NOP14 suppresses breast cancer progression by inhibiting NRIP1/Wnt/β-catenin pathway

NOP14, which is functionally conserved among eukaryotes, has been implicated in cancer development. Here, we show that NOP14 is poorly expressed in breast cancer cells and invasive breast cancer tissues. In vivo and in vitro studies indicated that NOP14 suppressed the tumorigenesis and metastasis of breast cancer cells. Further investigations revealed that NOP14 enhanced ERα expression and inhibited the Wnt/β-catenin pathway by up-regulating NRIP1 expression. Survival analysis indicated that low NOP14 expression was significantly associated with poor overall survival (P = 0.0006) and disease-free survival (P = 0.0007), suggesting that NOP14 is a potential prognostic factor in breast cancer. Taken together, our findings reveal that NOP14 may suppress breast cancer progression and provide new insights into the development of targeted therapeutic agents for breast cancer.     

Claudin-1 enhances tumor proliferation and metastasis by regulating cell anoikis in gastric cancer

Claudin-1 (CLDN1) is overexpressed in gastric cancer and correlated with tumor invasion, metastasis and poor outcome. Here, we both down and up regulated CLDN1 expression in gastric cancer cells to elucidate its role in gastric carcinogenesis and tumor progression. We found that deficiency of CLDN1 inhibited cells migration, invasion, and colony formation in vitro and tumorigenicity, metastasis in vivo. Also, CLDN1 promoted cell aggregation and increased anoikis resistance. Down or up regulation of CLDN1 was accompanied with changes of membrane β-catenin expression as well as Akt and Src activities. When β-catenin was up-regulated in CLDN1-KD cells, cell aggregation and anoikis resistance were restored, and Akt and Src signal pathways were re-activated. Taken together, these findings suggest that CLDN1 is oncogenic in gastric cancer and its malignant potential may be attributed in part to regulation of anoikis, by mediating membrane β-catenin-regulated cell-cell adhesion and cell survival.     

C1GALT1 overexpression promotes the invasive behavior of colon cancer cells through modifying O-glycosylation of FGFR2

Core 1 β1,3-galactosyltransferase (C1GALT1) transfers galactose (Gal) to N-acetylgalactosamine (GalNAc) to form Galβ1,3GalNAc (T antigen). Aberrant O-glycans, such as T antigen, are commonly found in colorectal cancer. However, the role of C1GALT1 in colorectal cancer remains unclear. Here we showed that C1GALT1 was frequently overexpressed in colorectal tumors and is associated with poor survival. C1GALT1 overexpression promoted cell survival, migration, invasion, and sphere formation as well as tumor growth and metastasis of colon cancer cells. Conversely, knockdown of C1GALT1 with small interference (si) RNA was sufficient to suppress these malignant phenotypes in vitro and in vivo. Moreover, we are the first to show that fibroblast growth factor receptor (FGFR) 2 carried O-glycans in colon cancer cells. Mechanistic investigations showed that C1GALT1 modified the O-glycans on FGFR2 and enhanced bFGF-triggered activation of FGFR2 as well as increased bFGF-mediated malignant phenotypes. In addition, BGJ398, a selective inhibitor of FGFR, blocked the effects of C1GALT1. These findings suggest that C1GALT1 overexpression modifies O-glycans on FGFR2 and enhances its phosphorylation to promote the invasive behavior and cancer stem-like property in colon cancer cells, indicating a critical role of O-glycosylation in the pathogenesis of colorectal cancer.     

Glycosylation of MUC6 by α1,4‐linked N‐acetylglucosamine enhances suppression of pancreatic cancer malignancy

Biomarkers for early diagnosis of pancreatic cancer are greatly needed, as the high fatality of this cancer is in part due to delayed detection. α1,4‐linked N‐acetylglucosamine (αGlcNAc), a unique O‐glycan specific to gastric gland mucus, is biosynthesized by α1,4‐N‐acetylglucosaminyltransferase (α4GnT) and primarily bound at the terminal glycosylated residue to scaffold protein MUC6. We previously reported that αGlcNAc expression decreases at early stages of neoplastic pancreatic lesions, followed by decreased MUC6 expression, although functional effects of these outcomes were unknown. Here, we ectopically expressed α4GnT, the αGlcNAc biosynthetic enzyme, together with MUC6 in the human pancreatic cancer cell lines MIA PaCa‐2 and PANC‐1, neither of which expresses α4GnT and MUC6. We observed significantly suppressed proliferation in both lines following coexpression of α4GnT and MUC6. Moreover, cellular motility decreased following MUC6 ectopic expression, an effect enhanced by cotransduction with α4GnT. MUC6 expression also attenuated invasiveness of both lines relative to controls, and this effect was also enhanced by additional α4GnT expression. We found αGlcNAc‐bound MUC6 formed a complex with trefoil factor 2. Furthermore, analysis of survival curves of patients with pancreatic ductal adenocarcinoma using a gene expression database showed that samples marked by higher A4GNT or MUC6 mRNA levels were associated with relatively favorable prognosis. These results strongly suggest that αGlcNAc and MUC6 function as tumor suppressors in pancreatic cancer and that decreased expression of both may serve as a biomarker of tumor progression to pancreatic cancer.     

Altered intracellular region of MUC1 and disrupted correlation of polarity‐related molecules in breast cancer subtypes

MUC1 glycoprotein is overexpressed and its intracellular localization altered during breast carcinoma tumorigenesis. The present study aimed to clarify the relationship of cytoplasmic localization of MUC1 with the breast cancer subtype and the correlation of 10 molecules associated with cell polarity in breast cancer subtypes. We immunostained 131 formalin-fixed and paraffin-embedded breast cancer specimens with an anti-MUC1 antibody (MUC1/CORE). For 48 of the 131 tumor specimens, laser-assisted microdissection and real-time quantitative RT-PCR were performed to analyze mRNA levels of MUC1 and 10 molecules, β-catenin, E-cadherin, claudin 3, claudin 4, claudin 7, RhoA, cdc42, Rac1, Par3 and Par6. Localization of MUC1 protein varied among breast cancer subtypes, that is, both the apical domain and cytoplasm in luminal A-like tumors (P < 0.01) and both the cytoplasm and cell membrane in luminal B-like (growth factor receptor 2 [HER2]+) tumors (P < 0.05), and no expression was found in triple negative tumors (P < 0.001). Estrogen receptor (ER)+ breast cancers showed higher MUC1 mRNA levels than ER− breast cancers (P < 0.01). The incidence of mutual correlations of expression levels between two of the 10 molecules (55 combinations) was 54.5% in normal breast tissue and 38.2% in luminal A-like specimens, 16.4% in luminal B-like (HER2+), 3.6% in HER2 and 18.2% in triple negative specimens. In conclusion, each breast cancer subtype has characteristic cytoplasmic localization patterns of MUC1 and different degrees of disrupted correlation of the expression levels between the 10 examined molecules in comparison with normal breast tissue.     

DACT1, an antagonist to Wnt/β-catenin signaling,suppresses tumor cell growth and is frequently silenced in breast cancer

Introduction

Aberrant activation of Wnt/β-catenin signaling plays an important role in the pathogenesis of breast cancer. DACT1 (Dapper/Frodo) has been identified as involved in antagonizing Wnt/β-catenin signaling through interacting with Dishevelled (Dvl), a central mediator of Wnt signaling, whereas its role in breast tumorigenesis remains unclear.

Methods

We examined DACT1 expression in breast cancer cell lines and primary tumors with semiquantitative or quantitative RT-PCR and immunochemistry, and further evaluated the promoter methylation of DACT1 with methylation-specific PCR (MSP). We also explored the tumor-suppressive functions of DACT1 in vivo and in vitro, and its related mechanism in breast cancer.

Results

We identified DACT1 as a methylated target in our breast cancer epigenome study. Here, we further investigated DACT1 expression in multiple breast cell lines and primary tumors, and further studied its function and molecular mechanisms. We found that DACT1 expression was silenced in eight (88.9%) of nine breast cancer cell lines, and its protein levels were obviously reduced in breast tumors compared with paired surgical-margin tissues. Promoter CpG methylation of DACT1 was detected in five (55.6%) of nine breast cancer cell lines and 40 (29.9%) of 134 primary tumors, but not in surgical-margin tissues and normal breast tissues. Demethylation treatment of breast cancer cell lines restored DACT1 expression along with promoter demethylation, suggesting that an epigenetic mechanism mediates DACT1 silencing in breast cancer. Functional assays showed that ectopic expression of DACT1 could inhibit breast tumor cell proliferation in vivo and in vitro through inducing apoptosis, and further suppress tumor cell migration through antagonizing the Wnt/β-catenin signaling pathway.

Conclusions

Our study demonstrates that DACT1 could function as a tumor suppressor but was frequently downregulated in breast cancer.     

Lysyl Oxidase Contributes to Mechanotransduction-Mediated Regulation of Transforming Growth Factor-β Signaling in Breast Cancer Cells

Transforming growth factor-β (TGF-β) regulates all stages of mammary gland development, including the maintenance of tissue homeostasis and the suppression of tumorigenesis in mammary epithelial cells (MECs). Interestingly, mammary tumorigenesis converts TGF-β from a tumor suppressor to a tumor promoter through molecular mechanisms that remain incompletely understood. Changes in integrin signaling and tissue compliance promote the acquisition of malignant phenotypes in MECs in part through the activity of lysyl oxidase (LOX), which regulates desmoplastic reactions and metastasis. TGF-β also regulates the activities of tumor reactive stroma and MEC metastasis. We show here that TGF-β1 stimulated the synthesis and secretion of LOX from normal and malignant MECs in vitro and in mammary tumors produced in mice. The ability of TGF-β1 to activate Smad2/3 was unaffected by LOX inactivation in normal MECs, whereas the stimulation of p38 MAPK by TGF-β1 was blunted by inhibiting LOX activity in malignant MECs or by inducing the degradation of hydrogen peroxide in both cell types. Inactivating LOX activity impaired TGF-β1-mediated epithelial-mesenchymal transition and invasion in breast cancer cells. We further show that increasing extracellular matrix rigidity by the addition of type I collagen to three-dimensional organotypic cultures promoted the proliferation of malignant MECs, a cellular reaction that was abrogated by inhibiting the activities of TGF-β1 or LOX, and by degrading hydrogen peroxide. Our findings identify LOX as a potential mediator that couples mechanotransduction to oncogenic signaling by TGF-β1 and suggest that measures capable of inactivating LOX function may prove effective in diminishing breast cancer progression stimulated by TGF-β1.     

Dietary compound isoliquiritigenin prevents mammary carcinogenesis by inhibiting breast cancer stem cells through WIF1 demethylation

Breast cancer stem cells (CSCs) are considered as the root of mammary tumorigenesis. Previous studies have demonstrated that ISL efficiently limited the activities of breast CSCs. However, the cancer prevention activities of ISL and its precise molecular mechanisms remain largely unknown. Here, we report a novel function of ISL as a natural demethylation agent targeting WIF1 to prevent breast cancer. ISL administration suppressed in vivo breast cancer initiation and progression, accompanied by reduced CSC-like populations. A global gene expression profile assay further identified WIF1 as the main response gene of ISL treatment, accompanied by the simultaneous downregulation of β-catenin signaling and G0/G1 phase arrest in breast CSCs. In addition, WIF1 inhibition significantly relieved the CSC-limiting effects of ISL and methylation analysis further revealed that ISL enhanced WIF1 gene expression via promoting the demethylation of its promoter, which was closely correlated with the inhibition of DNMT1 methyltransferase. Molecular docking analysis finally revealed that ISL could stably dock into the catalytic domain of DNMT1. Taken together, our findings not only provide preclinical evidence to demonstrate the use of ISL as a dietary supplement to inhibit mammary carcinogenesis but also shed novel light on WIF1 as an epigenetic target for breast cancer prevention.     

Wnt signaling through Snail1 and Zeb1 regulates bone metastasis in lung cancer

Wnt-β-catenin signaling participates in the epithelial-mesenchymal transition (EMT) in a variety of cancers; however, its role in lung cancer induced bone metastasis and the underlying mechanisms remain unclear. Here, we demonstrate that β-catenin, Snail1 and Zeb1 were significantly upregulated in bone metastasis tissues from human and mouse compared with the normal controls. E-cadherin expression is negatively regulated by Zeb1, Snail1 and β-catenin during bone metastasis tissues induced by lung cancer. Knocking down Zeb1 and Snail1 in lung cancer cell lines showed increased E-cadherin mRNA expression and less invasion compared with the original cell lines. In addition, β-catenin knockdown led to the increase of E-cadherin and the decrease of Zeb1 and Snail1, which in turn inhibited the invasive properties of lung cancer. Our results demonstrated that Wnt signaling through Snail1 and Zeb1 regulates bone metastasis in lung cancer.     

SIRT1 in B[a]P-induced lung tumorigenesis

Benzo[a]pyrene (B[a]P) is a carcinogen in cigarette smoke. We found that B[a]P induced SIRT1 in human bronchial epithelial BEAS-2B cell. SIRT1 was overexpressed in the lung of B[a]P-exposed mice and in human lung cancer biopsies. SIRT1 up-regulated TNF-α and β-catenin and down-regulated the membrane fraction of E-cadherin. In addition, SIRT1 promoted invasion, migration and tumorigenesis of BEAS-2B cells in nude mice upon B[a]P exposure. Thus, SIRT1 is involved in B[a]P-induced transformation associated with activation of the TNF-α/β-catenin axis and is as a potential therapeutic target for lung cancer.     

Cancer-associated fibroblast-secreted collagen triple helix repeat containing-1 promotes breast cancer cell migration,invasiveness and epithelial-mesenchymal transition by activating the Wnt/β-catenin pathway

Cancer-associated fibroblasts (CAFs) are continuously activated and are one of the most important cellular components of the tumor matrix. The role of CAFs in the tumor microenvironment has been widely recognized. However, the underlying molecular mechanism by which CAFs promote tumor characteristics in breast cancer (BC) remains poorly understood. The aim of the present study was to investigate the potential mechanisms and the possible pathways of collagen triple helix repeat containing-1 (CTHRC1) in the epithelial-mesenchymal transition (EMT) of BC cells. The level of CTHRC1 in BC tissues was found to be higher than that in adjacent-normal tissues. CAFs isolated from BC tissues secreted significantly greater amounts of CTHRC1 than normal fibroblasts. Furthermore, CAFs promoted the migration, invasiveness and EMT of BC cells by secreting CTHRC1, which activates the Wnt/β-catenin signaling pathway. However, the use of neutralizing antibodies towards CTHRC1, or the specific inhibitor Dickkopf-1, to inhibit the Wnt/β catenin pathway significantly alleviated the CAF-induced malignant phenotypes of BC cells. Collectively, the data indicate that CAFs in the tumor microenvironment promote BC cell malignant behaviors via the CTHRC1/Wnt/β-catenin signaling pathway. Furthermore, weakening CAF-BC cell communication by suppressing CTHRC1 expression may be a novel strategy for treating BC.     

Adiponectin promotes pancreatic cancer progression by inhibiting apoptosis via the activation of AMPK/Sirt1/PGC-1α signaling

Adiponectin is an adipocyte-secreted adipokine with pleiotropic actions. Clinical evidence has shown that serum adiponectin levels are increased and that adiponectin can protect pancreatic beta cells against apoptosis, which suggests that adiponectin may play an anti-apoptotic role in pancreatic cancer (PC). Here, we investigated the effects of adiponectin on PC development and elucidated the underlying molecular mechanisms. Adiponectin deficiency markedly attenuated pancreatic tumorigenesis in vivo. We found that adiponectin significantly inhibited the apoptosis of both human and mouse pancreatic cancer cells via adipoR1, but not adipoR2. Furthermore, adiponectin can increase AMP-activated protein kinase (AMPK) phosphorylation and NAD-dependent deacetylase sirtuin-1 (Sirt1) of PC cells. Knockdown of AMPK or Sirt1 can increase the apoptosis in PC cells. AMPK up-regulated Sirt1, and Sirt1 can inversely phosphorylate AMPK. Further studies have shown that Sirt1 can deacetylate peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), which can increase the expression levels of mitochondrial genes. Thus, adiponectin exerts potent anti-apoptotic effects on PC cells via the activation of AMPK/Sirt1/PGC1α signaling. Finally, adiponectin can elevate β-catenin levels. Taken together, these novel findings reveal an unconventional role of adiponectin in promoting pancreatic cancers, and suggest that the effects of adiponectin on tumorigenesis are highly tissue-dependent.     

β-catenin SUMOylation is involved in the dysregulated proliferation of myeloma cells

Over-activation of SUMOylation is correlated with poor prognosis in multiple myeloma (MM), with the mechanism unclear. Wnt signaling is one of the aberrantly regulated pathways related to cancer tumorigenesis and progression. Whether SUMOylation is involved in regulating the activity of Wnt/β-catenin pathway, however, has not been reported in MM. Here we found that the TOPflash reporter activity and the expression of Wnt/β-catenin target genes can be down-regulated after interference with SUMOylation through SUMO-1 small interfering RNA (siRNA). SUMOylation inhibition down-regulated β-catenin at protein level via promotion of ubiquitin-proteasomal mediated degradation. Furthermore, over-expression of β-catenin rescued Wnt/β-catenin pathway activity and partially prevented increased apoptosis and growth inhibition induced by SUMOylation inhibition, indicating that β-catenin was responsible for the observed effect on Wnt/β-catenin pathway. To gain a clearer view, we exploited the inter-protein interactions of β-catenin and SUMO-1 in myeloma cell lines. Immunoprecipitation and immunofluorescence assay proved that β-catenin is subjected to SUMOylation in vivo, which may, at least partially explain the impact of SUMOylation inhibition on β-catenin. The association of SUMO-1 and β-catenin was confirmed in myeloma patient samples. Taken together, our data proved that SUMOylation inhibition down-regulates Wnt/β-catenin pathway by promoting the ubiquitin-proteasomal mediated degradation of β-catenin. SUMOylation of β-catenin is part of the mechanisms involved in the dysregulated proliferation of myeloma cells.