Changes in expression of cell‐cycle‐related genes in PC‐3 prostate cancer cells caused by ovine uterine serpin

The hormonal‐regulated serpin, ovine uterine serpin (OvUS), also called uterine milk protein (UTMP), inhibits proliferation of lymphocytes and prostate cancer (PC‐3) cells by blocking cell‐cycle progression. The present aim was to identify cell‐cycle‐related genes regulated by OvUS in PC‐3 cells using the quantitative human cell‐cycle RT² Profiler? PCR array. Cells were cultured ±200 µg/ml recombinant OvUS (rOvUS) for 12 and 24 h. At 12 h, rOvUS increased expression of three genes related to cell‐cycle checkpoints and arrest (CDKN1A, CDKN2B, and CCNG2). Also, 14 genes were down‐regulated including genes involved in progression through S (MCM3, MCM5, PCNA), M (CDC2, CKS2, CCNH, BIRC5, MAD2L1, MAD2L2), G₁ (CDK4, CUL1, CDKN3) and DNA damage checkpoint and repair genes RAD1 and RBPP8. At 24 h, rOvUS decreased expression of 16 genes related to regulation and progression through M (BIRC5, CCNB1, CKS2, CDK5RAP1, CDC20, E2F4, MAD2L2) and G₁ (CDK4, CDKN3, TFDP2), DNA damage checkpoints and repair (RAD17, BRCA1, BCCIP, KPNA2, RAD1). Also, rOvUS down‐regulated the cell proliferation marker gene MKI67, which is absent in cells at G₀. Results showed that OvUS blocks cell‐cycle progression through upregulation of cell‐cycle checkpoint and arrest genes and down‐regulation of genes involved in cell‐cycle progression. J. Cell. Biochem. 107: 1182–1188, 2009. © 2009 Wiley‐Liss, Inc.     

Identification of mini‐chromosome maintenance 8 as a potential prognostic marker and its effects on proliferation and apoptosis in gastric cancer

Mini‐chromosome maintenance (MCM) proteins play important roles in initiating eukaryotic genome replication. The MCM family of proteins includes several members associated with the development and progression of certain cancers. We performed online data mining to assess the expression of MCMs in gastric cancer (GC) and the correlation between their expression and survival in patients with GC. Notably, MCM8 expression was undoubtedly up‐regulated in GC, and higher expression correlated with shorter overall survival (OS) and progression‐free survival (PFS) in patients with GC. However, the role of MCM8 in GC has not been previously explored. Our in vitro experiments revealed that MCM8 knockdown inhibited cell growth and metastasis. Moreover, MCM8 knockdown induced apoptosis. Mechanistically, the expression levels of Bax and cleaved caspase‐3 were increased, whereas Bcl‐2 expression decreased. Additionally, we demonstrated that MCM8 knockdown suppressed tumorigenesis in vivo. Overall, these results suggest that MCM8 plays a significant role in GC progression.     

Circular RNA circABCC4 as the ceRNA of miR‐1182 facilitates prostate cancer progression by promoting FOXP4 expression

In recent years, circular RNAs (circRNAs) have been identified to be essential regulators of various human cancers. However, knowledge of the functions of circRNAs in prostate cancer remains very limited. The correlation between circABCC4 and human cancer is largely unknown. This study aims to investigate the biological functions of circABCC4 in prostate cancer progression and illustrate the underlying mechanism. We found that circABCC4 was remarkably up‐regulated in prostate cancer tissues and cell lines and promoted FOXP4 expression by sponging miR‐1182 in prostate cancer cells. CircABCC4 knockdown markedly suppressed prostate cancer cell proliferation, cell‐cycle progression, migration and invasion in vitro. Furthermore, silencing of the circRNA also delayed tumor growth in vivo. Taken together, our findings indicated that circABCC4 facilitates the malignant behaviour of prostate cancer by promoting FOXP4 expression through sponging of miR‐1182. The circABCC4–miR‐1182‐FOXP4 regulatory loop may be a promising therapeutic target for prostate cancer intervention.     

NUCKS1 is a novel RAD51AP1 paralog important for homologous recombination and genome stability

NUCKS1 (nuclear casein kinase and cyclin-dependent kinase substrate 1) is a 27 kD chromosomal, vertebrate-specific protein, for which limited functional data exist. Here, we demonstrate that NUCKS1 shares extensive sequence homology with RAD51AP1 (RAD51 associated protein 1), suggesting that these two proteins are paralogs. Similar to the phenotypic effects of RAD51AP1 knockdown, we find that depletion of NUCKS1 in human cells impairs DNA repair by homologous recombination (HR) and chromosome stability. Depletion of NUCKS1 also results in greatly increased cellular sensitivity to mitomycin C (MMC), and in increased levels of spontaneous and MMC-induced chromatid breaks. NUCKS1 is critical to maintaining wild type HR capacity, and, as observed for a number of proteins involved in the HR pathway, functional loss of NUCKS1 leads to a slow down in DNA replication fork progression with a concomitant increase in the utilization of new replication origins. Interestingly, recombinant NUCKS1 shares the same DNA binding preference as RAD51AP1, but binds to DNA with reduced affinity when compared to RAD51AP1. Our results show that NUCKS1 is a chromatin-associated protein with a role in the DNA damage response and in HR, a DNA repair pathway critical for tumor suppression.     

Interaction between RAD51 and MCM complex is essential for RAD51 foci forming in colon cancer HCT116 cells

Colon cancer remains one of the most common digestive system malignancies in the World. This study investigated the possible interaction between RAD51 and minichromosome maintenance proteins (MCMs) in HCT116 cells, which can serve as a model system for forming colon cancer foci. The interaction between RAD51 and MCMs was detected by mass spectrometry. Silenced MCM vectors were transfected into HTC116 cells. The expressions of RAD51 and MCMs were detected using Western blotting. Foci forming and chromatin fraction of RAD51 in HCT116 cells were also analyzed. The results showed that RAD51 directly interacted with MCM2, MCM3, MCM5, and MCM6 in colon cancer HTC116 cells. Suppression of MCM2 or MCM6 by shRNA decreased the chromatin localization of RAD51 in HTC116 cells. Moreover, silenced MCM2 or MCM6 decreased the foci forming of RAD51 in HTC116 cells. Our study suggests that the interaction between MCMs and RAD51 is essential for the chromatin localization and foci forming of RAD51 in HCT116 cell DNA damage recovery, and it may be a theoretical basis for analysis of RAD51 in tumor samples of colon cancer patients.     

RAD51 paralogs: roles in DNA damage signalling, recombinational repair and tumorigenesis

Chromosomal double-strand breaks (DSBs) have the potential to permanently arrest cell cycle progression and endanger cell survival. They must therefore be efficiently repaired to preserve genome integrity and functionality. Homologous recombination (HR) provides an important error-free mechanism for DSB repair in mammalian cells. In addition to RAD51, the central recombinase activity in mammalian cells, a family of proteins known as the RAD51 paralogs and consisting of five proteins (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3), play an essential role in the DNA repair reactions through HR. The RAD51 paralogs act to transduce the DNA damage signal to effector kinases and to promote break repair. However, their precise cellular functions are not fully elucidated. Here we discuss recent advances in our understanding of how these factors mediate checkpoint responses and act in the HR repair process. In addition, we highlight potential functional similarities with the BRCA2 tumour suppressor, through the recently reported links between RAD51 paralog deficiencies and tumorigenesis triggered by genome instability.     

PC-１基因表达增强C4-2B前列腺癌细胞生存

建立稳定表达外源PC-１基因的人前列腺癌骨转移C4-2B细胞模型,初步探讨PC- １基因表达对前列腺癌发展的影响.通过脂质体介导的方法,将融合PC-１基因的真核表达载体pcDNA3.1PC-１稳定转染C4-2B细胞,Western 印迹和RT-PCR技术,分别从蛋白水平和RNA水平确定外源PC-１基因表达. MTT和软琼脂集落形成能力等一系列方法,研究PC-１基因的功能,RT-PCR和实时定量PCR检测前列腺癌发生发展相关基因表达的变化. 结果表明,PC-１基因的高表达能够诱导雄激素受体（AR）调控基因和一系列重要的信号通路成员基因PSA、PSMA、NKX31、Jagged1、EphA3、SGEF和 NOTCH3等表达发生变化. 实验结果初步证明,PC-１基因表达在晚期前列腺癌中,以及在雄激素非依赖的转变中可以发挥作用,PC-１基因表达可调控一些重要信号通路.对PC-１基因功能深入研究将有可能为发现新的前列腺癌的诊断治疗分子靶标提供线索.     

Cyr61 is up‐regulated in prostate cancer and associated with the p53 gene status

Cysteine‐rich 61 (Cyr61) is a member of the CCN protein family that has been implicated in diverse biological processes such as cell adhesion, proliferation, angiogenesis, and tumorigenesis. Altered expression of Cyr61 is found to be associated with human cancers. Here we show that Cyr61 was up‐regulated in prostate cancer cell lines and tumor tissues. A significant correlation of Cyr61 expression was found between benign prostatic hyperplasia and prostate cancer (P = 0.002). However, there was no significant correlation between levels of PSA and Cyr61 expression (P = 0.2). Cyr61 may represent an independent prostate cancer biomarker and potentially a useful therapeutic target for prostate cancer treatment. In addition, our analysis based on published data and data present in this report indicted that levels of Cyr61 expression associated with the status of the tumor suppressor gene p53 in 32 cancer cell lines analyzed, high levels of Cyr61 expression were found in cell lines with mutant or null p53 gene, whereas lower expression levels of Cyr61 in the cell lines with wild‐type p53. We further show that over‐expression of dominant negative p53 or down‐expression of endogenous wild‐type p53 resulted in up‐regulation of Cyr61 expression, suggesting a functional link between Cyr61 and p53 in cancers. J. Cell. Biochem. 106: 738–744, 2009. © 2009 Wiley‐Liss, Inc.     

Knockdown of SRPX2 inhibits the proliferation,migration, and invasion of prostate cancer cells through the PI3K/Akt/mTOR signaling pathway

Sushi repeat‐containing protein X‐linked 2 (SRPX2), a novel chondroitin sulfate proteoglycan, is reported to play a critical role in tumorigenesis. However, the expression and functional role of SRPX2 in prostate cancer have not been defined. Thus, the aim of this study was to investigate the expression and functional role of SRPX2 in human prostate cancer. Our results showed that the expression of SRPX2 was obviously increased in human prostate cancer tissues and cell lines. In addition, knockdown of SRPX2 inhibited the proliferation, migration, and invasion of prostate cancer cells, as well as prevented the epithelial‐mesenchymal transition process in prostate cancer cells. Mechanically, knockdown of SRPX2 efficiently inhibited the activation of PI3K/Akt/mTOR pathway in prostate cancer cells. Taken together, these data demonstrated that knockdown of SRPX2 inhibits the proliferation and metastasis in human prostate cancer cells, partly through the PI3K/Akt/mTOR signaling pathway. Thus, SRPX2 may be a novel therapeutic target for the treatment of prostate cancer.     

ZEB1‐mediated vasculogenic mimicry formation associates with epithelial–mesenchymal transition and cancer stem cell phenotypes in prostate cancer

The zinc finger E‐box‐binding homeobox 1 (ZEB1) induced the epithelial–mesenchymal transition (EMT) and altered ZEB1 expression could lead to aggressive and cancer stem cell (CSC) phenotypes in various cancers. Tissue specimens from 96 prostate cancer patients were collected for immunohistochemistry and CD34/periodic acid–Schiff double staining. Prostate cancer cells were subjected to ZEB1 knockdown or overexpression and assessment of the effects on vasculogenic mimicry formation in vitro and in vivo. The underlying molecular events of ZEB1‐induced vasculogenic mimicry formation in prostate cancer were then explored. The data showed that the presence of VM and high ZEB1 expression was associated with higher Gleason score, TNM stage, and lymph node and distant metastases as well as with the expression of vimentin and CD133 in prostate cancer tissues. Furthermore, ZEB1 was required for VM formation and altered expression of EMT‐related and CSC‐associated proteins in prostate cancer cells in vitro and in vivo. ZEB1 also facilitated tumour cell migration, invasion and clonogenicity. In addition, the effects of ZEB1 in prostate cancer cells were mediated by Src signalling; that is PP2, a specific inhibitor of the Src signalling, dose dependently reduced the p‐Src⁵²⁷ level but not p‐Src⁴¹⁶ level, while ZEB1 knockdown also down‐regulated the level of p‐Src⁵²⁷ in PC3 and DU‐145 cells. PP2 treatment also significantly reduced the expression of VE‐cadherin, vimentin and CD133 in these prostate cancer cells. Src signalling mediated the effects of ZEB1 on VM formation and gene expression.     

BRCA2 is epistatic to the RAD51 paralogs in response to DNA damage

Homologous recombination plays an important role in the high-fidelity repair of DNA double-strand breaks. A central player in this process, RAD51, polymerizes onto single-stranded DNA and searches for homology in a duplex donor DNA molecule, usually the sister chromatid. Homologous recombination is a highly regulated event in mammalian cells: some proteins have direct enzymatic functions, others mediate or overcome rate-limiting steps in the process, and still others signal cell cycle arrest to allow repair to occur. While the human BRCA2 protein has a clear role in delivering and loading RAD51 onto single-stranded DNA generated after resection of the DNA break, the mechanistic functions of the RAD51 paralogs remain unclear. In this study, we sought to determine the genetic interactions between BRCA2 and the RAD51 paralogs during DNA DSB repair. We utilized siRNA-mediated knockdown of these proteins in human cells to assess their impact on the DNA damage response. The results indicate that loss of BRCA2 alone imparts a more severe phenotype than the loss of any individual RAD51 paralog and that BRCA2 is epistatic to each of the four paralogs tested.     

RDM1 promotes critical processes in breast cancer tumorigenesis

Breast cancer is currently among the most common cancers in women, with almost 200,000 new cases diagnosed annually. Dysregulation of DNA repair pathways allows cells to accumulate damage and eventually mutations, with a subsequent reduction in DNA repair capacity in breast tissue, leading to tumorigenesis. One component of the DNA damage repair pathway is RAD52 motif‐containing 1 (RDM1), but the specific role of RDM1 in breast cancer and the underlying mechanism remain unclear. Here, we examined the role played by RDM1 in breast cancer cell culture using the HBL100 and MCF‐7 breast cancer cell lines. Disruption of RDM1 reduced in vitro cell proliferation and promoted apoptosis. Knockdown of RDM1 also induced up‐regulation of p53 levels, whereas RAD51 and RAD52, both involved in DNA repair, were down‐regulated. In addition, the in vivo growth of RDM1‐deficient cells was significantly repressed, suggesting that RDM1 is a novel oncogenic protein in human breast cancer cells. This study reveals a link between the DNA damage response pathway and oncogenic functionality in breast cancer. Accordingly, therapeutic targeting of RDM1 is a potential treatment strategy for breast cancer and overcoming drug resistance.     

MCM4 acts as a biomarker for LUAD prognosis

MCM4 forms the pre-replication complex (MCM2-7) with five other minichromosome maintenance (MCM) proteins. This complex binds to replication origins at G1 stage in cell cycle process, playing a critical role in DNA replication initiation. Recently, MCM4 is reported to have a complex interaction with multiple cancer progression, including gastric, ovarian and cervical cancer. Here, this study mainly focused on the expression of MCM4 and its values in lung adenocarcinoma (LUAD). MCM4 was highly expressed in LUAD tumours and cells, and had an important effect on the overall survival. Overexpression of MCM4 promoted the proliferation, and suppressed the apoptosis in LUAD cells. However, MCM4 silence led to the opposite results. In vivo, knockdown of MCM4 inhibited tumour volume and weight in xenograft mouse model. As a member of DNA helicase, knockdown of MCM4 caused cell cycle arrest at G1 stage through inducing the expression of P21, a CDK inhibitor. These findings indicate that MCM4 may be a possible new therapeutic target for LUAD in the future.     

The convergence of hormone regulation and cell cycle in prostate physiology and prostate tumorigenesis

Despite the intense research focused on prostate cancer, it remains the most frequently diagnosed malignancy in men over 40-yr-of-age, and the second most frequent cause of cancer-related deaths in men in the United States (1). In 1990, the National Cancer Institute convened 50 experts and leaders from various disciplines in the prostate cancer field to discuss research directions that would help elucidate the molecular basis of this disease and reduce the incidence and mortality of prostate cancer (2). Critical issues identified at this meeting included the role of androgens and the regulation of cell cycle in prostate tumorigenesis and its progression to androgen-independence. Hormones and cell cycle clearly play important roles in normal and cancerous prostate physiology; however, little information has emerged that clearly delineates their function in the etiology of prostate cancer. Some of the mutational events that occur during prostate tumorigenesis and its progression to androgen-independence involve alterations to normal growth, developmental and apoptotic programs regulated by androgen, and the cell cycle. As such, the delineation of events by which prostate cancer cells circumvent these regulatory mechanisms will be central to our understanding of prostate tumorigenesis and to the development of new modalities to treat this disease. This article is then intended to summarize the functional convergence of androgen regulation and cell cycle in normal prostate physiology and prostate tumorigenesis.