Epigenetic silencing of the WNT antagonist Dickkopf 3 disrupts normal Wnt/β‐catenin signalling and apoptosis regulation in breast cancer cells

Dickkopf‐related protein 3 (DKK3) is an antagonist of Wnt ligand activity. Reduced DKK3 expression has been reported in various types of cancers, but its functions and related molecular mechanisms in breast tumorigenesis remain unclear. We examined the expression and promoter methylation of DKK3 in 10 breast cancer cell lines, 96 primary breast tumours, 43 paired surgical margin tissues and 16 normal breast tissues. DKK3 was frequently silenced in breast cell lines (5/10) by promoter methylation, compared with human normal mammary epithelial cells and tissues. DKK3 methylation was detected in 78% of breast tumour samples, whereas only rarely methylated in normal breast and surgical margin tissues, suggesting tumour‐specific methylation of DKK3 in breast cancer. Ectopic expression of DKK3 suppressed cell colony formation through inducing G0/G1 cell cycle arrest and apoptosis of breast tumour cells. DKK3 also induced changes of cell morphology, and inhibited breast tumour cell migration through reversing epithelial‐mesenchymal transition (EMT) and down‐regulating stem cell markers. DKK3 inhibited canonical Wnt/β‐catenin signalling through mediating β‐catenin translocation from nucleus to cytoplasm and membrane, along with reduced active‐β‐catenin, further activating non‐canonical JNK signalling. Thus, our findings demonstrate that DKK3 could function as a tumour suppressor through inducing apoptosis and regulating Wnt signalling during breast tumorigenesis.     

Disabled‐1 is down‐regulated in clinical breast cancer and regulates cell apoptosis through NF‐κB/Bcl‐2/caspase‐9

Disabled‐1 (Dab1) is best known as an adaptor protein regulating neuron migration and lamination during development. However, the exact function of Dab1 in breast cancer is unknown. In this study, we examined the expression of Dab1 in 38 breast cancer paraffin sections, as well as 60 paired frozen breast cancer and their adjacent tissues. Our results showed that Dab1 was reduced in breast cancer, and its compromised expression correlated with triple negative breast cancer phenotype, poor differentiation, as well as lymph node metastasis. Functional analysis in breast cancer cell lines demonstrated that Dab1 promoted cell apoptosis, which, at least partially, depended on its regulation of NF‐κB/Bcl‐2/caspase‐9 pathway. Our study strongly suggests that Dab1 may be a potential tumour suppressor gene in breast cancer.     

The 3p14.2 tumour suppressor ADAMTS9 is inactivated by promoter CpG methylation and inhibits tumour cell growth in breast cancer

Chromosome region 3p12‐14 is an important tumour suppressor gene (TSG) locus for multiple cancers. ADAMTS9, a member of the metalloprotease large family, has been identified as a candidate 3p14.2 TSG inactivated by aberrant promoter CpG methylation in several carcinomas, but little known about its expression and function in breast cancer. In this report, ADAMTS9 expression and methylation was analysed in breast cancer cell lines and tissue samples. ADAMTS9 RNA was significantly down‐regulated in breast cancer cell lines (6/8). After treating the cells with demethylation agent Aza and TSA, ADAMTS9 expression was dramatically increased. Bisulphite genomic sequencing and methylation‐specific PCR detected promoter methylation, which was associated with decreased ADAMTS9 expression. Hypermethylation was also detected in 130/219 (59.4%) of primary tumours but only in 4.5% (2/44) of paired surgical margin tissues. Ectopic expression of ADAMTS9 in tumor cells induced significant growth suppression, cell cycle arrest at the G0/G1 phase, enhanced apoptosis and reduced cell migration and invasion. Conditioned culture medium from ADAMTS9‐transfected BT549 cells markedly disrupted tube formation ability of human umbilical vein endothelial cell (HUVEC) in Matrigel. Furthermore, ADAMTS9 inhibited AKT signaling and its downstream targets (MDM2, p53, p21, p27, E‐cadherin, VIM, SNAIL, VEGFA, NFκB‐p65 and MMP2). In addition, we demonstrated, for the first time, that ADAMTS9 inhibits AKT signaling, through suppressing its upstream activators EGFR and TGFβ1/TβR(I/II) in breast cancer cells. Our results suggest that ADAMTS9 is a TSG epigenetically inactivated in breast cancer, which functions through blocking EGFR‐ and TGFβ1/TβR(I/II)‐activated AKT signaling.     

Cyclooxygenase‐2 up‐regulates CCR7 expression via AKT‐mediated phosphorylation and activation of Sp1 in breast cancer cells

Up‐regulation of cyclooxygenase‐2 (COX‐2) is frequently found in human cancers and is significantly associated with tumor metastasis. Our previous results demonstrate that COX‐2 and its metabolite prostaglandin E2 (PGE2) stimulate the expression of CCR7 chemokine receptor via EP2/EP4 receptors to promote lymphatic invasion in breast cancer cells. In this study, we address the underlying mechanism of COX‐2/PGE2‐induced CCR7 expression. We find that COX‐2/PGE2 increase CCR7 expression via the AKT signaling pathway in breast cancer cells. Promoter deletion and mutation assays identify the Sp1 site located at the −60/−57 region of CCR7 gene promoter is critical for stimulation. Chromatin immunoprecipitation (ChIP) assay confirms that in vivo binding of Sp1 to human CCR7 promoter is increased by COX‐2 and PGE2. Knockdown of Sp1 by shRNA reduces the induction of CCR7 by PGE2. We demonstrate for the first time that AKT may directly phosphorylate Sp1 at S42, T679, and S698. Phosphorylation‐mimic Sp1 protein harboring S42D, T679D, and S698D mutation strongly activates CCR7 expression. In contrast, change of these three residues to alanine completely blocks the induction of CCR7 by PGE2. Pathological investigation demonstrates that CCR7 expression is strongly associated with phospho‐AKT and Sp1 in 120 breast cancer tissues. Collectively, our results demonstrate that COX‐2 up‐regulates CCR7 expression via AKT‐mediated phosphorylation and activation of Sp1 and this pathway is highly activated in metastatic breast cancer. J. Cell. Physiol. 228: 341–348, 2013. © 2012 Wiley Periodicals, Inc.     

R5020 and RU486 act as progesterone receptor agonists to enhance Sp1/Sp4-dependent gene transcription by an indirect mechanism

It has been suggested that ligand-dependent gene activation by the progesterone receptor (PR) can result from recruitment of PR by the promoter bound Sp1. A detailed investigation of the Sp1-dependent agonistic activity of RU486 and R5020 on the folate receptor (FR) type alpha, p27, thymidine kinase 1 and p21 genes reveals a different mechanism. The FR-alpha P4 promoter and the endogenous FR-alpha gene were up-regulated by the PR agonist R5020 through either PR-A or PR-B. The classical antagonist RU486 also activated the promoter but only through PR-B. The most proximal (essential) G/C-rich (Sp1 binding) element and the initiator region constituted the minimal promoter responsive to PR regulation; substitution with a stronger cluster of G/C-rich elements enhanced the magnitude of the PR response. In contrast, substitution of the G/C-rich element with a TATA box resulted in the loss of regulation by PR. Overexpression of Sp1 and Sp4 but not Sp3 enhanced activation of the FR-alpha promoter by PR, knocking down Sp1 decreased the activation in a manner that was reversed by ectopic Sp1 or Sp4. The ligand-dependent action of PR on the promoter was delayed compared with its activation of a classical glucocorticoid response element-driven promoter and activation of both the promoter and the endogenous FR-alpha gene by PR required new protein synthesis. Activation by PR paralleled RNA polymerase II recruitment but was not accompanied by either association of PR or a change in the association of Sp1 with the endogenous FR-alpha P4 promoter. Similar observations were made for PR regulation of the genes encoding p27, thymidine kinase 1, and p21. The results contradict the current view of Sp1-dependent gene regulation by PR and point to the existence of one or more PR target genes whose promoter and cell context(s) must thus be key determinants of the agonistic activity of RU486 on a large group of important Sp1-dependent downstream target genes.     

DKK3 blocked translocation of β‐catenin/EMT induced by hypoxia and improved gemcitabine therapeutic effect in pancreatic cancer Bxpc‐3 cell

The Wnt/β‐catenin signalling pathway is activated in pancreatic cancer initiation and progression. Dickkopf‐related protein 3 (DKK3) is a member of the human Dickkopf family and an antagonist of Wnt ligand activity. However, the function of DKK3 in this pathway in pancreatic cancer is rarely known. We examined the expression of DKK3 in six human pancreatic cancer cell lines, 75 pancreatic cancer and 75 adjacent non‐cancerous tissues. Dickkopf‐related protein 3 was frequently silenced and methylation in pancreatic cancer cell lines (3/6). The expression of DKK3 was significantly lower in pancreatic cancer tissues than in adjacent normal pancreas tissues. Further, ectopic expression of DKK3 inhibits nuclear translocation of β‐catenin induced by hypoxia in pancreatic cancer Bxpc‐3 cell. The forced expression of DKK3 markedly suppressed migration and the stem cell‐like phenotype of pancreatic cancer Bxpc‐3 cell in hypoxic conditions through reversing epithelial–mesenchymal transition (EMT). The stable expression of DKK3 sensitizes pancreatic cancer Bxpc‐3 cell to gemcitabine, delays tumour growth and augments gemcitabine therapeutic effect in pancreatic cancer xenotransplantation model. Thus, we conclude from our finding that DKK3 is a tumour suppressor and improved gemcitabine therapeutic effect through inducing apoptosis and regulating β‐catenin/EMT signalling in pancreatic cancer Bxpc‐3 cell.     

Paired box 5 is a frequently methylated lung cancer tumour suppressor gene interfering β‐catenin signalling and GADD45G expression

Recent studies suggest that paired box 5 (PAX5) is down‐regulated in multiple tumours through its promoter methylation. However, the role of PAX5 in non‐small cell lung cancer (NSCLC) pathogenesis remains unclear. The aim of this study is to examine PAX5 expression, its methylation status, biological functions and related molecular mechanism in NSCLC. We found that PAX5 was widely expressed in normal adult tissues but silenced or down‐regulated in 88% (7/8) of NSCLC cell lines. PAX5 expression level was significantly lower in NSCLC than that in adjacent non‐cancerous tissues (P = 0.0201). PAX5 down‐regulation was closely associated with its promoter hypermethylation status and PAX5 expression could be restored by demethylation treatment. Frequent PAX5 promoter methylation in primary tumours (70%) was correlated with lung tumour histological types (P = 0.006). Ectopic expression of PAX5 in silenced lung cancer cell lines (A549 and H1975) inhibited their colony formation and cell viability, arrested cell cycle at G2 phase and suppressed cell migration/invasion as well as tumorigenicity in nude mice. Restoration of PAX5 expression resulted in the down‐regulation of β‐catenin and up‐regulation of tissue inhibitors of metalloproteinase 2, GADD45G in lung tumour cells. In summary, PAX5 was found to be an epigenetically inactivated tumour suppressor that inhibits NSCLC cell proliferation and metastasis, through down‐regulating the β‐catenin pathway and up‐regulating GADD45G expression.     

INPP4B restrains cell proliferation and metastasis via regulation of the PI3K/AKT/SGK pathway

Cervical cancer continues to be among the most frequent gynaecologic cancers worldwide. The phosphoinositide 3‐kinase (PI3K)/protein kinase B (AKT) pathway is constitutively activated in cervical cancer. Inositol polyphosphate 4‐phosphatase type II (INPP4B) is a phosphoinositide phosphatase and considered a negative regulatory factor of the PI3K/AKT pathway. INPP4B has diverse roles in various tumours, but its role in cervical cancer is largely unknown. In this study, we investigated the role of INPP4B in cervical cancer. Overexpression of INPP4B in HeLa, SiHa and C33a cells inhibited cell proliferation, metastasis and invasiveness in CCK‐8, colony formation, anchorage‐independent growth in soft agar and Transwell assay. INPP4B reduced the expression of some essential proteins in the PI3K/AKT/SGK3 pathway including p‐AKT, p‐SGK3, p‐mTOR, phospho‐p70S6K and PDK1. In addition, overexpression of INPP4B decreased xenograft tumour growth in nude mice. Loss of INPP4B protein expression was found in more than 60% of human cervical carcinoma samples. In conclusion, INPP4B impedes the proliferation and invasiveness of cervical cancer cells by inhibiting the activation of two downstream molecules of the PI3K pathway, AKT and SGK3. INPP4B acts as a tumour suppressor in cervical cancer cells.     

Diallyl trisulfide inhibits migration,invasion and angiogenesis of human colon cancer HT‐29 cells and umbilical vein endothelial cells,and suppresses murine xenograft tumour growth

Angiogenesis inhibitors are beneficial for the prevention and treatment of angiogenesis‐dependent diseases including cancer. We examined the cytotoxic, anti‐metastatic, anti‐cancer and anti‐angiogenic effects of diallyl trisulfide (DATS). In HT29 cells, DATS inhibited migration and invasion through the inhibition of focal adhesion kinase (FAK), extracellular signal‐regulated kinase, c‐Jun N‐terminal kinase and p38 which was associated with inhibition of matrix metalloproteinases‐2, ‐7 and ‐9 and VEGF. In human umbilical vein endothelial cells (HUVEC), DATS inhibited the migration and angiogenesis through FAK, Src and Ras. DATS also inhibited the secretion of VEGF. The capillary‐like tube structure formation and migration by HUVEC was inhibited by DATS. The chicken egg chorioallantoic membrane (CAM) assay indicated that DATS treatment inhibited ex‐vivo angiogenesis. We investigated the anti‐tumour effects of DATS against human colon cancer xenografts in BALB/c^nu/nu mice and its anti‐angiogenic activity in vivo. In this in‐vivo study, DATS also inhibited the tumour growth, tumour weight and angiogenesis (decreased the levels of haemoglobin) in HT29 cells. In conclusion, the present results suggest that the inhibition of angiogenesis may be an important mechanism in colon cancer chemotherapy by DATS.     

Insulin-like growth factor-II regulates PTEN expression in the mammary gland

The tumor suppressor PTEN is altered in many cancers, including breast cancer, but only a handful of factors are known to control its expression. PTEN plays a vital role in cell survival and proliferation by regulating Akt phosphorylation, a key component of the phosphatidylinositol 3 kinase (PI3K) pathway. Here we show that insulin-like growth factor-II (IGF-II), which signals through PI3K, regulates PTEN expression in the mammary gland. IGF-II injection into mouse mammary gland significantly increased PTEN expression. Transgenic IGF-II expression also increased mammary PTEN protein, leading to reductions in Akt phosphorylation, epithelial proliferation, and mammary morphogenesis. IGF-II induced PTEN promoter activity and protein levels and this involved the immediate early gene egr-1. Thus, we have identified a novel negative feedback loop within the PI3K pathway where IGF-II induces PTEN expression to modulate its physiologic effects.