Low dose histone deacetylase inhibitor,LBH589, potentiates anticancer effect of docetaxel in epithelial ovarian cancer via PI3K/Akt pathway in vitro

The purpose of this study was to investigate the effect of combination of LBH589 with docetaxel (DTX) on the growth and survival of epithelial ovarian cancer (EOC) cells in vitro and the possible mechanisms of chemo-sensitization of LBH589 in the combination treatment. The effect of LBH589 alone or in combination with DTX on four EOC cell lines (OVCAR-3, IGROV-1, A2780 and SKOV-3) was studied by MTT and clonogenic assays, acridine orange (AO)/ethidium bromide (EB) staining for apoptosis, Western blotting for apoptosis-related proteins, histone H3 and H4 proteins, DNA double strand break (DSB) repair marker and phosphorylation of Akt. LBH589 alone inhibited EOC cell proliferation in a time and dose-dependent manner. Low-dose of LBH589 (IC₂₀) combined with DTX had an additive effect and greatly improved efficacy of DTX cell killing in EOC cells. Compared to DTX alone, the combination treatment with LBH589 and DTX induced more apoptosis and led to an increased and persistent DSB. Cell death following single or combined treatment was associated with the release of cytochrome c activity, increased caspase-3 (active) and PARP-1(cleaved), histone acetylation-related proteins and PI3k/Akt signaling pathway. Our results suggest that LBH589 enhances DTX-induced apoptosis in human EOC cells, and can be used in combination with DTX as an attractive strategy for treating human EOC.     

Association of gamma-glutamyltransferase with severity of disease at diagnosis and prognosis of ovarian cancer

Background:

Gamma-glutamyltransferase (GGT) – a membrane-bound enzyme crucially involved in the cell''s detoxification pathway and apoptotic balance – is involved in tumour development, progression and chemotherapy resistance. Elevated GGT serum levels are associated with increased cancer risk in women and worse prognosis in gynaecologic cancers. The present study investigated the prognostic role of GGT in ovarian cancer patients.

Methods:

In this multicenter study, pre-therapeutic GGT levels were ascertained in 634 consecutive patients with epithelial ovarian cancer (EOC, n=567) and borderline tumour of the ovary (BTO, n=67). Gamma-glutamyltransferase serum levels were associated with clinicopathological parameters and uni- and multivariate survival analyses were performed. Immunohistochemistry of GGT was performed in ovarian cancer tissue and correlated with GGT serum levels.

Results:

Pre-therapeutic GGT serum levels were higher in patients with EOC (28.56 (38.24) U l⁻¹) than in patients with BTO (20.01 (12.78) U l⁻¹, P=0.01). High GGT serum levels were associated with advanced FIGO stage (P<0.001) and with worse overall survival in univariate (P<0.001) and multivariable analysis (P=0.02, HR 1.2 (1.1–1.5)). We further investigated the association between systemic GGT serum levels and local GGT expression in EOC tumour tissue and observed an association between these two parameters (P=0.03).

Conclusion:

High pre-therapeutic GGT serum levels are associated with advanced tumour stage and serve as an independent prognostic marker for worse overall survival in patients with EOC. Gamma-glutamyltransferase expression in ovarian cancer tissue is reflected in GGT serum levels.     

Lysophosphatidic acid‐induced expression of periostin in stromal cells: Prognoistic relevance of periostin expression in epithelial ovarian cancer

Lysophosphatidic acid (LPA) is a bioactive lipid crucial for the initiation and progression of ovarian cancer. Identification of LPA‐induced biomarkers is necessary for predicting prognosis of ovarian cancer patients. Here we report periostin, an extracellular matrix protein, as an LPA‐induced protein in stromal cells and as a prognostic marker in patients with epithelial ovarian cancer (EOC). In human EOC tissues, periostin was mainly expressed in cancer‐associated stromal fibroblasts, but not in cancer cells. The expression levels of periostin highly correlated with poor survival and tumor recurrence of ovarian cancer patients. Treatment of human adipose tissue‐derived stromal cells with LPA or conditioned media from human ovarian adenocarcinoma cell lines, such as SK‐OV‐3 and OVCAR‐3, induced expression of periostin. The periostin expression induced by cancer‐conditioned media was abrogated by silencing of the LPA receptor 1 expression using small hairpin RNA lentivirus. Recombinant periostin stimulated adhesion and invasion of SK‐OV‐3 human ovarian adenocarcinoma cells and induced expression of matrix metalloprotease‐2 in the cancer cells. These results suggest that LPA is associated with the expression of periostin in cancer‐associated fibroblasts of EOC.     

BCAT1 expression associates with ovarian cancer progression: possible implications in altered disease metabolism

Previously, we have identified the branched chain amino-acid transaminase 1 (BCAT1) gene as notably hypomethylated in low-malignant potential (LMP) and high-grade (HG) serous epithelial ovarian tumors, compared to normal ovarian tissues. Here we show that BCAT1 is strongly overexpressed in both LMP and HG serous epithelial ovarian tumors, which probably correlates with its hypomethylated status. Knockdown of the BCAT1 expression in epithelial ovarian cancer (EOC) cells led to sharp decrease of cell proliferation, migration and invasion and inhibited cell cycle progression. BCAT1 silencing was associated with the suppression of numerous genes and pathways known previously to be implicated in ovarian tumorigenesis, and the induction of some tumor suppressor genes (TSGs). Moreover, BCAT1 suppression resulted in downregulation of numerous genes implicated in lipid production and protein synthesis, suggesting its important role in controlling EOC metabolism. Further metabolomic analyses were indicative for significant depletion of most amino acids and different phospho- and sphingolipids following BCAT1 knockdown. Finally, BCAT1 suppression led to significantly prolonged survival time in xenograft model of advanced peritoneal EOC. Taken together, our findings provide new insights about the functional role of BCAT1 in ovarian carcinogenesis and identify this transaminase as a novel EOC biomarker and putative EOC therapeutic target.     

溶血磷脂信号在上皮性卵巢癌微环境中作用的研究进展

卵巢癌是妇科恶性肿瘤死亡的主要原因,发生在肿瘤微环境(TME)中的包括脂质代谢改变在内的代谢重编程是其主要特征。脂质中的几种溶血磷脂（也称癌脂）如溶血磷脂酸（LPA)是TME的重要组成部分,参与了肿瘤发生、发展的各个方面。本文综述了上皮性卵巢癌（EOC）TME中脂质代谢的改变,包括脂肪酸氧化增强、其它几种脂肪酸含量的改变,其次是溶血磷脂信号在EOC中的研究进展,重点介绍了LPA在EOC的TME中的作用:促进卵巢癌细胞的增殖、削弱免疫监测、侵袭转移、对化疗药物的抵抗等。     

Identification of glucocorticoid-induced leucine zipper as a key regulator of tumor cell proliferation in epithelial ovarian cancer

Background  

Little is known about the molecules that contribute to tumor progression of epithelial ovarian cancer (EOC), currently a leading cause of mortality from gynecological malignancies. Glucocorticoid-Induced Leucine Zipper (GILZ), an intracellular protein widely expressed in immune tissues, has been reported in epithelial tissues and controls some of key signaling pathways involved in tumorigenesis. However, there has been no report on GILZ in EOC up to now. The objectives of the current study were to examine the expression of GILZ in EOC and its effect on tumor cell proliferation.     

Role of the polypeptide N-acetylgalactosaminyltransferase 3 in ovarian cancer progression: possible implications in abnormal mucin O-glycosylation

Previously, we have identified the polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3) gene as notably hypomethylated in low-malignant potential (LMP) and high-grade (HG) serous epithelial ovarian tumors, compared to normal ovarian tissues. Here we show that GALNT3 is strongly overexpressed in HG serous EOC tumors as compared to normal ovarian tissue. Moreover, the GALNT3 expression significantly correlated with shorter progression-free survival (PFS) intervals in epithelial ovarian cancer (EOC) patients with advanced disease.Knockdown of the GALNT3 expression in EOC cells led to sharp decrease of cell proliferation and induced S-phase cell cycle arrest. Additionally, GALNT3 suppression significantly inhibited EOC cell migration and invasion. Gene expression profiling and consecutive network and pathway analyses confirmed these findings, as numerous genes and pathways known previously to be implicated in ovarian tumorigenesis, including EOC tumor invasion and metastasis, were found to be downregulated upon GALNT3 suppression, while some tumor suppressor genes were induced. Moreover, GALNT3 downregulation was associated with reduced MUC1 protein expression in EOC cells, probably related to destabilization of the MUC1 protein due to lack of GALNT3 glycosylation activity. GALNT3 knockdown was also accompanied with increase of the cell adhesion molecules β-catenin and E-cadherin, which are normally suppressed by MUC1 in cancer, thus supporting the role of the GALNT3-MUC1 axis in EOC invasion.Taken together, our data are indicative for a strong oncogenic potential of the GALNT3 gene in advanced EOC and identify this transferase as a novel EOC biomarker and putative EOC therapeutic target. Our findings also suggest that GALNT3 overexpression might contribute to EOC progression through aberrant mucin O-glycosylation     

Cyclooxygenase‐2 inhibition inhibits PI3K/AKT kinase activity in epithelial ovarian cancer

Cyclooxygenase‐2 (COX‐2) expression contributes to tumor growth and invasion in epithelial ovarian cancer (EOC). COX‐2 inhibitors exhibit important anticarcinogenic potential against EOC, but the molecular mechanisms underlying this effect and relation with PI3‐kinase/AKT signaling remain the subject of intense investigations. Therefore, the role of COX‐2 in EOC and its cross talk with PI3‐kinase/AKT pathway were investigated using a large series of EOC tissues in a tissue micro array (TMA) format followed by in vitro and in vivo studies using EOC cell lines and NUDE mice. Clinically, COX‐2 was overexpressed in 60.3% of EOC and was significantly associated with activated AKT (p < 0.0001). Cox‐1 expression was seen in 59.9% but did not associate with AKT. Our in vitro data using EOC cell line showed that inhibition of COX‐2 by aspirin, selective inhibitor NS398 and gene silencing by COX‐2 specific siRNA impaired phosphorylation of AKT resulting decreased downstream signaling leading to cell growth inhibition and induction of apoptosis. Finally, treatment of MDAH2774 cell line xenografts with aspirin resulted in growth inhibition of tumors in NUDE mice via down‐regulation of COX‐2 and AKT activity. These data identify COX‐2 as a potential biomarker and therapeutic target in distinct molecular subtypes of ovarian cancer.     

Coexpression of invasive markers (uPA, CD44) and multiple drug-resistance proteins (MDR1, MRP2) is correlated with epithelial ovarian cancer progression

Background:

Invasion and metastases of cancer cells and the development of resistance to anticancer therapies are the main causes of treatment failure and mortality in cancer patients.

Methods:

We evaluated invasive markers of urokinase plasminogen activator (uPA) and CD44 and multiple drug-resistance (MDR) markers of MDR1 and MRP2 in four epithelial ovarian cancer (EOC) cell lines, primary tumours (n=120) and matched metastatic lesions (n=40) by immunofluoresence labelling. We correlated uPA and CD44 with MDR markers in primary and metastatic cells using confocal microscope. We also investigated the relationship of the expression of uPA, CD44 and MDR1 with various progression parameters.

Results:

The coexpression of uPA and CD44 with MDR markers was found in primary and metastatic cells. The overexpression of uPA, CD44 and MDR1 was found in most primary and matched metastatic lesions of EOC, and was significantly associated with tumour stage, grade, residual disease status, relapse and presence of ascites (P<0.05), but not with histology type (P>0.05).

Conclusions:

Our results suggest that the overexpression of uPA, CD44 and MRD1 is correlated with EOC progression; both uPA and CD44 are related with drug resistance during EOC metastasis and could be useful therapeutically.     

Midkine as a potential diagnostic marker in epithelial ovarian cancer for cisplatin/paclitaxel combination clinical therapy

The paclitaxel/cisplatin combination therapy commonly is used as the first-line treatment for advanced ovarian cancer patients. Midkine (MK), known as a novel tumor biomarker, has been elevated in the serum of patients with epithelial ovarian cancer (EOC). In this study, we aimed to detect the expression of MK in EOC tissues and evaluate clinical value of MK in diagnosis and therapy of EOC. We perform immunohistochemistry analysis to detect MK in EOC sample with postoperative platinum/paclitaxel combination therapy, we found that 71.4% (85 in 119 samples) of these samples were MK positive (> 10% of the cells were stained), and the expression of MK was significantly associated with disease histology (P = 0.038) as well as differentiation grade (P < 0.001). Moreover, MK positive samples show much more sensitive to cisplatin/paclitaxel combination therapy, compared with MK negative samples (P = 0.029). Those results indicated that MK expression might correlate with paclitaxel and/or cisplatin cytotoxicity in clinical therapy of EOC. Then, we evaluated the sensitivity to cisplatin and paclitaxel in 5 ovarian cancer cell lines (ES2, A2870, HO-8910, SKOV3 and SW626), and ES2, the highest MK expression among those cell lines, show the most sensitive to paclitaxel and cisplatin. Further, we confirmed this correlation between MK and paclitaxel and/or cisplatin cytotoxicity with the gain- and lost- of function. Finally, we demonstrated that MK enhanced the cytotoxicity of paclitaxel and/or cisplatin by accumulated cisplatin and paclitaxel through inhibited the expression of multidrug resistance-associated protein 3 (MRP3). In conclusion, MK could be an effective biomarker in diagnosis and therapy of EOC, especially for the drug selection at the time of initial diagnosis.     

Cytotoxicity induced by manipulation of signal transduction pathways is associated with down-regulation of Bcl-2 but not Mcl-1 in MCF-7 human breast cancer

We examined the role of Mcl-1 and Bcl-2 expression in the induction of apoptosis, through blocking protein tyrosine kinase (PTK), protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3-K) and mitogen-activated protein kinase (MAPK)/Erk kinase (MEK) signaling pathways by various kinase inhibitors in MCF-7 breast cancer cells. The PTK inhibitor genistein (GEN) and PKC inhibitor staurosporine (STP) down-regulated Mcl-1 and Bcl-2 expression, and induced growth inhibition by blocking at the G₂/M phase of cell cycle, followed by apoptosis, leading to chromatin condensation and DNA fragmentation. LY294002 (LY)-mediated inhibition of PI3-K activity down-regulated Bcl-2 but not Mcl-1 expression, triggered growth arrest at the G₁/G₀ phase of cell cycle and also led to apoptosis marked with chromatin condensation and DNA fragmentation. The MEK inhibitor U0126 (U0) decreased Bcl-2 expression but not Mcl-1 expression, inhibited cells growth and induced G₁/G₀ arrest, but in this case cell death occurred without significant apoptotic features. The kinase inhibitor concentration dependence of cytotoxicity correlated well with down-regulation of Bcl-2 but not with changes in Mcl-1 levels. This suggests that Bcl-2 plays a predominant role in the regulation of cell death induced by cell signaling alterations whereas Mcl-1 does not appear to control cell survival under these conditions in MCF-7 cells. Further studies showed that the combination of GEN, STP and LY with U0 can produce synergetic cytotoxic effects on MCF-7 cells. Our results suggest that PTK, PKC, PI3-K and MEK signaling pathways can regulate Bcl-2 expression and form an integrated network that plays a critical role in cell survival.     

Autotaxin and LPA receptor signaling in cancer

Lysophosphatidic acid (LPA; monoacyl-glycerol-3-phosphate) is a lipid mediator that functions as a mitogen and motility factor for many cell types. LPA signals through six specific G protein-coupled receptors, named LPA_1–6, which trigger both overlapping and distinct signaling pathways. LPA is produced from extracellular lysophosphatidylcholine by a secreted lysophospholipase D, named autotaxin (ATX), originally identified as an “autocrine motility factor” for tumor cells. ATX–LPA signaling is vital for embryonic development and promotes tumor formation, angiogenesis, and experimental metastasis in mice. Elevated expression of ATX and/or aberrant expression of LPA receptors are found in several human malignancies, while loss of LPA₆ function has been implicated in bladder cancer. In this review, we summarize our present understanding of ATX and LPA receptor signaling in cancer.     

Lysophosphatidic acid (LPA)‐induced vasodilator‐stimulated phosphoprotein mediates lamellipodia formation to initiate motility in PC‐3 prostate cancer cells

Prostate cancer remains the most frequently diagnosed malignancy and the second leading cause of cancer mortality among men in the United States. Hormone refractory, metastatic disease has no molecular therapeutics to date and survival is poor. Lysophosphatidic acid (LPA) is a bioactive lipid exhibiting motility, invasive, growth, proliferative and survival effects in multiple cancer cell lineages. Cells express different combinations of LPA‐specific G protein‐coupled receptors, LPA1, LPA2 LPA3, and LPA4 as well as other LPA receptors, which bind LPA and thereby regulate lipid signaling. The role of specific LPA receptors in functional outcomes of lysolipid signaling remains to be fully elucidated in prostate cancer. We hypothesized that LPA can initiate cell migration through specific LPA receptors by activating actin‐associating proteins involved in motility, including the vasodilator‐stimulated phosphoprotein (VASP). In the present study, we demonstrate that LPA‐induced lamellipodia formation in cells is dependent on LPA receptor‐mediated phosphorylation of VASP, demonstrating a previously unknown regulation by LPA. LPA induces phosphorylation of VASP at Ser(157), through protein kinase A (PKA) since the stimulation was abrogated by PKA inhibition. In addition, we found that the effects of LPA‐induced lamellipodia formation and migration were reduced by knockdown of either VASP or LPA receptor expression, suggesting that LPA receptor‐induced VASP phosphorylation is a critical mediator of migration initiation. Thus the LPA2 and LPA3 receptors, in addition to the previously implicated LPA1 receptor, play a role in cellular motility potentially contributing to invasion and metastases. Emerging drugs targeting the LPA pathway may be beneficial for the treatment of metastatic progression in prostate cancer.     

Diagnostic and prognostic potential of serum miR-7, miR-16, miR-25, miR-93, miR-182, miR-376a and miR-429 in ovarian cancer patients

Background:

Owing to late diagnosis in advanced disease stages, prognosis of patients with epithelial ovarian cancer (EOC) is poor. The quantification of deregulated levels of microRNAs could facilitate earlier diagnosis and improve prognosis of EOC.

Methods:

Seven microRNAs (miR-7, miR-16, miR-25, miR-93, miR-182, miR-376a and miR-429) were quantified in the serum of 180 EOC patients and 66 healthy women by TaqMan PCR microRNA assays. Median follow-up time was 21 months. The effects of miR-7 and miR-429 on apoptosis, cell proliferation, migration and invasion were investigated in two (EOC) cell lines.

Results:

Serum levels of miR-25 (P=0.0001) and miR-93 (P=0.0001) were downregulated, whereas those of miR-7 (P=0.001) and miR-429 (P=0.0001) were upregulated in EOC patients compared with healthy women. The four microRNAs discriminated EOC patients from healthy women with a sensitivity of 93% and a specificity of 92%. The levels of miR-429 positively correlated with CA125 values (P=0.0001) and differed between FIGO I–II and III–IV stages (P=0.001). MiR-429 was an independent predictor of overall survival (P=0.011). Overexpressed miR-429 in SKOV3 cells led to suppression of cell migration (P=0.037) and invasion (P=0.011). Increased levels of miR-7 were associated with lymph node metastases (P=0.0001) and FIGO stages III–IV (P=0.0001). Overexpressed miR-7 in SKOV3 cells resulted in increased cell migration (P=0.001) and invasion (P=0.011). Additionally, the increased levels of miR-376a correlated with FIGO stages III–IV (P=0.02).

Conclusions:

Our data indicate the diagnostic potential of miR-7, miR-25, miR-93 and miR-429 in EOC and the prognostic potential of miR-429. This microRNA panel may be promising molecules to be targeted in the treatment of EOC.