S-nitrosylation-triggered unfolded protein response maintains hematopoietic progenitors in Drosophila.

The Drosophila lymph gland houses blood progenitors that give rise to myeloid-like blood cells. Initially, blood progenitors proliferate, but later, they become quiescent to maintain multipotency before differentiation. Despite the identification of various factors involved in multipotency maintenance, the cellular mechanism controlling blood progenitor quiescence remains elusive. Here, we identify the expression of nitric oxide synthase in blood progenitors, generating nitric oxide for post-translational S-nitrosylation of protein cysteine residues. S-nitrosylation activates the Ire1-Xbp1-mediated unfolded protein response, leading to G2 cell-cycle arrest. Specifically, we identify the epidermal growth factor receptor as a target of S-nitrosylation, resulting in its retention within the endoplasmic reticulum and blockade of its receptor function. Overall, our findings highlight developmentally programmed S-nitrosylation as a critical mechanism that induces protein quality control in blood progenitors, maintaining their undifferentiated state by inhibiting cell-cycle progression and rendering them unresponsive to paracrine factors.

CYR61 confers chemoresistance by upregulating survivin expression in triple-negative breast cancer.

Cysteine-rich angiogenic inducer 61 (CYR61) is a protein from the CCN family of matricellular proteins that play diverse regulatory roles in the extracellular matrix. CYR61 is involved in cell adhesion, migration, proliferation, differentiation, apoptosis, and senescence. Here, we show that CYR61 induces chemoresistance in triple-negative breast cancer (TNBC). We observed that CYR61 is overexpressed in TNBC patients, and CYR61 expression correlates negatively with the survival of patients who receive chemotherapy. CYR61 knockdown reduced cell migration, sphere formation and the cancer stem cell (CSC) population and increased the chemosensitivity of TNBC cells. Mechanistically, CYR61 activated Wnt/ß-catenin signaling and increased survivin expression, which are associated with chemoresistance, the epithelial-mesenchymal transition, and CSC-like phenotypes. Altogether, our study demonstrates a novel function of CYR61 in chemotherapy resistance in breast cancer.

CCN3/NOV promotes metastasis and tumor progression via GPNMB-induced EGFR activation in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. TNBC patients typically exhibit unfavorable outcomes due to its rapid growth and metastatic potential. Here, we found overexpression of CCN3 in TNBC patients. We identified that CCN3 knockdown diminished cancer stem cell formation, metastasis, and tumor growth in vitro and in vivo. Mechanistically, ablation of CCN3 reduced activity of the EGFR/MAPK pathway. Transcriptome profiling revealed that CCN3 induces glycoprotein nonmetastatic melanoma protein B (GPNMB) expression, which in turn activates the EGFR pathway. An interrogation of the TCGA dataset further supported the transcriptional regulation of GPNMB by CCN3. Finally, we showed that CCN3 activates Wnt signaling through a ligand-dependent or -independent mechanism, which increases microphthalmia-associated transcription factor (MITF) protein, a transcription factor inducing GPNMB expression. Together, our findings demonstrate the oncogenic role of CCN3 in TNBC, and we propose CCN3 as a putative therapeutic target for TNBC.

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.

YAP, CTGF and Cyr61 are overexpressed in tamoxifen-resistant breast cancer and induce transcriptional repression of ERα.

About 70% of breast cancers overexpress estrogen receptor α (ERα, encoded by ESR1). Tamoxifen, a competitive inhibitor of estrogen that binds to ER, has been widely used as a treatment for ER-positive breast cancer. However, 20-30% of breast cancer is resistant to tamoxifen treatment. The mechanisms underlying tamoxifen resistance remain elusive. We found that Yes-associated protein (YAP; also known as YAP1), connective tissue growth factor (CTGF; also known as CCN2) and cysteine-rich angiogenic inducer 61 (Cyr61; also known as CCN1) are overexpressed, while ERα is downregulated in tamoxifen-resistant breast cancer. Inhibition of YAP, CTGF and Cyr61 restored ERα expression and increased sensitivity to tamoxifen. Overexpression of YAP, CTGF, and Cyr61 led to downregulation of ERα and conferred resistance to tamoxifen in ER-positive breast cancer cells. Mechanistically, CTGF and Cyr61 downregulated ERα expression at the transcriptional level by directly binding to the regulatory regions of the ERα-encoding gene, leading to increased tamoxifen resistance. Also, CTGF induced Glut3 (also known as SLC2A3) expression, leading to increased glycolysis, which enhanced cell proliferation and migration in tamoxifen-resistant cells. Together, these results demonstrate a novel role of YAP, CTGF and Cyr61 in tamoxifen resistance and provide a molecular basis for their function in tamoxifen-resistant breast cancer.

CTGF regulates cell proliferation, migration, and glucose metabolism through activation of FAK signaling in triple-negative breast cancer.

Connective tissue growth factor (CTGF), also known as CCN2, is a member of the CCN protein family of secreted proteins with roles in diverse biological processes. CTGF regulates biological functions such as cell proliferation, migration, adhesion, wound healing, and angiogenesis. In this study, we demonstrate a mechanistic link between CTGF and enhanced aerobic glycolysis in triple-negative breast cancer (TNBC). We found that CTGF is overexpressed in TNBC and high CTGF expression is correlated with a poor prognosis. Also, CTGF was required for in vivo tumorigenesis and in vitro proliferation, migration, invasion, and adhesion of TNBC cells. Our results indicate that extracellular CTGF binds directly to integrin αvß3, activating the FAK/Src/NF-κB p65 signaling axis, which results in transcriptional upregulation of Glut3. Neutralization of CTGF decreased cell proliferation, migration, and invasion through downregulation of Glut3-mediated glycolytic phenotypes. Overall, our work suggests a novel function for CTGF as a modulator of cancer metabolism, indicating that CTGF is a potential therapeutic target in TNBC.

Silencing of CD133 inhibits GLUT1-mediated glucose transport through downregulation of the HER3/Akt/mTOR pathway in colon cancer.

Cluster of differentiation 133 (CD133) is a transmembrane glycoprotein that has been reported as a marker of cancer stem cells or cancer-initiating cells in various cancers. However, its contribution to tumorigenesis and differentiation remains to be elucidated. To determine the role of CD133 in colon cancer, we silenced CD133 in human colon cancer cells. Silencing of CD133 results in decreased cell proliferation, survival, migration, invasion, and glucose transport. These effects are mediated by downregulation of the human epidermal growth factor receptor 3 (HER3)/Akt/mTOR signaling pathway, culminating in reduced expression of the glucose transporter GLUT1. We also confirm that the cellular phenotypes of CD133-silenced cells are mediated by GLUT1 downregulation. We conclude that CD133 is a potential tumor initiator that positively regulates GLUT1 expression through modulation of HER3/Akt/mTOR signaling.

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).

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.

Role of the CCN protein family in cancer.

The CCN protein family is composed of six matricellular proteins, which serve regulatory roles rather than structural roles in the extracellular matrix. First identified as secreted proteins which are induced by oncogenes, the acronym CCN came from the names of the first three members: CYR61, CTGF, and NOV. All six members of the CCN family consist of four cysteine-rich modular domains. CCN proteins are known to regulate cell adhesion, proliferation, differentiation, and apoptosis. In addition, CCN proteins are associated with cardiovascular and skeletal development, injury repair, inflammation, and cancer. They function either through binding to integrin receptors or by regulating the expression and activity of growth factors and cytokines. Given their diverse roles related to the pathology of certain diseases such as fibrosis, arthritis, atherosclerosis, diabetic nephropathy, retinopathy, and cancer, there are many emerging studies targeting CCN protein signaling pathways in attempts to elucidate their potentials as therapeutic targets. [BMB Reports 2018; 51(10): 486-493].

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.