FAM129B, an antioxidative protein, reduces chemosensitivity by competing with Nrf2 for Keap1 binding.

BackgroundThe transcription factor Nrf2 is a master regulator of antioxidant response. While Nrf2 activation may counter increasing oxidative stress in aging, its activation in cancer can promote cancer progression and metastasis, and confer resistance to chemotherapy and radiotherapy. Thus, Nrf2 has been considered as a key pharmacological target. Unfortunately, there are no specific Nrf2 inhibitors for therapeutic application. Moreover, high Nrf2 activity in many tumors without Keap1 or Nrf2 mutations suggests that alternative mechanisms of Nrf2 regulation exist.MethodsInteraction of FAM129B with Keap1 is demonstrated by immunofluorescence, colocalization, co-immunoprecipitation and mammalian two-hybrid assay. Antioxidative function of FAM129B is analyzed by measuring ROS levels with DCF/flow cytometry, Nrf2 activation using luciferase reporter assay and determination of downstream gene expression by qPCR and wester blotting. Impact of FAM129B on in vivo chemosensitivity is examined in mice bearing breast and colon cancer xenografts. The clinical relevance of FAM129B is assessed by qPCR in breast cancer samples and data mining of publicly available databases.FindingsWe have demonstrated that FAM129B in cancer promotes Nrf2 activity by reducing its ubiquitination through competition with Nrf2 for Keap1 binding via its DLG and ETGE motifs. In addition, FAM129B reduces chemosensitivity by augmenting Nrf2 antioxidative signaling and confers poor prognosis in breast and lung cancer.InterpretationThese findings demonstrate the important role of FAM129B in Nrf2 activation and antioxidative response, and identify FMA129B as a potential therapeutic target. FUND: The Chang Gung Medical Foundation (Taiwan) and the Ministry of Science and Technology (Taiwan)

Fucosyltransferase 1 and 2 play pivotal roles in breast cancer cells.

FUT1 and FUT2 encode alpha 1, 2-fucosyltransferases which catalyze the addition of alpha 1, 2-linked fucose to glycans. Glycan products of FUT1 and FUT2, such as Globo H and Lewis Y, are highly expressed on malignant tissues, including breast cancer. Herein, we investigated the roles of FUT1 and FUT2 in breast cancer. Silencing of FUT1 or FUT2 by shRNAs inhibited cell proliferation in vitro and tumorigenicity in mice. This was associated with diminished properties of cancer stem cell (CSC), including mammosphere formation and CSC marker both in vitro and in xenografts. Silencing of FUT2, but not FUT1, significantly changed the cuboidal morphology to dense clusters of small and round cells with reduced adhesion to polystyrene and extracellular matrix, including laminin, fibronectin and collagen. Silencing of FUT1 or FUT2 suppressed cell migration in wound healing assay, whereas FUT1 and FUT2 overexpression increased cell migration and invasion in vitro and metastasis of breast cancer in vivo. A decrease in mesenchymal like markers such as fibronectin, vimentin, and twist, along with increased epithelial like marker, E-cadherin, was observed upon FUT1/2 knockdown, while the opposite was noted by overexpression of FUT1 or FUT2. As expected, FUT1 or FUT2 knockdown reduced Globo H, whereas FUT1 or FUT2 overexpression showed contrary effects. Exogenous addition of Globo H-ceramide reversed the suppression of cell migration by FUT1 knockdown but not the inhibition of cell adhesion by FUT2 silencing, suggesting that at least part of the effects of FUT1/2 knockdown were mediated by Globo H. Our results imply that FUT1 and FUT2 play important roles in regulating growth, adhesion, migration and CSC properties of breast cancer, and may serve as therapeutic targets for breast cancer

GPER-induced signaling is essential for the survival of breast cancer stem cells.

G protein-coupled estrogen receptor-1 (GPER), a member of the G protein-coupled receptor (GPCR) superfamily, mediates estrogen-induced proliferation of normal and malignant breast epithelial cells. However, its role in breast cancer stem cells (BCSCs) remains unclear. Here we showed greater expression of GPER in BCSCs than non-BCSCs of three patient-derived xenografts of ER- /PR+ breast cancers. GPER silencing reduced stemness features of BCSCs as reflected by reduced mammosphere forming capacity in vitro, and tumor growth in vivo with decreased BCSC populations. Comparative phosphoproteomics revealed greater GPER-mediated PKA/BAD signaling in BCSCs. Activation of GPER by its ligands, including tamoxifen (TMX), induced phosphorylation of PKA and BAD-Ser118 to sustain BCSC characteristics. Transfection with a dominant-negative mutant BAD (Ser118Ala) led to reduced cell survival. Taken together, GPER and its downstream signaling play a key role in maintaining the stemness of BCSCs, suggesting that GPER is a potential therapeutic target for eradicating BCSCs

Sialylation of vasorin by ST3Gal1 facilitates TGF-β1-mediated tumor angiogenesis and progression.

ST3Gal1 is a key sialyltransferase which adds α2,3-linked sialic acid to substrates and generates core 1 O-glycan structure. Upregulation of ST3Gal1 has been associated with worse prognosis of breast cancer patients. However, the protein substrates of ST3Gal1 implicated in tumor progression remain elusive. In our study, we demonstrated that ST3GAL1-silencing significantly reduced tumor growth along with a notable decrease in vascularity of MCF7 xenograft tumors. We identified vasorin (VASN) which was shown to bind TGF-β1, as a potential candidate that links ST3Gal1 to angiogenesis. LC-MS/MS analysis of VASN secreted from MCF7, revealed that more than 80% of its O-glycans are sialyl-3T and disialyl-T. ST3GAL1-silencing or desialylation of VASN by neuraminidase enhanced its binding to TGF-β1 by 2- to 3-fold and thereby dampening TGF-β1 signaling and angiogenesis, as indicated by impaired tube formation of HUVECs, suppressed angiogenesis gene expression and reduced activation of Smad2 and Smad3 in HUVEC cells. Examination of 114 fresh primary breast cancer and their adjacent normal tissues showed that the expression levels of ST3Gal1 and TGFB1 were high in tumor part and the expression of two genes was positively correlated. Kaplan Meier survival analysis showed a significantly shorter relapse-free survival for those with lower expression VASN, notably, the combination of low VASN with high ST3GAL1 yielded even higher risk of recurrence (p = 0.025, HR = 2.967, 95% CI = 1.14-7.67). Since TGF-β1 is known to transcriptionally activate ST3Gal1, our findings illustrated a feedback regulatory loop in which TGF-β1 upregulates ST3Gal1 to circumvent the negative impact of VASN

Curcumin-Mediated HDAC Inhibition Suppresses the DNA Damage Response and Contributes to Increased DNA Damage Sensitivity

<div><p>Chemo- and radiotherapy cause multiple forms of DNA damage and lead to the death of cancer cells. Inhibitors of the DNA damage response are candidate drugs for use in combination therapies to increase the efficacy of such treatments. In this study, we show that curcumin, a plant polyphenol, sensitizes budding yeast to DNA damage by counteracting the DNA damage response. Following DNA damage, the Mec1-dependent DNA damage checkpoint is inactivated and Rad52 recombinase is degraded by curcumin, which results in deficiencies in double-stand break repair. Additive effects on damage-induced apoptosis and the inhibition of damage-induced autophagy by curcumin were observed. Moreover, <i>rpd3</i> mutants were found to mimic the curcumin-induced suppression of the DNA damage response. In contrast, <i>hat1</i> mutants were resistant to DNA damage, and Rad52 degradation was impaired following curcumin treatment. These results indicate that the histone deacetylase inhibitor activity of curcumin is critical to DSB repair and DNA damage sensitivity.</p></div

Rad52 protein expression was inhibited by curcumin following DNA damage.

<p><i>RAD52-HA</i> cells (YAY013) were arrested in G2 with nocodazole, and HO endonuclease was induced by the addition of galactose to generate a DSB. After 30 min, the cultures were divided equally and treated with or without 200 μM curcumin. (A) Immunoblotting analyses of Rad52 using HA antibodies at the indicated time points. (B) The recruitment of Rad52 to DSBs was analyzed by ChIP. Error bars represent the standard deviations of three independent experiments. *P<0.05. (C) Samples were processed for reverse transcription to generate cDNA and analyzed by PCR or (D) quantitative PCR. Error bars represent the standard deviations of three independent experiments. (E) The deletion of <i>SEM1</i> counteracts the disappearance of Rad52 after curcumin treatment following an HO-induced break. Immunoblotting analysis of the <i>RAD52-MYC</i> (YAY028) and <i>sem1△ RAD52-MYC</i> (RLY006) strains was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134110#pone.0134110.g003" target="_blank">Fig 3A</a>. Amido black staining of total proteins and Pgk1 protein levels serve as loading controls.</p

Curcumin sensitizes wild-type cells to the DNA-damaging drugs.

<p>Five-fold serial dilution analysis of the indicated isogenic strains, including WT (BY4741), <i>rpd3∆</i> (BY4741-rpd3), <i>hda1∆</i> (BY4741-hda1), <i>sir2∆</i> (BY4741-sir2), <i>gcn5∆</i> (BY4741-gcn5), and <i>rtt109∆</i> (BY4741-rtt109) shows sensitivity to (A) MMS, (B) 4NQO, and (C) HU with or without curcumin. The cells were allowed to grow at 30°C for 3 days and photographed to record colony formation.</p

Curcumin increases DNA damage sensitivity and inhibits DNA double-strand break repair in SSA strains.

<p>(A) A schematic diagram of the SSA system. Galactose was used to induce HO endonuclease to generate a specific HO lesion. Repair of the HO lesion at the HO cleavage site (black box) requires 5-kb or 30-kb of resection back to the uncleavable HO cleavage site (gray box). Three PCR primers were used to measure the DNA damage and repair. (B) Curcumin inhibits DNA double-strand break repair in the 5-kb resection strains. The repair of the HO lesion was analyzed by PCR. The DSB was induced by the addition of galactose to the 5-kb resection strain (YMV045). After 30 min, cultures were treated with 50 μM and 200 μM curcumin. (C) Curcumin inhibits DNA double-strand break repair in the 30-kb resection strain. PCR analysis of the 30-kb resection strain (YMV002) as described in (B). (D) Five-fold serial dilution analysis of WT-1 (YMV002), <i>rad52∆</i> (YMV037), WT-2 (YMV045), <i>srs2∆</i> (YMV057), and <i>rad52∆</i> (YMV046) shows the sensitivity to galactose and curcumin. The cells were allowed to grow at 30°C for 3 days and photographed to record colony formation.</p