Collectively, these results indicate that the CD44 silencing-induced metabolic shift is mediated by the regulation of c-Src/Akt/LKB1/AMPKα/HIF-1α signaling in human breast cancer cells.

EGFR negates the proliferative effect of oncogenic HER2 in MDA-MB-231 cells.

Members of the EGFR family are potent mediators of normal cell growth and development. HER2 possesses an active tyrosine kinase domain, but no direct ligand has been identified. To investigate the differential effect of HER2 in breast cell lines, HER2 was overexpressed in MCF-10A, MCF7 and MDA-MB-231 cells. HER2 overexpression promoted proliferation, survival and migration in MCF-10A and MCF-7 cells. No significant differences were seen in proliferation, survival or migration between MDA-MB-231 vec and HER2 cells. The activity of downstream HER2 proteins increased in MCF-10A HER2 and MCF-7 HER2 cells but not in MDA-MB-231 HER2 cells. Exogenously expressed HER2 failed to associate with EGFR or HER3 in MDA-MB-231 cells, while overexpression of HER2 enhanced HER family dimerization in MCF-10A and MCF-7 cells.

Extracellular matrix protein 1 regulates cell proliferation and trastuzumab resistance through activation of epidermal growth factor signaling.

INTRODUCTION: Extracellular matrix protein 1 (ECM1) is a secreted glycoprotein with putative functions in cell proliferation, angiogenesis and differentiation. Expression of ECM1 in several types of carcinoma suggests that it may promote tumor development. In this study, we investigated the role of ECM1 in oncogenic cell signaling in breast cancer, and potential mechanisms for its effects. METHODS: In order to find out the functional role of ECM1, we used the recombinant human ECM1 and viral transduction systems which stably regulated the expression level of ECM1. We examined the effect of ECM1 on cell proliferation and cell signaling in vitro and in vivo. Moreover, tissues and sera of patients with breast cancer were used to confirm the effect of ECM1. RESULTS: ECM1 protein was increased in trastuzumab-resistant (TR) cells, in association with trastuzumab resistance and cell proliferation. Through physical interaction with epidermal growth factor receptor (EGFR), ECM1 potentiated the phosphorylation of EGFR and extracellular signal-regulated kinase upon EGF treatment. Moreover, ECM1-induced galectin-3 cleavage through upregulation of matrix metalloproteinase 9 not only improved mucin 1 expression, but also increased EGFR and human epidermal growth factor receptor 3 protein stability as a secondary signaling. CONCLUSIONS: ECM1 has important roles in both cancer development and trastuzumab resistance in breast cancer through activation of EGFR signaling.

Protein kinase B/Akt1 inhibits autophagy by down-regulating UVRAG expression.

Autophagy, or autophagocytosis, is a selective intracellular degradative process involving the cell's own lysosomal apparatus. An essential component in cell development, homeostasis, repair and resistance to stress, autophagy may result in either cell death or survival. The targeted region of the cell is sequestered within a membrane structure, the autophagosome, for regulation of the catabolic process. A key factor in both autophagosome formation and autophagosome maturation is a protein encoded by the ultraviolet irradiation resistance-associated gene (UVRAG). Conversely, the serine/threonine-specific protein kinase B (PKB, also known as Akt), which regulates survival in various cancers, inhibits autophagy through mTOR activation. We found that Akt1 may also directly inhibit autophagy by down-regulating UVRAG both in a 293T transient transfection system and breast cancer cells stably expressing Akt1. The UVRAG with mutations at putative Akt1-phosphorylation sites were still inhibited by Akt1, and dominant-negative Akt1 also inhibited UVRAG expression, suggesting that Akt1 down-regulates UVRAG by a kinase activity-independent mechanism. We showed that Akt1 overexpression in MDA-MB-231 breast cancer cells down-regulated UVRAG transcription. Cells over-expressing Akt1 were more resistant than control cells to ultraviolet light-induced autophagy and exhibited the associated reduction in cell viability. Levels of the autophagosome indicator protein LC3B-II and mRFP-GFP-LC3 were reduced in cells that over-expressing Akt1. Inhibiting Akt1 by siRNA or reintroducing UVRAG gene rescued the level of LC3B-II in UV-irradiation. Altogether, these data suggest that Akt1 may inhibit autophagy by decreasing UVRAG expression, which also sensitizes cancer cells to UV irradiation.

HER2 stabilizes survivin while concomitantly down-regulating survivin gene transcription by suppressing Notch cleavage.

In breast cancer, the HER2 (human epidermal growth factor receptor 2) receptor tyrosine kinase is associated with extremely poor prognosis and survival. Notch signalling has a key role in cell-fate decisions, especially in cancer-initiating cells. The Notch intracellular domain produced by Notch cleavage is translocated to the nucleus where it activates transcription of target genes. To determine the combinatory effect of HER2 and Notch signalling in breast cancer, we investigated the effect of HER2 on Notch-induced cellular phenomena. We found the down-regulation of Notch-dependent transcriptional activity by HER2 overexpression. Also, the HER2/ERK (extracellular-signal-regulated kinase) signal pathway down-regulated the activity of γ-secretase. When we examined the protein level of Notch target genes in HER2-overexpressing cells, we observed that the level of survivin, downstream of Notch, increased in HER2 cells. We found that activation of ERK resulted in a decrease in XAF1 [XIAP (X-linked inhibitor of apoptosis)-associated factor 1] which reduced the formation of the XIAP-XAF1 E3 ligase complex to ubiquitinate survivin. In addition, Thr(34) of survivin was shown to be the most important residue in determining survivin stability upon phosphorylation after HER2/Akt/CDK1 (cyclin-dependent kinase 1)-cyclin B1 signalling. The results of the present study show the combinatorial effects of HER2 and Notch during breast oncogenesis.

Protein kinase Cδ negatively regulates Notch1-dependent transcription via a kinase-independent mechanism in vitro.

Protein kinase Cδ (PKCδ) plays a significant role in the regulation of growth, apoptosis, and differentiation in a diversity of cell types. We investigated the effect of PKCδ on Notch1 intracellular domain (NICD)-mediated transcription with Notch transcription reporter constructs. The results indicate that co-expression of PKCδ down-regulated NICD-dependent transcription. Co-expression of a dominant negative PKCδ (K376R) variant lacking kinase activity was also able to downregulate NICD-dependent transcription, suggesting that PKCδ exerts its inhibitory effect via a kinase-independent mechanism(s). Interestingly, expression of PKCδ as well as K376R induced NICD up-regulation by inhibiting proteasome-mediated degradation of NICD, indicating that NICD protein quantity is not proportional to its transcriptional activity. When the subcellular distribution of NICD was investigated by both subcellular fractionation and immunocytochemistry, it was found that PKCδ and K376R effectively impaired proper nuclear localization of NICD, possibly via a physical association between NICD and PKCδ, which was confirmed by co-immunoprecipitation experiments. Chromatin immunoprecipitation assays revealed that both PKCδ and K376R inhibit the association of NICD with the promoter region of its target gene, Hes1. Furthermore, silencing of PKCδ resulted in increased NICD nuclear localization and NICD transcriptional activity in MCF-7 cells. PKCδ silencing-induced increase in anti-apoptotic survivin could not rescue apoptosis induced by doxorubicin. The data herein indicate that PKCδ can induce down-regulation of NICD transcriptional activity via a kinase-independent inhibition of NICD nuclear targeting and dissociation of NICD from target gene promoters.

Brain type of creatine kinase induces doxorubicin resistance via TGF-ß signaling in MDA-MB-231 breast cancer cells.

Brain type of creatine kinase (CKB) regulates energy homeostasis by reversibly transferring phosphate groups between phosphocreatine and ATP at sites of high energy demand. Several types of cancer cells exhibit upregulated CKB expression, but the function of CKB in cancer cells remains unclear. In this study, we investigated the function of CKB in breast cancer by overexpressing CKB in MDA-MB-231 cells. The overexpression of CKB did not affect cell growth rate, cell cycle distribution, ATP level or key mediators of aerobic glycolysis and lactate dehydrogenase isoform levels. Meanwhile, CKB overexpression did increase resistance to doxorubicin. TGF-ß-induced Smad phosphorylation and Smad-dependent transcriptional activity were significantly up-regulated by CKB expression without changes in inhibitory Smad protein levels. Moreover, treatment with TGF-ß considerably enhanced cell viability during doxorubicin treatment and decreased doxorubicin-induced apoptosis in CKB-expressing MDA-MB-231 cells compared to control cells. These results suggest that CKB attenuates doxorubicin-induced apoptosis and potentiates resistance to doxorubicin by enhancing TGF-ß signaling in MDA-MB-231 cells.

Egr-1 is required for neu/HER2-induced mammary tumors.

Egr-1 is known to function mainly as a tumor suppressor through direct regulation of multiple tumor suppressor genes. To determine the role of Egr-1 in breast tumors in vivo, we used mouse models of breast cancer induced by HER2/neu. We compared neu-overexpressing Egr-1 knockout mice (neu/Egr-1 KO) to neu-overexpressing Egr-1 wild type or heterozygote mice (neu/Egr-1 WT or neu/Egr-1 het) with regard to onset of tumor appearance and number of tumors per mouse. In addition, to examine the role of Egr-1 in vitro, we established neu/Egr-1 WT and KO tumor cell lines derived from breast tumors developed in each mouse. Egr-1 deletion delayed tumor development in vivo and decreased the rate of cell growth in vitro. These results suggest that Egr-1 plays an oncogenic role in HER2/neu-driven mammary tumorigenesis.

Glut1 promotes cell proliferation, migration and invasion by regulating epidermal growth factor receptor and integrin signaling in triple-negative breast cancer cells.

Elevated glucose levels in cancer cells can be attributed to increased levels of glucose transporter (GLUT) proteins. Glut1 expression is increased in human malignant cells. To investigate alternative roles of Glut1 in breast cancer, we silenced Glut1 in triple-negative breast-cancer cell lines using a short hairpin RNA (shRNA) system. Glut1 silencing was verified by Western blotting and qRT-PCR. Knockdown of Glut1 resulted in decreased cell proliferation, glucose uptake, migration, and invasion through modulation of the EGFR/ MAPK signaling pathway and integrin ß1/Src/FAK signaling pathways. These results suggest that Glut1 not only plays a role as a glucose transporter, but also acts as a regulator of signaling cascades in the tumorigenesis of breast cancer. [BMB Reports 2017; 50(3): 132-137].

Cytotoxic Effect of Nano-SiO2 in Human Breast Cancer Cells via Modulation of EGFR Signaling Cascades.

BACKGROUND/AIM: Silica nanoparticles (nano-SiO2) are widely used in many industrial areas and there is much controversy surrounding cytotoxic effects of such nanoparticles. In order to determine the toxicity and possible molecular mechanisms involved, we conducted several tests with two breast cancer cell lines, MDA-MB-231 and Hs578T. MATERIALS AND METHODS: After exposure to nano-SiO2, growth, apoptosis, motility of breast cancer cells were monitored. In addition, modulation of signal transduction induced by nano-SiO2 was detected through western blot analysis. RESULTS: Treatment of nano-SiO2 repressed the growth of breast cancer cell lines. It also increased apoptosis and reduced cell motility. Moreover, exposure to nano-SiO2 significantly disturbed the dimerization of epidermal growth factor receptor (EGFR), followed by down-regulation of its downstream cellular sarcoma kinase (c-SRC) and signal transducer and activator of transcription 3 (STAT3) signaling cascades. CONCLUSION: Nano-SiO2 has a cytotoxic effect on MDA-MB-231 and Hs578T breast cancer cells via modulation of EGFR signaling cascades.

Binding of galectin-1 to integrin ß1 potentiates drug resistance by promoting survivin expression in breast cancer cells.

Galectin-1 is a ß-galactoside binding protein secreted by many types of aggressive cancer cells. Although many studies have focused on the role of galectin-1 in cancer progression, relatively little attention has been paid to galectin-1 as an extracellular therapeutic target. To elucidate the molecular mechanisms underlying galectin-1-mediated cancer progression, we established galectin-1 knock-down cells via retroviral delivery of short hairpin RNA (shRNA) against galectin-1 in two triple-negative breast cancer (TNBC) cell lines, MDA-MB-231 and Hs578T. Ablation of galectin-1 expression decreased cell proliferation, migration, invasion, and doxorubicin resistance. We found that these effects were caused by decreased galectin-1-integrin ß1 interactions and suppression of the downstream focal adhesion kinase (FAK)/c-Src pathway. We also found that silencing of galectin-1 inhibited extracellular signal-regulated kinase (ERK)/signal transducer and activator of transcription 3 (STAT3) signaling, thereby down-regulating survivin expression. This finding implicates STAT3 as a transcription factor for survivin. Finally, rescue of endogenous galectin-1 knock-down and recombinant galectin-1 treatment both recovered signaling through the FAK/c-Src/ERK/STAT3/survivin pathway. Taken together, these results suggest that extracellular galectin-1 contributes to cancer progression and doxorubicin resistance in TNBC cells. These effects appear to be mediated by galectin-1-induced up-regulation of the integrin ß1/FAK/c-Src/ERK/STAT3/survivin pathway. Our results imply that extracellular galectin-1 has potential as a therapeutic target for triple-negative breast cancer.

ECM1 promotes the Warburg effect through EGF-mediated activation of PKM2.

The Warburg effect is an oncogenic metabolic switch that allows cancer cells to take up more glucose than normal cells and favors anaerobic glycolysis. Extracellular matrix protein 1 (ECM1) is a secreted glycoprotein that is overexpressed in various types of carcinoma. Using two-dimensional digest-liquid chromatography-mass spectrometry (LC-MS)/MS, we showed that the expression of proteins associated with the Warburg effect was upregulated in trastuzumab-resistant BT-474 cells that overexpressed ECM1 compared to control cells. We further demonstrated that ECM1 induced the expression of genes that promote the Warburg effect, such as glucose transporter 1 (GLUT1), lactate dehydrogenase A (LDHA), and hypoxia-inducible factor 1 α (HIF-1α). The phosphorylation status of pyruvate kinase M2 (PKM-2) at Ser37, which is responsible for the expression of genes that promote the Warburg effect, was affected by the modulation of ECM1 expression. Moreover, EGF-dependent ERK activation that was regulated by ECM1 induced not only PKM2 phosphorylation but also gene expression of GLUT1 and LDHA. These findings provide evidence that ECM1 plays an important role in promoting the Warburg effect mediated by PKM2.

CD44 regulates cell proliferation, migration, and invasion via modulation of c-Src transcription in human breast cancer cells.

CD44 was recently identified as a cancer initiation marker on the cell membrane. The cytoplasmic tail of CD44 is known to bind ERM (ezrin, radixin, moesin) proteins, cytoskeletal proteins like ankyrin, and the non-receptor tyrosine kinase c-Src. CD44 transmits its oncogenic signaling via c-Src and its downstream effectors. To investigate the role of CD44 in breast cancer cells, we generated CD44 knock-down cells via retroviral delivery of shRNA against CD44. We found that silencing of CD44 decreased the proliferation, migration, and invasion of breast cancer cells. The expression and activity of cell migration-related proteins, including c-Src, paxillin, and FAK were decreased by CD44 silencing. We also found that the c-Jun protein level was negatively regulated via induction of a GSK-3ß-dependent degradation pathway in CD44 knock-down cells. The expression level of Sp1, a target gene product of c-Jun, was also decreased in these cells. Finally, CD44 knock-down suppressed both mRNA and protein levels of c-Src and its downstream MAPK pathway as a result of down-regulation of Sp1 as a transcription factor for c-Src. Collectively, these results indicate that biological changes induced by CD44 silencing are mediated by cumulative down-regulation of c-Jun, Sp1, and c-Src in human breast cancer cells.

S100A4 negatively regulates ß-catenin by inducing the Egr-1-PTEN-Akt-GSK3ß degradation pathway.

S100A4, also known as the mts1 gene, has been reported as an invasive and metastatic marker for many types of cancers. S100A4 interacts with various target genes that affect tumor cell metastasis; however, little is known about cellular signaling pathways elicited by S100A4. In the current study, we demonstrate an inhibitory effect of S100A4 on ß-catenin signaling in breast cancer cells. By overexpressing S100A4 in MCF-7, MDA-MB-231 and MDA-MB-453 breast cancer cells, we observed the down-regulation of ß-catenin expression and ß-catenin-dependent TCF/LEF transcriptional activities. The activity of GSK3ß, which phosphorylates ß-catenin and induces proteasomal degradation of ß-catenin, was increased in S100A4-overexpressing cell lines. Blocking Glycogen Synthase Kinase (GSK3ß) activity by lithium chloride or Dvl gene overexpression restored ß-catenin expression. We also found that increased GSK3ß activity was due to decrease in Akt activity resulting from Egr-1-induced phosphatase and tensin homolog (PTEN) expression. S100A4 induced Egr-1 nuclear localization by increasing the association between Egr-1 and importin-7 and this effect was reduced in S100A4 mutants that harbored a defect in nuclear localization signals. Collectively, we verify herein that S100A4 may act as a tumor suppressor in breast cancers by down-regulating the central signaling axis for tumor cell survival.

Regulation of cell proliferation and migration by keratin19-induced nuclear import of early growth response-1 in breast cancer cells.

PURPOSE: Keratin19 (KRT19) is the smallest known type I intermediate filament and is used as a marker for reverse transcriptase PCR-mediated detection of disseminated tumors. In this study, we investigated the functional analysis of KRT19 in human breast cancer. EXPERIMENTAL DESIGN: Using a short hairpin RNA system, we silenced KRT19 in breast cancer cells. KRT19 silencing was verified by Western blot analysis and immunocytochemistry. We further examined the effect of KRT19 silencing on breast cancer cells by cell proliferation, migration, invasion, colony formation assay, cell-cycle analysis, immunocytochemistry, immunohistochemistry, and mouse xenograft assay. RESULTS: Silencing of KRT19 resulted in increased cell proliferation, migration, invasion, and survival. These effects were mediated by upregulation of Akt signaling as a result of reduced PTEN mRNA expression. Silencing of KRT19 decreased the nuclear import of early growth response-1 (Egr1), a transcriptional factor for PTEN transcription, through reduced association between Egr1 and importin-7. We also confirmed that silencing of KRT19 increased tumor formation in a xenograft model. CONCLUSIONS: KRT19 is a potential tumor suppressor that negatively regulates Akt signaling through modulation of Egr1 nuclear localization.