15-hydroxyprostaglandin dehydrogenase is a tumor suppressor of human breast cancer

Prostaglandin E(2) plays a growth-stimulatory role in breast cancer, and the rate-limiting enzyme in its synthesis, cyclooxygenase-2, is often overexpressed in these cancers. Little is known about the role of the key prostaglandin catabolic enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH) in breast cancer pathogenesis. Using a pharmacologically based screen for epigenetically silenced genes, we found low levels of 15-PGDH in MDA-MB-231 cells [estrogen receptor (ER) negative] but high levels in MCF-7 cells (ER positive) and observed its up-regulation following demethylation treatment. Further analysis revealed methylation of the 15-PGDH promoter in one breast cancer cell line and 30% of primary tumors. Analysis of 15-PGDH expression revealed low levels in 40% of primary breast tumors and identified a correlation between 15-PGDH and ER expression. Transfection assays showed that transient up-regulation of 15-PGDH levels in MDA-MB-231 cells resulted in a decreased clonal growth, and stable up-regulation significantly decreased the ability of these cells to form tumors in athymic mice. In contrast, transient silencing of 15-PGDH in MCF-7 cells resulted in their enhanced proliferation, and a stable silencing in these cells enhanced cell cycle entry in vitro and tumorigenicity in vivo. Forced expression of 15-PGDH inhibited the ER pathway and silencing of 15-PGDH up-regulated expression of aromatase. In addition, 15-PGDH levels were down-regulated by estrogen but up-regulated by the tumor suppressor gene CAAT/enhancer binding protein alpha. Our results indicate for the first time that 15-PGDH may be a novel tumor suppressor gene in breast cancer, and suggest that this enzyme can modulate the ER pathway.     

Epigenetic silencing of the tumor suppressor klotho in human breast cancer

Klotho is a single pass transmembrane protein, associated with premature aging. We identified tumor suppressor activities for klotho, associated with reduced expression in breast cancer. We now aimed to analyze klotho expression in early stages of breast tumorigenesis and elucidate mechanisms leading to klotho silencing in breast tumors. We studied klotho expression, using immunohistochemistry, and found high klotho expression in all normal and mild hyperplasia samples, whereas reduced expression was associated with moderate and atypical ductal hyperplasia. Promoter methylation and histone deacetylation were studied as possible mechanisms for klotho silencing. Using bisulfite sequencing, and methylation-specific PCR, we identified KLOTHO promoter methylation in five breast cancer cell lines and in hyperplastic MCF-12A cells, but not in the non-tumorous mammary cell line HB2. Importantly, methylation status inversely correlated with klotho mRNA levels, and treatment of breast caner cells with 5-aza-2-deoxycytidine elevated klotho expression by up to 150-fold. KLOTHO promoter methylation was detected in 8/23 of breast cancer samples but not in normal breast samples. Chromatin immunoprecipitation revealed that in HB2 KLOTHO promoter was enriched with AcH3K9; however, in breast cancer cells, H3K9 was deacetylated, and treatment with the histone deacetylase inhibitor suberoylanilide bishydroxamide (SAHA) restored H3K9 acetylation. Taken together, these data indicate loss of klotho expression as an early event in breast cancer development, and suggest a role for DNA methylation and histone deacetylation in klotho silencing. Klotho expression and methylation may, therefore, serve as early markers for breast tumorigenesis.     

SHP-1 acts as a tumor suppressor by interacting with EGFR and predicts the prognosis of human breast cancer

Objective:The aims of this study were to examine the prognostic value of SHP-1 in breast cancer, its roles in the regulation of breast cancer cell growth and metastasis, and the underlying mechanisms.Methods:Tumor specimens from 160 patients with breast cancer and 160 noncancerous tissues were used to examine the expression of SHP-1 and to analyze its association with overall survival through Kaplan–Meier and multivariate Cox regression analyses. RNA sequencing data and the expression and clinical importance of SHP-1 in breast cancer were evaluated with data from The Cancer Genome Atlas. In vitro and in vivo assays were performed to elucidate the effects of SHP-1 on breast cancer cell proliferation and invasion. Confocal immunofluorescence and GST pulldown assays were used to demonstrate the interaction between SHP-1 and epidermal growth factor receptor, as well as its downstream pathways. Immunohistochemistry and The Cancer Genome Atlas database were used to investigate the clinical association between SHP-1 and EGFR in human breast cancer.Results:SHP-1 expression was associated with better survival in patients with breast cancer, whereas SHP-1 expression was negatively correlated with EGFR in human breast cancer. Ectopic SHP-1 expression significantly suppressed breast cancer cell proliferation, migration, and invasion. SHP-1 knockdown induced a more invasive phenotype and accelerated cell growth. Mechanistically, EGFR, a protein directly interacting with SHP-1, mediates the SHP-1-induced inactivation of Ras/Erk/GSK3β signaling and its downstream effectors.Conclusions:SHP-1 is an important prognostic biomarker in patients with breast cancer, and the SHP-1-EGFR axis is a promising target for treatment.     

Altered expression of the IGF-1 receptor in a tamoxifen-resistant human breast cancer cell line

The relationship between oestrogen (E2) and insulin-like growth factor-one (IGF-1) was examined in both tamoxifen-sensitive (MCF 7/5-21) and tamoxifen-resistant (MCF 7/5-23) subclones of the MCF 7 cell line. Both subclones were grown in defined, serum-free (SF) medium over a period of 7 days with the addition of E2 or IGF-1 or a combination of both agents. Growth of both MCF 7/5-21 and 7/5-23 cells was stimulated (245% and 350%, respectively) by E2. However, only the growth of MCF 7/5-23 cells was stimulated (266%) by IGF-1. A combination of E2 and IGF-1 significantly enhanced MCF 7/5-21 and 7/5-23 cell growth (581% and 695%, respectively). E2-induced IGF-1 receptor (IGF-1R) levels (as measured by 125I-IGF-1 binding and Northern analyses) in only MCF 7/5-23 cells. This effect was partially inhibited by tamoxifen. In medium containing serum, the growth of only the MCF 7/5-23 cells was significantly inhibited by the IGF-1R monoclonal antibody, alphaIR-3. The detection of E2-induced expression of IGF-2 using RT-PCR was demonstrated in the MCF 7/5-23 cells. These experiments indicate that E2 may sensitize tamoxifen-resistant MCF 7/5-23 cells to the growth stimulatory actions of IGF-2 via up-regulation of the IGF-1R and describes a cell-survival mechanism that may manifest itself as tamoxifen resistance.     

Signalling pathways in UHRF1-dependent regulation of tumor suppressor genes in cancer

Epigenetic silencing of tumor suppressor genes (TSGs) through DNA methylation and histone changes is a main hallmark of cancer. Ubiquitin-like with PHD and RING Finger domains 1 (UHRF1) is a potent oncogene overexpressed in various solid and haematological tumors and its high expression levels are associated with decreased expression of several TSGs including p16 ^INK4A , BRCA1, PPARG and KiSS1. Using its several functional domains, UHRF1 creates a strong coordinated dialogue between DNA methylation and histone post-translation modification changes causing the epigenetic silencing of TSGs which allows cancer cells to escape apoptosis. To ensure the silencing of TSGs during cell division, UHRF1 recruits several enzymes including histone deacetylase 1 (HDAC1), DNA methyltransferase 1 (DNMT1) and histone lysine methyltransferases G9a and Suv39H1 to the right place at the right moment. Several in vitro and in vivo works have reported the direct implication of the epigenetic player UHRF1 in tumorigenesis through the repression of TSGs expression and suggested UHRF1 as a promising target for cancer treatment. This review describes the molecular mechanisms underlying UHRF1 regulation in cancer and discusses its importance as a therapeutic target to induce the reactivation of TSGs and subsequent apoptosis.     

Testin is a tumor suppressor and prognostic marker in breast cancer

The testin (TES) gene was previously identified in the fragile chromosomal region FRA7G at 7q31.2. In the present study, we aimed to investigate the candidate tumor suppressor function of TES and explore its correlations to clinicopathologic features and prognosis in breast cancer. In clinical samples, we showed that the expression of TES decreased gradually from normal through ductal hyperplasia without atypia, atypical ductal hyperplasia, and ductal carcinoma in situ, to invasive ductal carcinoma. To explore the possible tumor suppressing function of TES, the expression of TES in breast cancer cells was manipulated by ectopic expression or by RNAi. We revealed that ectopic TES expression significantly inhibited cell proliferation, invasive ability, and angiogenesis, whereas knockdown of TES by RNAi enhanced cell proliferation, invasive ability, and angiogenesis. In an animal model, TES markedly inhibited breast cancer cell xenograft formation in athymic nude mice and reduced breast cancer cell metastasis to lung. Moreover, we revealed that TES inhibited the invasion and angiogenesis of breast cancer partially through miR‐29b‐mediated MMP‐2 inhibition. Using the tissue microarray of breast cancer from Yale University, we found that lower TES expression was an independent prognostic factor for shorter overall survival and disease‐free survival with univariate and multivariate analyses. Taken together, these data suggest that TES, as a valuable marker of breast cancer prognosis, plays an important role in the development and progression of breast cancer. TES may be an effective novel target in breast cancer prevention and treatment.     

Light at night activates IGF-1R/PDK1 signaling and accelerates tumor growth in human breast cancer xenografts

Regulation of diurnal and circadian rhythms and cell proliferation are coupled in all mammals, including humans. However, the molecular mechanisms by which diurnal and circadian rhythms regulate cell proliferation are relatively poorly understood. In this study, we report that tumor growth in nude rats bearing human steroid receptor-negative MCF-7 breast tumors can be significantly accelerated by exposing the rats to light at night (LAN). Under normal conditions of an alternating light/dark cycle, proliferating cell nuclear antigen (PCNA) levels in tumors were maximal in the early light phase but remained at very low levels throughout the daily 24-hour cycle period monitored. Surprisingly, PCNA was expressed in tumors continually at a high level throughout the entire 24-hour period in LAN-exposed nude rats. Daily fluctuations of Akt and mitogen activated protein kinase activation in tumors were also disrupted by LAN. These fluctuations did not track with PCNA changes, but we found that activation of the Akt stimulatory kinase phosphoinositide-dependent protein kinase 1 (PDK1) directly correlated with PCNA levels. Expression of insulin-like growth factor 1 receptor (IGF-1R), an upstream signaling molecule for PDK1, also correlated with fluctuations of PDK1/PCNA in the LAN group. In addition, circulating IGF-1 concentrations were elevated in LAN-exposed tumor-bearing nude rats. Finally, RNAi-mediated knockdown of PDK1 led to a reduction in PCNA expression and cell proliferation in vitro and tumor growth in vivo, indicating that PDK1 regulates breast cancer growth in a manner correlated with PCNA expression. Taken together, our findings demonstrate that LAN exposure can accelerate tumor growth in vivo, in part through continuous activation of IGF-1R/PDK1 signaling.     

乳腺癌转移抑制基因1抑制肿瘤转移的作用机制

乳腺癌转移抑制基因1 (BRMS1)在多种恶性肿瘤细胞中表达降低或缺失,具有明显降低癌细胞侵袭和转移的作用.BRMS1基因通过磷酸肌醇信号及核转录因子-κB(NF-κB)信号通路等调节基因转录和蛋白翻译,还可与mSin3-组蛋白去乙酰化酶(HDAC)复合体、雌激素受体等蛋白相互作用、修复细胞间隙通讯等途径抑制肿瘤细胞的侵袭及转移.BRMS1基因将可能成为有效抑制肿瘤转移基因治疗的新靶点.     

Chromodomain helicase DNA binding protein 5 plays a tumor suppressor role in human breast cancer

Introduction

The chromodomain helicase DNA binding protein 5 (CHD5) has recently been identified as a tumor suppressor in a mouse model. The CHD5 locus at 1p36 is deleted, and its mutation has been detected in breast cancer. We, therefore, evaluated whether CHD5 plays a role in human breast cancer.

Methods

We screened mutations in 55 tumors, determined promoter methylation in 39 tumors, measured RNA expression in 90 tumors, analyzed protein expression in 289 tumors, and correlated expression changes with clinicopathological characteristics of breast cancer. Functional effects of CHD5 on cell proliferation, invasion and tumorigenesis were also tested.

Results

Although only one mutation was detected, CHD5 mRNA expression was significantly reduced, accompanied by frequent genomic deletion and promoter methylation, in breast cancer. The extent of methylation was significantly associated with reduced mRNA expression, and demethylating treatment restored CHD5 expression. Lower CHD5 mRNA levels correlated with lymph node metastasis (P = 0.026). CHD5 protein expression was also reduced in breast cancer, and lack of CHD5 expression significantly correlated with higher tumor stage, ER/PR-negativity, HER2 positivity, distant metastasis and worse patient survival (P ≤ 0.01). Functionally, ectopic expression of CHD5 in breast cancer cells inhibited cell proliferation and invasion in vitro and tumorigenesis in nude mice. Consistent with the inhibition of invasion, CHD5 down-regulated mesenchymal markers vimentin, N-cadherin and ZEB1 in breast cancer cells.

Conclusion

Down-regulation of CHD5, mediated at least in part by promoter methylation, contributes to the development and progression of human breast cancer.     

Role of the putative tumor metastasis suppressor gene Drg-1 in breast cancer progression

The differentiation-related gene-1 (Drg-1) was first identified as a gene strongly upregulated by induction of differentiation in colon carcinoma cells in vitro, and later the same gene was shown to suppress tumorigenicity of human bladder cancer cells in vivo. On the other hand, we and others have demonstrated that the Drg-1 gene suppresses prostate and colon cancer metastases in mouse models. In the context of such potential organ-specific differential function of the Drg-1 gene, the present study was designed to clarify the expression status, regulation and function of Drg-1 in the case of human breast cancer. We found that the expression of the Drg-1 protein was significantly reduced in breast tumor cells, particularly in patients with lymph node or bone metastasis as compared to those with localized breast cancer. Drg-1 expression also exhibited significant inverse correlation with the disease-free survival rate of patients and emerged as an independent prognostic factor. The downregulation of the Drg-1 gene appeared to be largely at the RNA level, and the DNA methylation inhibitor, 5-Azacytidine, significantly elevated the Drg-1 gene expression in various breast tumor cell lines. Furthermore, we found that overexpression of the Drg-1 gene suppresses the invasiveness of breast cancer cells in vitro, and this suppression was also achieved by treatment of cells with 5-Azacytidine. Together, our results strongly suggest functional involvement of the Drg-1 gene in suppressing the metastatic advancement of human breast cancer.     

ELOVL2: a novel tumor suppressor attenuating tamoxifen resistance in breast cancer

Epigenetic events have successfully explained the cause of various cancer types, but little is known about tamoxifen resistance (TamR) that induces cancer recurrence. In this study, via genome-wide methylation analysis in MCF-7/TamR cells we show that elongation of very-long chain fatty acid protein 2 (ELOVL2) was hypermethylated and downregulated in the samples from TamR breast cancer patients (n = 28) compared with those from Tam-sensitive (TamS) patients (n = 33) (P < 0.001). Strikingly, in addition to having tumor suppressor activity, ELOVL2 was shown to recover Tam sensitivity up to 70% in the MCF-7/TamR cells and in a xenograft mouse model. A group of genes in the AKT and ERa signaling pathways, e.g., THEM4, which play crucial roles in drug resistance, were found to be regulated by ELOVL2. This study implies that the deregulation of a gene in fatty acid metabolism can lead to drug resistance, giving insight into the development of a new therapeutic strategy for drug-resistant breast cancer.     

HYAL1 hyaluronidase in prostate cancer: a tumor promoter and suppressor

Hyaluronidases degrade hyaluronic acid, which promotes metastasis. HYAL1 type hyaluronidase is an independent prognostic indicator of prostate cancer progression and a biomarker for bladder cancer. However, it is controversial whether hyaluronidase (e.g., HYAL1) functions as a tumor promoter or as a suppressor. We stably transfected prostate cancer cells, DU145 and PC-3 ML, with HYAL1-sense (HYAL1-S), HYAL1-antisense (HYAL1-AS), or vector DNA. HYAL1-AS transfectants were not generated for PC-3 ML because it expresses little HYAL1. HYAL1-S transfectants produced < or = 42 milliunits (moderate overproducers) or > or = 80 milliunits hyaluronidase activity (high producers). HYAL1-AS transfectants produced <10% hyaluronidase activity when compared with vector transfectants (18-24 milliunits). Both blocking HYAL1 expression and high HYAL1 production resulted in a 4- to 5-fold decrease in prostate cancer cell proliferation. HYAL1-AS transfectants had a G2-M block due to decreased cyclin B1, cdc25c, and cdc2/p34 expression and cdc2/p34 kinase activity. High HYAL1 producers had a 3-fold increase in apoptotic activity and mitochondrial depolarization when compared with vector transfectants and expressed activated proapoptotic protein WOX1. Blocking HYAL1 expression inhibited tumor growth by 4- to 7-fold, whereas high HYAL1 producing transfectants either did not form tumors (DU145) or grew 3.5-fold slower (PC-3 ML). Whereas vector and moderate HYAL1 producers generated muscle and blood vessel infiltrating tumors, HYAL1-AS tumors were benign and contained smaller capillaries. Specimens of high HYAL1 producers were 99% free of tumor cells. This study shows that, depending on the concentration, HYAL1 functions as a tumor promoter and as a suppressor and provides a basis for anti-hyaluronidase and high-hyaluronidase treatments for cancer.     

miR-125b is methylated and functions as a tumor suppressor by regulating the ETS1 proto-oncogene in human invasive breast cancer

The microRNA miR-125b is dysregulated in various human cancers but its underlying mechanisms of action are poorly understood. Here, we report that miR-125b is downregulated in invasive breast cancers where it predicts poor patient survival. Hypermethylation of the miR-125b promoter partially accounted for reduction of miR-125b expression in human breast cancer. Ectopic restoration of miR-125b expression in breast cancer cells suppressed proliferation, induced G(1) cell-cycle arrest in vitro, and inhibited tumorigenesis in vivo. We identified the ETS1 gene as a novel direct target of miR-125b. siRNA-mediated ETS1 knockdown phenocopied the effect of miR-125b in breast cell lines and ETS1 overexpression in invasive breast cancer tissues also correlated with poor patient prognosis. Taken together, our findings point to an important role for miR-125b in the molecular etiology of invasive breast cancer, and they suggest miR-125b as a potential theranostic tool in this disease.     

乳腺癌转移抑制基因1抑制肿瘤转移的机制

乳腺癌转移抑制基因1(BRMS1)具有抑制肿瘤细胞转移的能力,可明显减少转移灶的发生,但不影响肿瘤的生长.其作用机制复杂,与细胞间通讯、磷酸肌醇信号转导以及与转录核因子-KB(NF-KB)的相互作用等有关.故探讨BRMS1抑制肿瘤转移的机制,希望用于肿瘤基因治疗.