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-ß1-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-ß1, 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-ß1 by 2- to 3-fold and thereby dampening TGF-ß1 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-ß1 is known to transcriptionally activate ST3Gal1, our findings illustrated a feedback regulatory loop in which TGF-ß1 upregulates ST3Gal1 to circumvent the negative impact of VASN.

Effective suppression of tumor growth and hepatic metastasis of neuroblastoma by NKT-stimulatory phenyl glycolipid.

Invariant natural killer T cell (iNKT) cells produce large amounts of cytokines in response to α-Galactosylceramide (α-GalCer) stimulation. An analog containing two phenyl rings on the acyl chain, C34, was previously found to be more Th1-biased than α-GalCer and triggered greater anticancer activities against breast cancer, melanoma and lung cancer in mice. Since liver is enriched in iNKT cells, we investigated anticancer efficacy of C34 on neuroblastoma with hepatic metastasis. C34 induced Th1-biased cytokine secretions in the liver, significantly suppressed neuroblastoma growth/metastasis and prolonged mouse survival. The anti-tumor efficacy might be attributed to greater expansions of hepatic NKT, NK, CD4+ T, and CD8+ T cells as well as reduction of the number of SSCloGr1intCD11b+ subset of myeloid-derived suppressor cells (MDSCs) in the liver of tumor-bearing mice, as compared to DMSO control group. C34 also upregulated expression of CD1d and CD11c, especially in the SSCloGr1intCD11b+ subset of MDSCs, which might be killed by C34-activated NKT cells, attributing to their reduced number. In addition, C34 also induced expansion of CD4+ T, CD8+ T, and NK cells, which might eliminate neuroblastoma cells. These immune-modulating effects of C34 might act in concert in the local milieu of liver to suppress the tumor growth. Further analysis of database of neuroblastoma revealed that patients with high CD11c expression in the monocytic MDSCs in the tumor had longer survival, suggesting the potential clinical application of C34 for treatment of neuroblastoma.

O-Acetyl-GD2 as a Therapeutic Target for Breast Cancer Stem Cells.

Synopsis: A sugar-lipid molecule called OAcGD2 is a novel marker for breast cancer stem cells. Treatment with anti-OAcGD2 mAb8B6 may have superior anticancer efficacy by targeting cancer stem cells, thereby reducing metastasis and recurrence of cancer. Background: Cancer stem cells (CSCs) that drive tumor progression and disease recurrence are rare subsets of tumor cells. CSCs are relatively resistant to conventional chemotherapy and radiotherapy. Eradication of CSCs is thus essential to achieve durable responses. GD2 was reported to be a CSC marker in human triple-negative breast cancer, and anti-GD2 immunotherapy showed reduced tumor growth in cell lines. Using a specific anti-OAcGD2 antibody, mAb8D6, we set out to determine whether OAcGD2+ cells exhibit stem cell properties and mAb8D6 can inhibit tumor growth by targeting OAcGD2+CSCs. Method: OAcGD2 expression in patient-derived xenografts (PDXs) of breast cancer was determined by flow cytometric analyses using mAb8D6. The stemness of OAcGD2+ cells isolated by sorting and the effects of mAb8B6 were assessed by CSC growth and mammosphere formation in vitro and tumor growth in vivo using PDX models. Result: We found that the OAcGD2 expression levels in six PDXs of various molecular subtypes of breast cancer highly correlated with their previously defined CSC markers in these PDXs. The sorted OAcGD2+ cells displayed a greater capacity for mammosphere formation in vitro and tumor initiation in vivo than OAcGD2- cells. In addition, the majority of OAcGD2+ cells were aldehyde dehydrogenase (ALDH+) or CD44hiCD24lo, the known CSC markers in breast cancer. Treatment of PDXs-bearing mice with mAb8B6, but not doxorubicin, suppressed the tumor growth, along with reduced CSCs as assessed by CSC markers and in vivo tumorigenicity. In vitro, mAb8B6 suppressed proliferation and mammosphere formation and induced apoptosis of OAcGD2+ breast cancer cells harvested from PDXs, in a dose-dependent manner. Finally, administration of mAb8B6 in vivo dramatically suppressed tumor growth of OAcGD2+ breast CSCs (BCSCs) with complete tumor abrogation in 3/6 mice. Conclusion: OAcGD2 is a novel marker for CSC in various subtypes of breast cancer. Anti-OAcGD2 mAb8B6 directly eradicated OAcGD2+ cells and reduced tumor growth in PDX model. Our data demonstrate the potential of mAb8B6 as a promising immunotherapeutic agent to target BCSCs.

Transmembrane and coiled-coil domain family 3 (TMCC3) regulates breast cancer stem cell and AKT activation.

Cancer stem cells (CSC) play a pivotal role in cancer metastasis and resistance to therapy. Previously, we compared the phosphoproteomes of breast cancer stem cells (BCSCs) enriched subpopulation and non-BCSCs sorted from breast cancer patient-derived xenograft (PDX), and identified a function unknown protein, transmembrane and coiled-coil domain family 3 (TMCC3) to be a potential enrichment marker for BCSCs. We demonstrated greater expression of TMCC3 in BCSCs than non-BCSCs and higher expression of TMCC3 in metastatic lymph nodes and lungs than in primary tumor of breast cancer PDXs. TMCC3 silencing suppressed mammosphere formation, ALDH activity and cell migration in vitro, along with reduced tumorigenicity and metastasis in vivo. Mechanistically, we found that AKT activation was reduced by TMCC3 silencing, but enhanced by TMCC3 overexpression. We further demonstrated that TMCC3 interacted directly with AKT through its 1-153 a.a. domain by cell-free biochemical assay in vitro and co-immunoprecipitation and interaction domain mapping assays in vivo. Based on domain truncation studies, we showed that the AKT-interacting domain of TMCC3 was essential for TMCC3-induced AKT activation, self-renewal, and metastasis. Clinically, TMCC3 mRNA expression in 202 breast cancer specimens as determined by qRT-PCR assay showed that higher TMCC3 expression correlated with poorer clinical outcome of breast cancer, including early-stage breast cancer. Multivariable analysis identified TMCC3 expression as an independent risk factor for survival. These findings suggest that TMCC3 is crucial for maintenance of BCSCs features through AKT regulation, and TMCC3 expression has independent prognostic significance in breast cancer. Thus, TMCC3 may serve as a new target for therapy directed against CSCs.

Design and synthesis of glyco-peptides as anti-cancer agents targeting thrombin-protease activated receptor-1 interaction.

Thrombin activates protease-activated receptor-1 (PAR-1) through binding to exosite I and the active site to promote tumor growth. We have developed a new class of anti-cancer glyco-peptides to target exosite I selectively without affecting the active-site-mediated coagulation activity and showed the importance of glycans for the stability and anti-cancer activity of the glyco-peptides.

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.

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

BACKGROUND: The 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. METHODS: Interaction 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. FINDINGS: We 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. INTERPRETATION: These 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).

Phenyl Glycolipids with Different Glycosyl Groups Exhibit Marked Differences in Murine and Human iNKT Cell Activation.

Glycosphingolipids (GSLs) bearing the α-galactosyl headgroup and the acyl chain terminated with a phenyl derivative were found to be more potent than α-galactosyl ceramide (αGalCer) to stimulate both murine and human invariant natural killer T (iNKT) cells and to induce an antibody isotope switch to IgG. In this study, we replaced the galactosyl group with glucose (αGlc) and its fluoro-analogs and found that phenyl GSLs with αGlc (C34-Glc) and its fluoro-analog 6F-C34-Glc were stronger than those with αGal in stimulating human iNKT cells but weaker in mice. Their activities have a strong correlation with the binding avidities of the ternary interaction between the iNKT-cell receptor (iNKTCR) and CD1d-GSL complex. It was the iNKTCR rather than CD1d that dictated the species-specific responses. C34-Glc was further used as an adjuvant for a SSEA4-crm-197 vaccine, and after immunization in mice, the vaccine was highly effective against Lewis lung carcinoma.

Antioxidant N-acetylcysteine blocks nerve growth factor-induced H2O2/ERK signaling in PC12 cells.

We investigated whether H2O2, superoxide, and ERK participate in nerve growth factor (NGF)-induced signaling cascades and whether antioxidant N-acetylcysteine (NAC) regulates these NGF-induced responses. PC12 cells were cultured in medium containing NGF or vehicle with or without NAC pretreatment, and the intracellular H2O2 and superoxide levels and the amount of phosphorylated ERK were evaluated by flow cytometry and Western blotting, respectively. We found that NGF increased intracellular H2O2 concentration and activated ERK but failed to affect intracellular superoxide level. Moreover, NAC counteracted these NGF-induced responses. These findings demonstrate that NAC blocks the NGF-induced H2O2/ERK signaling in PC12 cells.

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

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, rpd3 mutants were found to mimic the curcumin-induced suppression of the DNA damage response. In contrast, hat1 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.

Targeting of Topoisomerase I for Prognoses and Therapeutics of Camptothecin-Resistant Ovarian Cancer.

DNA topoisomerase I (TOP1) levels of several human neoplasms are higher than those of normal tissues. TOP1 inhibitors are widely used in treating conventional therapy-resistant ovarian cancers. However, patients may develop resistance to TOP1 inhibitors, hampering chemotherapy success. In this study, we examined the mechanisms associated with the development of camptothecin (CPT) resistance in ovarian cancers and identified evodiamine (EVO), a natural product with TOP1 inhibiting activity that overcomes the resistance. The correlations among TOP1 levels, cancer staging, and overall survival (OS) were analyzed. The effect of EVO on CPT-resistant ovarian cancer was evaluated in vitro and in vivo. TOP1 was associated with poor prognosis in ovarian cancers (p = 0.024). EVO induced apoptosis that was detected using flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The tumor size decreased significantly in the EVO treatment group compared with the control group (p < 0.01) in a xenograft mouse model. Effects of drugs targeting TOP1 for prognosis and therapy in CPT-resistant ovarian cancer are anticipated. EVO with TOP1 can be developed as an antiproliferative agent for overcoming CPT resistance in ovarian cancers.

Globo-H ceramide shed from cancer cells triggers translin-associated factor X-dependent angiogenesis.

Tumor angiogenesis is a critical element of cancer progression, and strategies for its selective blockade are still sought. Here, we examine the angiogenic effects of Globo-H ceramide (GHCer), the most prevalent glycolipid in a majority of epithelial cancers and one that acts as an immune checkpoint. Here, we report that GHCer becomes incorporated into endothelial cells through the absorption of microvesicles shed from tumor cells. In endothelial cells, GHCer addition induces migration, tube formation, and intracellular Ca(2+) mobilization in vitro and angiogenesis in vivo. Breast cancer cells expressing high levels of GHCer displayed relatively greater tumorigenicity and angiogenesis compared with cells expressing low levels of Globo-H. Clincally, GHCer(+) breast cancer specimens contained higher vessel density than GHCer(-) breast cancer specimens. Mechanistic investigations linked the angiogenic effects of GHCer to its endocytosis and binding to TRAX, with consequent release of PLCß1 from TRAX to trigger Ca(2+) mobilization. Together, our findings highlight the importance of GHC as a target for cancer therapy by providing new information on its key role in tumor angiogenesis.

JNK signaling pathway is required for bFGF-mediated surface cadherin downregulation on HUVEC.

Angiogenesis, the process of new blood vessel formation, is important in wound healing, inflammation, tumorigenesis and metastases. During this process, it is a critical step of the loosening of cellular interactions between endothelial cells, which are dependent on the architecture of adherens junction constructed by homophilic interactions of cell surface cadherins. Several studies suggested that the dynamic changes of cadherins are necessary during angiogenesis. However, the mechanism of cadherins regulation on endothelial cells requires further delineation. Here, we showed that basic fibroblast growth factor (bFGF), a pivotal pro-angiogenic factor, can downregulate typical cadherins (E-, N-, P- and VE-cadherin) expression on the surface of human umbilical vein endothelial cells (HUVECs) via FGF receptor 1 (FGFR1) signaling. The bFGF-mediated surface cadherin downregulation was significantly reversed only when the HUVECs were treated with JNK inhibitor (SP600125), but not ERK (PD98059) or p38 inhibitor (SB203580). Infecting HUVECs with a dominant negative H-Ras mutant (Ras(S17N)) interferes bFGF-mediated cadherin downregulation, and the result suggests that bFGF attenuates surface cadherin expression on HUVECs via FGFR1 and intracellular Ras-JNK signaling. However, after growth factors withdrawal, FGFR1 blockade or JNK inhibition for 16 h, cadherins were re-expressed on cell surface of HUVECs. But the mRNA or total protein of cadherins had no significant change, suggesting that the effect of bFGF on cadherin expression may work through a post-translational control. Our data first suggest that JNK participates in bFGF-mediated surface cadherin downregulation. Loss of surface cadherins may affect the cell-cell interaction between endothelial cells and facilitate angiogenesis.

Protective role of programmed death 1 ligand 1 (PD-L1)in nonobese diabetic mice: the paradox in transgenic models.

OBJECTIVE: Coinhibitory signals mediated via programmed death 1 (PD-1) receptor play a critical role in downregulating immune responses and in maintaining peripheral tolerance. Programmed death 1 ligand 1 (PD-L1), the interacting ligand for PD-1, widely expressed in many cell types, acts as a tissue-specific negative regulator of pathogenic T-cell responses. We investigated the protective potential of PD-L1 on autoimmune diabetes by transgenically overexpressing PD-L1 in pancreatic beta-cells in nonobese diabetic (NOD) mice. RESEARCH DESIGN AND METHODS: We established an insulin promoter-driven murine PD-L1 transgenic NOD mouse model to directly evaluate the protective effect of an organ-specific PD-L1 transgene against autoimmune diabetes. Transgene expression, insulitis, and diabetic incidence were characterized in these transgenic NOD mice. Lymphocyte development, Th1 cells, and regulatory T-cells were analyzed in these transgenic mice; and T-cell proliferation, adoptive transfer, and islet transplantation were performed to evaluate the PD-L1 transgene-mediated immune-protective mechanisms. RESULTS: The severity of insulitis in these transgenic mice is significantly decreased, disease onset is delayed, and the incidence of diabetes is markedly decreased compared with littermate controls. NOD/SCID mice that received lymphocytes from transgenic mice became diabetic at a slower rate than mice receiving control lymphocytes. Moreover, lymphocytes collected from recipients transferred by lymphocytes from transgenic mice revealed less proliferative potential than lymphocytes obtained from control recipients. Transgenic islets transplanted in diabetic recipients survived moderately longer than control islets. CONCLUSIONS: Our results demonstrate the protective potential of transgenic PD-L1 in autoimmune diabetes and illustrate its role in downregulating diabetogenic T-cells in NOD mice.

The protective effect of adenosine triphosphate-MgCl2 on ischemia-reperfusion lung injury is leukocyte dependent.

Adenosine triphosphate (ATP)-MgCl(2) attenuates ischemia-reperfusion (I-R)-induced lung injury in rats. A previous study indirectly suggests that Mg(2+)-dependent ecto-ATPases on the surface of leukocytes are responsible for the hydrolysis of ATP-MgCl(2) to adenosine, which then contributes to the protective effect of ATP-MgCl(2). This study investigated the role of leukocytes in I-R injury and the protective effect of ATP-MgCl(2) in our buffer-perfused isolated rat lung model. After isolating the lung blood flow of adult male Sprague-Dawley rats, the lungs were perfused through the pulmonary artery cannula with a physiologic salt solution containing human serum albumin. The protective effect of ATP-MgCl(2) pretreatment with or without leukocytes was investigated. Capillary permeability (K(fc)), lung weight gain (LWG), lung wet weight/body weight ratio (LW/BW), lung lavage protein concentration (LPC) and pulmonary artery pressure (PAP) were measured. I-R produced a significant increase in K(fc), LWG, LW/BW, LPC, and PAP. The increases in these indices were significantly attenuated by pretreatment with ATP-MgCl(2) (1 x 10(-6)M) together with leukocytes (2.9 x 10(6)/ml in the perfusate) but not with ATP-MgCl(2) alone. Our data suggest that I-R-induced acute lung injury is not dependent on circulating leukocytes. Pretreatment with ATP-MgCl(2) plus leukocytes but not ATP-MgCl(2) alone had protective effects against I-R lung injury. Whether these findings occur in vivo could not be determined in this study. In our isolated lung red blood cell-free perfusate system, the protective effect of ATP-MgCl(2) requires the presence of leukocytes.