The association between vitamin D receptor expression and prolonged overall survival in breast cancer

In this study, we analyzed vitamin D receptor (VDR) expression and survival in a breast cancer patient cohort of 82 breast cancer patients. Immunohistochemical analysis was possible in 91.5% of the patients (75/82). Staining was evaluated using the semi-quantitative assay according to Remmele and Stegner (immunoreactivity score [IRS]). IRS 0-1 was negative/very low, IRS 2-4 was moderate to high, and IRS 6-12 was high. Statistical analysis was performed by Spearman's correlation test (p<0.05 significant). Overall survival was analyzed using Kaplan-Meier estimations. Only 6 patients had a negative IRS. Moderate IRS values were present in 20 patients. Most of the patients had a high IRS (49). For survival analysis, data were dichotomized (IRS 0-4: negative to moderate and IRS 6-12: high VDR expression). In univariate analysis, VDR expression showed significant differences in progression-free survival (PFS) and overall survival (OS). Patients with high IRS scores showed significantly better PFS and OS than patients with moderate/negative IRS scores for VDR expression. Tumor size was significantly correlated to PFS. When analyzed separately, the three different IRS groups showed significant differences in VDR expression. The present data suggest that VDR expression in breast cancer tissue may be of clinical significance, and the results provide evidence that VDR may be a factor with prognostic relevance.     

Aberrant expression of Arpin in human breast cancer and its clinical significance

Arpin (Arp2/3 complex inhibitor), a novel protein found in 2013, plays a pivotal role in cell motility and migration. However, the precise role of Arpin in cancer is unclear. This study investigated the expression of Arpin in breast cancer and evaluated its correlation with the characteristics of clinical pathology and prognosis of breast cancer patients. Immunohistochemistry (IHC) for Arpin protein was performed on formalin‐fixed, paraffin‐embedded 176 breast cancer tissues and 43 normal breast tissues while qRT‐PCR for Arpin mRNA with 104 paired tumour and paratumoural tissues from breast cancer patients respectively. The association of Arpin expression with clinical pathological features and survival was assessed in a retrospective cohort analysis of patients. The results showed that the expression of Arpin protein in cancer tissues was lower compared to that in normal breast and the expression of Arpin mRNA was also lower in cancer tissues than that in the matched paratumoural tissues. Among the 176 breast cancer patients, the lower expression of Arpin was significantly associated with advanced tumour, nodes and metastasis system stage, and the reduced Arpin expression was strongly associated with axillary lymph node metastasis using univariate and multivariate logistic regression analysis [odds ratio: 3.242; 95% confidence interval (CI): 1.526, 6.888; P < 0.05]. Furthermore, Arpin expression was an independent risk factor for recurrence‐free survival (HR: 0.373; 95% CI: 0.171, 0.813; P < 0.05). As Arpin expression was first examined in human breast cancer tissues with qRT‐PCR and IHC, our results suggest that Arpin downregulation may contribute to the initiation and development of breast cancer metastasis. Therefore, as a potential predictive marker, Arpin deserves future studies.     

Dopamine and cAMP‐regulated phosphoprotein 32kDa (DARPP‐32), protein phosphatase‐1 and cyclin‐dependent kinase 5 expression in ovarian cancer

Dopamine and cyclic‐AMP activated phosphoprotein Mr32kDa (DARPP‐32) is a central signalling protein in neurotransmission. Following DARPP‐32 phosphorylation by protein kinase A (PKA), DARPP‐32 becomes a potent protein phosphatase 1 (PP1) inhibitor. DARPP‐32 can itself inhibit PKA following DARPP‐32 phosphorylation by cyclin‐dependent kinase 5 (Cdk5). Increasing evidence indicates a role for DARPP‐32 and its associated signalling pathways in cancer; however, its role in ovarian cancer remains unclear. Using immunohistochemistry, expression of DARPP‐32, PP1 and Cdk5 was determined in a large cohort of primary tumours from ovarian cancer patients (n = 428, 445 and 434 respectively) to evaluate associations between clinical outcome and clinicopathological criteria. Low cytoplasmic and nuclear DARPP‐32 expression was associated with shorter patient overall survival and progression‐free survival (P = .001, .001, .004 and .037 respectively). Low nuclear and cytoplasmic DARPP‐32 expression remained significantly associated with overall survival in multivariate Cox regression (P = .045, hazard ratio (HR) = 0.734, 95% confidence interval (CI) = 0.542‐0.993 and P = .001, HR = 0.494, 95% CI = 0.325‐0.749, respectively). High cytoplasmic and nuclear PP1 expression was associated with shorter patient overall survival and high cytoplasmic PP1 expression with shorter progression‐free survival (P = .005, .033, and .037, respectively). High Cdk5 expression was associated with shorter progression‐free survival (P = .006). These data suggest a role for DARPP‐32 and associated signalling kinases as prognostic markers with clinical utility in ovarian cancer.     

Aberrant Keap1 methylation in breast cancer and association with clinicopathological features

Keap1 (Kelch-like ECH-associated protein 1) is an adaptor protein that mediates the ubiquitination/degradation of genes regulating cell survival and apoptosis under oxidative stress conditions. We determined methylation status of the KEAP1 promoter in 102 primary breast cancers, 14 pre-invasive lesions, 38 paired normal breast tissues and 6 normal breast from reductive mammoplasty by quantitative methylation specific PCR (QMSP). Aberrant promoter methylation was detected in 52 out of the 102 primary breast cancer cases (51%) and 10 out of 14 pre-invasive lesions (71%). No mutations of the KEAP1 gene were identified in the 20 breast cancer cases analyzed by fluorescence based direct sequencing. Methylation was more frequent in the subgroup of patients identified as ER positive-HER2 negative tumors (66.7%) as compared with triple-negative breast cancers (35%) (p = 0.05, Chi-square test). The impact of the interactions between Er, PgR, Her2 expression and KEAP1 methylation on mortality was investigated by RECPAM multivariable statistical analysis, identifying four prognostic classes at different mortality risks. Triple-negative breast cancer patients with KEAP1 methylation had higher mortality risk than patients without triple-negative breast cancer (HR = 14.73, 95%CI: 3.65–59.37). Both univariable and multivariable COX regressions analyses showed that KEAP1 methylation was associated with a better progression free survival in patients treated with epirubicin/cyclophosfamide and docetaxel as sequential chemotherapy (HR = 0.082; 95%CI: 0.007–0.934). These results indicate that aberrant promoter methylation of the KEAP1 gene is involved in breast cancerogenesis. In addition, identifying patients with KEAP1 epigenetic abnormalities may contribute to disease progression prediction in breast cancer patients.     

E-cadherin deregulation in breast cancer

E-cadherin protein (CDH1 gene) integrity is fundamental to the process of epithelial polarization and differentiation. Deregulation of the E-cadherin function plays a crucial role in breast cancer metastases, with worse prognosis and shorter overall survival. In this narrative review, we describe the inactivating mechanisms underlying CDH1 gene activity and its possible translation to clinical practice as a prognostic biomarker and as a potential targeted therapy.     

Regulation of androgen receptor and prostate cancer growth by cyclin-dependent kinase 5

Prostate cancer is the most frequently diagnosed male malignancy. The normal prostate development and prostate cancer progression are mediated by androgen receptor (AR). Recently, the roles of cyclin-dependent kinase 5 (Cdk5) and its activator, p35, in cancer biology are explored one after another. We have previously demonstrated that Cdk5 may regulate proliferation of thyroid cancer cells. In addition, we also identify that Cdk5 overactivation can be triggered by drug treatments and leads to apoptosis of prostate cancer cells. The aim of this study is to investigate how Cdk5 regulates AR activation and growth of prostate cancer cells. At first, the data show that Cdk5 enables phosphorylation of AR at Ser-81 site through direct biochemical interaction and, therefore, results in the stabilization of AR proteins. The Cdk5-dependent AR stabilization causes accumulation of AR proteins and subsequent activation. Besides, the positive regulations of Cdk5-AR on cell growth are also determined in vitro and in vivo. S81A mutant of AR diminishes its interaction with Cdk5, reduces its nuclear localization, fails to stabilize its protein level, and therefore, decreases prostate cancer cell proliferation. Prostate carcinoma specimens collected from 177 AR-positive patients indicate the significant correlations between the protein levels of AR and Cdk5 or p35. These findings demonstrate that Cdk5 is an important modulator of AR and contributes to prostate cancer growth. Therefore, Cdk5-p35 may be suggested as diagnostic and therapeutic targets for prostate cancer in the near future.     

Association of cyclin-dependent kinase inhibitor 2A/B with increased risk of developing breast cancer

There is a growing body of data reporting the association of genetic alterations in chromosome 9P21 with the risk of developing cancer. In the current study, we studied the association of a genetic variant in CDKN2A/B, rs1333049, with the risk of developing breast cancer. A total of 339 participants with and without breast cancer entered to the study. Genotyping was done by the TaqMan real-time polymerase chain reaction (RT-PCR) method and gene expression analysis was ran by RT-PCR. Our data showed that the minor allele homozygote in the total population was 10%, whereas for heterozygote was 38%. The dominant genetic model demonstrated that individuals with breast cancer had advanced TNM classification. Moreover, the logistic regression revealed that individuals who had CC/CG genotypes might have an enhanced risk of developing breast cancer when compared to the holders of GG genotype (e.g., OR = 2.8; 95% CI,1.4–5.4; p = .001), after regulated for confounders; age and body mass index. Furthermore, our analysis showed that the CDKN2A/B gene was downregulated in patients (p < .001). We showed a meaningful relationship of CDKN2A/B with the risk of breast cancer, cancer, showing the importance of studies in great sample size and several centers for studying the value of the marker as a risk classification in the management of patients with breast cancer.     

Evidence for amylase release by cyclin-dependent kinase 5 in the rat parotid

Cyclin-dependent kinase 5 (Cdk5) plays no apparent role in cell cycle regulation, and Cdk5 is not activated by cyclins but only p35 or p39. Although the enzymatic activity of Cdk5 is highest in the central nervous system, recent reports indicate that it also has important functions in non-neuronal cells. In the present study, we investigated whether Cdk5 and its activators are expressed in rat parotid acinar cells, whether a β-adrenergic agonist enhances the expression of Cdk5, and whether Cdk5 mediates amylase release. We found that Cdk5 and its activator, cyclin I, were expressed in rat parotid acinar cells, and that the expression of Cdk5 was enhanced by treatment of the cells with isoproterenol. Amylase release stimulated by isoproterenol was depressed by the addition of olomoucine, a Cdk5 inhibitor, or by the introduction of an anti-Cdk5 antibody. Cdk5 activity was enhanced by treatment with isoproterenol and this enhanced activity was attenuated by the addition of olomoucine. Olomoucine also attenuated both phosphorylation of Munc18c and translocation of Munc18c from the plasma membrane induced by isoproterenol. These results indicated that β-stimulation of rat parotid acinar cells enhanced the expression of Cdk5, and that this Cdk5 activation may mediate amylase release through phosphorylation of Munc18c.     

Snail mRNA及其蛋白在乳腺癌中的表达研究

目的观察Snail蛋白及mRNA在人乳腺癌组织中的表达及其与临床病理特征的关系,并探讨它在乳腺癌发生、发展及转移中的作用。方法应用SP免疫组织化学和原位分子杂交方法检测Snail蛋白和Snail mRNA在70例乳腺浸润性导管癌、30例乳腺导管内癌、30例乳腺单纯性增生组织中的表达。结果①70例乳腺癌中,Snail蛋白和Snail mRNA阳性率分别为87.2%(61/70)、81.4%(57/70),分别高于乳腺导管内癌组织53.3%(16/30)、46.7%(14/30)和乳腺单纯性增生组织26.7%(8/30)、23.3%(7/30),三者相比有统计学意义(χ2=36.4,P<0.01;χ2=32.4,P<0.01)。②Snail蛋白和SnailmRNA在有淋巴结转移组中的阳性率为97.6%(40/41)、95.1%(39/41),无转移组阳性率为72.4%(21/29)、62.1%(18/29),两者相比有统计学意义(χ2=8.29,P<0.01);组织学分级Ⅲ级组明显高于Ⅰ、Ⅱ级组表达,但无统计学意义(χ2=0.72,P>0.05;χ2=0.98,P>0.05)。③Snail蛋白与Snail mRNA的表达与年龄、肿瘤大小均无关(P>0.05)。结论 Snail蛋白与Snail mRNA的表达呈正相关,与乳腺癌的发生发展、淋巴结转移密切相关,可作为判断乳腺癌预后、转移的生物学标志。     

miR-122-5p promotes aggression and epithelial-mesenchymal transition in triple-negative breast cancer by suppressing charged multivesicular body protein 3 through mitogen-activated protein kinase signaling

Triple-negative breast cancer (TNBC) is highly metastatic and frequently has a poor prognosis. The lack of comprehension of TNBC and gene therapy targets has led to limitedly effective treatment for TNBC. This study was conducted to better understand the molecular mechanism behind TNBC progression, and to find out promising gene therapy targets for TNBC. Herein the influence of miR-122-5p's binding charged multivesicular body protein 3 (CHMP3) 3′-untranslated region (3′-UTR) on in TNBC cells was investigated. in vitro experiments quantitative real-time polymerase chain reaction, immunoblot analysis, dual-luciferase reporter gene assay, cell counting assay, transwell invasion assay, and flow cytometry-determined cell apoptosis assay were employed. We also used TargetScan Human 7.2 database to find out the target relationship between miR-122-5p and CHMP3 3′-UTR. TImer algorithm was used to provide an overview of the expression of CHMP3 gene across human pan-cancer, to predict the survival outcome of breast cancer patients, and to predict the correlation between CHMP3 gene expression and epithelial-mesenchymal transition (EMT) and mitogen-activated protein kinase (MAPK)-related gene expression. CHMP3 gene was significantly downregulated across a wide range of human cancers including breast cancer (BRCA). A higher level of CHMP3 gene predicted a better 3- and 5-year survival outcome of patients with BRCA. In our experiments, miR-122-5p was significantly upregulated and CHMP3 gene was significantly downregulated in TNBC cells compared with normal cell line. miR-122-5p mimics enhanced TNBC cell viability, proliferation, and invasion whereas the upregulation of CHMP3 gene led to an opposite outcome. Forced expression of miR-122-5p suppressed cell apoptosis, compelled EMT and MAPK signaling whereas forced expression of CHMP3 did the opposite. We then conclude that miR-122-5p promotes aggression and EMT in TNBC by suppressing CHMP3 through MAPK signaling.     

A prognostic eight-lncRNA expression signature in predicting recurrence of ER-positive breast cancer receiving endocrine therapy

Long noncoding RNAs (lncRNAs) have the main role in the tumorigenesis of breast cancer. In the present study, lncRNA expression profiling was collected to identify a lncRNA expression signature from the Gene Expression Omnibus database. An eight-lncRNA signature was established to predict the survival of patients with estrogen receptor (ER)-positive breast cancer receiving endocrine therapy. Patients were separated into a low-risk group and a high-risk group based on this signature. Patients in high-risk group have worse survival compared to those in low-risk group using Kaplan–Meier curve analysis with log-rank test. Receiver operating characteristic analysis suggested good diagnostic efficiency of the eight-lncRNA signature. When adjusting the clinical features, including age, grade, lymph node status, and tumor size, this signature was independently associated with the relapse-free survival. The prognostic value of the lncRNA prognostic model was then validated in validation sets. When validated in a cohort of patients treated with neoadjuvant chemotherapy and endocrine therapy, this signature demonstrated good performance as well. Besides, we have built a nomogram that integrated the conventional clinicopathological features and the eight-lncRNA-based signature. To sum up, our results indicated that the eight-lncRNA prognostic model was a reliable tool to group patients at high and low risk of disease relapse. This signature may have possible implication in prognostic evaluations of patients with ER-positive breast cancer receiving endocrine therapy.     

Nuclear NHERF1 expression as a prognostic marker in breast cancer

Our purpose was to investigate whether Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1) expression could be linked to prognosis in invasive breast carcinomas. NHERF1, an ezrin-radixin-moesin (ERM) binding phosphoprotein 50, is involved in the linkage of integral membrane proteins to the cytoskeleton. It is therefore believed to have an important role in cell signaling associated with changes in cell cytoarchitecture. NHERF1 expression is observed in various types of cancer and is related to tumor aggressiveness. To date the most extensive analyses of the influence of NHERF1 in cancer development have been performed on breast cancer. However, the underlying mechanism and its prognostic significance are still undefined. NHERF1 expression was studied by immunohistochemistry (IHC) in a cohort of 222 breast carcinoma patients. Association of cytoplasmic and nuclear NHERF1 expression with survival was analyzed. Disease-free survival (DFS) and overall survival (OS) were determined based on the Kaplan–Meier method. Cytoplasmic NHERF1 expression was associated with negative progesterone receptor (PgR) (P=0.017) and positive HER2 expression (P=0.023). NHERF1 also showed a nuclear localization and this correlated with small tumor size (P=0.026) and positive estrogen receptor (ER) expression (P=0.010). Multivariate analysis identified large tumor size (P=0.011) and nuclear NHERF1 expression (P=0.049) to be independent prognostic variables for DFS. Moreover, the nuclear NHERF1(−)/ER(−) immunophenotype (27%) was statistically associated with large tumor size (P=0.0276), high histological grade (P=0.0411), PgR-negative tumors (P<0.0001) and high proliferative activity (P=0.0027). These patients had worse DFS compared with patients with nuclear NHERF1(+)/ER(+) tumors (75.4% versus 92.6% P=0.010). These results show that the loss of nuclear NHERF1 expression is associated with reduced survival, and the link between nuclear NHERF1 and ER expression may serve as a prognostic marker for the routine clinical management of breast cancer patients.     

The cyclin-dependent kinase inhibitor roscovitine inhibits kinase activity, cell proliferation, multicellular development, and Cdk5 nuclear translocation in Dictyostelium discoideum

Roscovitine, a cyclin-dependent kinase (Cdk) inhibitor, inhibited kinase activity and the axenic growth of Dictyostelium discoideum at micromolar concentrations. Growth was almost fully rescued in 50 μM and ≈ 50% rescued in 100 μM roscovitine-treated cultures by the over-expression of Cdk5-GFP. This supports the importance of Cdk5 function during cell proliferation in Dictyostelium and indicates that Cdk5 is a primary target of the drug. Roscovitine did not affect the expression of Cdk5 protein during axenic growth but did inhibit its nuclear translocation. This novel result suggests that the effects of roscovitine could be due in part to altering Cdk5 translocation in other systems as well. Kinase activity was inhibited by roscovitine in assays using AX3 whole cell lysates, but not in assays using lysates from Cdk5-GFP over-expressing cells. At higher concentrations, roscovitine impaired slug and fruiting body formation. Fruiting bodies that did form were small and produced relatively fewer spores many of which were round. However, roscovitine did not affect stalk cell differentiation. Together with previous findings, these data reveal that roscovitine inhibits Cdk5 during growth and as yet undefined Cdks during mid-late development.     

Twice switched at birth: cell cycle-independent roles of the "neuron-specific" cyclin-dependent kinase 5 (Cdk5) in non-neuronal cells

Cdk5 (cyclin-dependent kinase 5 or initially NCLK for neuronal CDC2-like kinase) was switched twice at its birth nearly twenty years ago: first it was thought to be cyclin-dependent, second it was assumed to be primarily of importance in neuronal cells—both turned out not to be the case. In this review we want to discuss issues of pharmacological inhibition, to highlight the versatile roles, and to summarize the growing evidence for the functional importance of Cdk5 in non-neuronal tissues, such as blood cells, tumor cells, epithelial cells, the vascular endothelium, testis, adipose and endocrine tissues. The organizing principles we follow are apoptosis/cell death, migration/motility, aspects of inflammation, and, finally, secretion/metabolism.     

Identification of a 4‐mRNA metastasis‐related prognostic signature for patients with breast cancer

Metastasis‐related mRNAs have showed great promise as prognostic biomarkers in various types of cancers. Therefore, we attempted to develop a metastasis‐associated gene signature to enhance prognostic prediction of breast cancer (BC) based on gene expression profiling. We firstly screened and identified 56 differentially expressed mRNAs by analysing BC tumour tissues with and without metastasis in the discovery cohort (GSE102484, n = 683). We then found 26 of these differentially expressed genes were associated with metastasis‐free survival (MFS) in the training set (GSE20685, n = 319). A metastasis‐associated gene signature built using a LASSO Cox regression model, which consisted of four mRNAs, can classify patients into high‐ and low‐risk groups in the training cohort. Patients with high‐risk scores in the training cohort had shorter MFS (hazard ratio [HR] 3.89, 95% CI 2.53‐5.98; P < 0.001), disease‐free survival (DFS) (HR 4.69, 2.93‐7.50; P < 0.001) and overall survival (HR 4.06, 2.56‐6.45; P < 0.001) than patients with low‐risk scores. The prognostic accuracy of mRNAs signature was validated in the two independent validation cohorts (GSE21653, n = 248; GSE31448, n = 246). We then developed a nomogram based on the mRNAs signature and clinical‐related risk factors (T stage and N stage) that predicted an individual's risk of disease, which can be assessed by calibration curves. Our study demonstrated that this 4‐mRNA signature might be a reliable and useful prognostic tool for DFS evaluation and will facilitate tailored therapy for BC patients at different risk of disease.     

Protein kinase CK2 modulates IL-6 expression in inflammatory breast cancer

Inflammatory breast cancer is driven by pro-angiogenic and pro-inflammatory cytokines. One of them Interleukin-6 (IL-6) is implicated in cancer cell proliferation and survival, and promotes angiogenesis, inflammation and metastasis. While IL-6 has been shown to be upregulated by several oncogenes, the mechanism behind this phenomenon is not well characterized. Here we demonstrate that the pleotropic Serine/Threonine kinase CK2 is implicated in the regulation of IL-6 expression in a model of inflammatory breast cancer. We used siRNAs targeted toward CK2 and a selective small molecule inhibitor of CK2, CX-4945, to inhibit the expression and thus suppress the secretion of IL-6 in in vitro as well as in vivo models. Moreover, we report that in a clinical trial, CX-4945 was able to dramatically reduce IL-6 levels in plasma of an inflammatory breast cancer patient. Our data shed a new light on the regulation of IL-6 expression and position CX-4945 and potentially other inhibitors of CK2, for the treatment of IL-6-driven cancers and possibly other diseases where IL-6 is instrumental, including rheumatoid arthritis.     

Clinical significance of miR-140-5p and miR-193b expression in patients with breast cancer and relationship to IGFBP5

The functional role of IGFBP5 in breast cancer is complicated. Experimental and bioinformatics studies have shown that IGFBP5 is targeted by miR-140-5p and miR-193b, although this has not yet been proven in clinical samples. The aim of this study was to evaluate the expression of miR-140-5p and miR-193b in breast cancer and adjacent normal tissue and assess its correlation with IGFBP5 and the clinicopathological characteristics of the tumors. IGFBP5 protein expression was analyzed immunohistochemically and IGFBP5, miR-140 and miR-193b mRNA expression levels were analyzed with real-time RT-PCR. Tumor tissue had higher miR-140-5p expression than adjacent normal tissue (p = 0.015). Samples with no immunohistochemical staining for IGFBP5 showed increased miR-140-5p expression (p = 0.009). miR-140-5p expression was elevated in invasive ductal carcinomas (p = 0.002), whereas basal-like tumors had decreased expression of miR-140-5p compared to other tumors (p = 0.008). Lymph node-positive samples showed an approximately 13-fold increase in miR-140-5p expression compared to lymph node-negative tissue (p = 0.049). These findings suggest that miR-140-5p, but not miR-193b, could be an important determinant of IGFBP5 expression and clinical phenotype in breast cancer patients. Further studies are needed to clarify the expressional regulation of IGFBP5 by miR-140-5p.     

Eg5 high expression predicts dismal prognosis in epithelial ovarian cancer

The expression of kinesin spindle protein (Eg5) and its significance of clinical prognosis of patients with epithelial ovarian cancer (EOC) were evaluated in this study. Forty-five fresh frozen tissue samples for quantitative real-time polymerase chain reaction (qPCR) and 196 samples for immunohistochemistry (IHC) analysis with tissue microarray (TMA) were applied to characterize Eg5 mRNA and protein expressions in EOC. The correlation between clinical parameters and Eg5 protein expression was investigated using statistical analysis. The expression of Eg5 protein was significantly higher in EOC tissues compared with that in corresponding non-cancerous tissues (P < 0.05). The high Eg5 expression was significantly correlated with older age (P = 0.003), higher stage (P = 0.001), presence of metastasis (P = 0.041) and higher CA125 serum level (P = 0.013). For univariate analysis, associated prognostic markers in patients with ovarian cancer were analyzed for correlations with poor overall survival, including Eg5 (P = 0.011), age (P = 0.001), FIGO stage (P = 0.011), CA125 serum level (P = 0.001), lymph nodes (P = 0.012), and metastasis (P = 0.001). For multivariate analysis, Eg5 expression, FIGO stage and age were independent factors found contributing to a largely unfavorable prognosis in patients with ovarian cancer. In conclusion, the high expression of Eg5 is correlated with an unfavorable prognosis in EOC.     

Cdk5 promotes DNA replication stress checkpoint activation through RPA-32 phosphorylation,and impacts on metastasis free survival in breast cancer patients

Cyclin dependent kinase 5 (Cdk5) is a determinant of PARP inhibitor and ionizing radiation (IR) sensitivity. Here we show that Cdk5-depleted (Cdk5-shRNA) HeLa cells show higher sensitivity to S-phase irradiation, chronic hydroxyurea exposure, and 5-fluorouracil and 6-thioguanine treatment, with hydroxyurea and IR sensitivity also seen in Cdk5-depleted U2OS cells. As Cdk5 is not directly implicated in DNA strand break repair we investigated in detail its proposed role in the intra-S checkpoint activation. While Cdk5-shRNA HeLa cells showed altered basal S-phase dynamics with slower replication velocity and fewer active origins per DNA megabase, checkpoint activation was impaired after a hydroxyurea block. Cdk5 depletion was associated with reduced priming phosphorylations of RPA32 serines 29 and 33 and SMC1-Serine 966 phosphorylation, lower levels of RPA serine 4 and 8 phosphorylation and DNA damage measured using the alkaline Comet assay, gamma-H2AX signal intensity, RPA and Rad51 foci, and sister chromatid exchanges resulting in impaired intra-S checkpoint activation and subsequently higher numbers of chromatin bridges. In vitro kinase assays coupled with mass spectrometry demonstrated that Cdk5 can carry out the RPA32 priming phosphorylations on serines 23, 29, and 33 necessary for this checkpoint activation. In addition we found an association between lower Cdk5 levels and longer metastasis free survival in breast cancer patients and survival in Cdk5-depleted breast tumor cells after treatment with IR and a PARP inhibitor. Taken together, these results show that Cdk5 is necessary for basal replication and replication stress checkpoint activation and highlight clinical opportunities to enhance tumor cell killing.