Maria Prencipe

Docetaxel-Resistance in Prostate Cancer: Evaluating Associated Phenotypic Changes and Potential for Resistance Transfer via Exosomes

Background Hormone-refractory prostate cancer remains hindered by inevitable progression of resistance to first-line treatment with docetaxel. Recent studies suggest that phenotypic changes associated with cancer may be transferred from cell-to-cell via microvesicles/exosomes. Here we aimed to investigate phenotypic changes associated with docetaxel-resistance in order to help determine the complexity of this problem and to assess the relevance of secreted exosomes in prostate cancer. Methodology/Principal Findings Docetaxel-resistant variants of DU145 and 22Rv1 were established and characterised in terms of cross-resistance, morphology, proliferation, motility, invasion, anoikis, colony formation, exosomes secretion their and functional relevance. Preliminary analysis of exosomes from relevant serum specimens was also performed. Acquired docetaxel-resistance conferred cross-resistance to doxorubicin and induced alterations in motility, invasion, proliferation and anchorage-independent growth. Exosomes expelled from DU145 and 22Rv1 docetaxel-resistant variants (DU145RD and 22Rv1RD) conferred docetaxel-resistance to DU145, 22Rv1 and LNCap cells, which may be partly due to exosomal MDR-1/P-gp transfer. Exosomes from prostate cancer patients’ sera induced increased cell proliferation and invasion, compared to exosomes from age-matched controls. Furthermore, exosomes from sera of patients undergoing a course of docetaxel treatment compared to matched exosomes from the same patients prior to commencing docetaxel treatment, when applied to both DU145 and 22Rv1 cells, showed a correlation between cellular response to docetaxel and patients’ response to treatment with docetaxel. Conclusions/Significance Our studies indicate the complex and multifaceted nature of docetaxel-resistance in prostate cancer. Furthermore, our in vitro observations and preliminary clinical studies indicate that exosomes may play an important role in prostate cancer, in cell-cell communication, and thus may offer potential as vehicles containing predictive biomarkers and new therapeutic targets.

Nonrandom Distribution of Aberrant Promoter Methylation of Cancer-Related Genes in Sporadic Breast Tumors

Purpose: In an effort to additionally determine the global patterns of CpG island hypermethylation in sporadic breast cancer, we searched for aberrant promoter methylation at 10 gene loci in 54 primary breast cancer and 10 breast benign lesions. Experimental Design: Genomic DNA sodium bisulfate converted from benign and malignant tissues was used as template in methyl-specific PCR for BRCA1, p16, ESR1, GSTP1, TRβ1, RARβ2, HIC1, APC, CCND2, and CDH1 genes. Results: The majority of the breast cancer (85%) showed aberrant methylation in at least 1 of the loci tested with half of them displaying 3 or more methylated genes. The highest frequency of aberrant promoter methylation was found for HIC1 (48%) followed by ESR1 (46%), and CDH1 (39%). Similar methylation frequencies were detected for breast benign lesions with the exception of the CDH1 gene (P = 0.02). The analysis of methylation distribution indicates a statistically significant association between methylation of the ESR1 promoter, and methylation at CDH1, TRβ1, GSTP1, and CCND2 loci (P < 0.03). Methylated status of the BRCA1 promoter was inversely correlated with methylation at the RARβ2 locus (P < 0.03). Conclusions: Our results suggest a nonrandom distribution for promoter hypermethylation in sporadic breast cancer, with tumor subsets characterized by aberrant methylation of specific cancer-related genes. These breast cancer subgroups may represent separate biological entities with potential differences in sensitivity to therapy, occurrence of metastasis, and overall prognosis.

Characterisation and manipulation of docetaxel resistant prostate cancer cell lines

BackgroundThere is no effective treatment strategy for advanced castration-resistant prostate cancer. Although Docetaxel (Taxotere®) represents the most active chemotherapeutic agent it only gives a modest survival advantage with most patients eventually progressing because of inherent or acquired drug resistance. The aims of this study were to further investigate the mechanisms of resistance to Docetaxel. Three Docetaxel resistant sub-lines were generated and confirmed to be resistant to the apoptotic and anti-proliferative effects of increasing concentrations of Docetaxel.ResultsThe resistant DU-145 R and 22RV1 R had expression of P-glycoprotein and its inhibition with Elacridar partially and totally reversed the resistant phenotype in the two cell lines respectively, which was not seen in the PC-3 resistant sublines. Resistance was also not mediated in the PC-3 cells by cellular senescence or autophagy but multiple changes in pro- and anti-apoptotic genes and proteins were demonstrated. Even though there were lower basal levels of NF-κB activity in the PC-3 D12 cells compared to the Parental PC-3, docetaxel induced higher NF-κB activity and IκB phosphorylation at 3 and 6 hours with only minor changes in the DU-145 cells. Inhibition of NF-κB with the BAY 11-7082 inhibitor reversed the resistance to Docetaxel.ConclusionThis study confirms that multiple mechanisms contribute to Docetaxel resistance and the central transcription factor NF-κB plays an immensely important role in determining docetaxel-resistance which may represent an appropriate therapeutic target.

Changes in CpG Islands Promoter Methylation Patterns during Ductal Breast Carcinoma Progression

Aberrant promoter methylation of several known or putative tumor suppressor genes occurs frequently during carcinogenesis, and this epigenetic change has been considered as a potential molecular marker for cancer. We examined the methylation status of nine genes (APC, CDH1, CTNNB1, TIMP3, ESR1, GSTP1, MGMT, THBS1, and TMS1), by quantitative methylation specific PCR. Synchronous preinvasive lesions (atypical ductal hyperplasia and/or ductal carcinoma in situ) and invasive ductal breast carcinoma from 52 patients, together with pure lesions from 24 patients and 12 normal tissues paired to tumor and 20 normal breast distant from tumor were analyzed. Aberrant promoter methylation was detected in both preinvasive and invasive lesions for genes APC, CDH1, CTNNB1, TIMP3, ESR1, and GSTP1. However, hierarchical mixed model and Generalized Estimating Equations model analyses showed that only APC, CDH1, and CTNNB1 promoter regions showed a higher frequency and methylation levels in pathologic samples when compared with normal breast. Whereas APC and CTNNB1 did not show differences in methylation levels or frequencies, CDH1 showed higher methylation levels in invasive tumors as compared with preinvasive lesions (P < 0.04, Mann-Whitney test with permutation correction). The analysis of APC, CDH1, and CTNNB1 methylation status was able to distinguish between normal and pathologic samples with a sensitivity of 67% (95% confidence interval, 60-71%) and a specificity of 75% (95% confidence interval, 69-81%). Our data point to the direct involvement of APC, CDH1, and CTNNB1 promoter methylation in the early stages of breast cancer progression and suggest that they may represent a useful tool for the detection of tumor cells in clinical specimens. (Cancer Epidemiol Biomarkers Prev 2009;18(10):2694–700)

Low MAD2 expression levels associate with reduced progression-free survival in patients with high-grade serous epithelial ovarian cancer

Epithelial ovarian cancer (EOC) has an innate susceptibility to become chemoresistant. Up to 30% of patients do not respond to conventional chemotherapy [paclitaxel (Taxol®) in combination with carboplatin] and, of those who have an initial response, many patients relapse. Therefore, an understanding of the molecular mechanisms that regulate cellular chemotherapeutic responses in EOC cells has the potential to impact significantly on patient outcome. The mitotic arrest deficiency protein 2 (MAD2), is a centrally important mediator of the cellular response to paclitaxel. MAD2 immunohistochemical analysis was performed on 82 high‐grade serous EOC samples. A multivariate Cox regression analysis of nuclear MAD2 IHC intensity adjusting for stage, tumour grade and optimum surgical debulking revealed that low MAD2 IHC staining intensity was significantly associated with reduced progression‐free survival (PFS) (p = 0.0003), with a hazard ratio of 4.689. The in vitro analyses of five ovarian cancer cell lines demonstrated that cells with low MAD2 expression were less sensitive to paclitaxel. Furthermore, paclitaxel‐induced activation of the spindle assembly checkpoint (SAC) and apoptotic cell death was abrogated in cells transfected with MAD2 siRNA. In silico analysis identified a miR‐433 binding domain in the MAD2 3′ UTR, which was verified in a series of experiments. Firstly, MAD2 protein expression levels were down‐regulated in pre‐miR‐433 transfected A2780 cells. Secondly, pre‐miR‐433 suppressed the activity of a reporter construct containing the 3′‐UTR of MAD2. Thirdly, blocking miR‐433 binding to the MAD2 3′ UTR protected MAD2 from miR‐433 induced protein down‐regulation. Importantly, reduced MAD2 protein expression in pre‐miR‐433‐transfected A2780 cells rendered these cells less sensitive to paclitaxel. In conclusion, loss of MAD2 protein expression results in increased resistance to paclitaxel in EOC cells. Measuring MAD2 IHC staining intensity may predict paclitaxel responses in women presenting with high‐grade serous EOC. Copyright

Cellular senescence induced by aberrant MAD2 levels impacts on paclitaxel responsiveness in vitro.

Background:The mitotic arrest deficiency protein 2 (MAD2) is a key component of the mitotic spindle assembly checkpoint, monitoring accurate chromosomal alignment at the metaphase plate before mitosis. MAD2 also has a function in cellular senescence and in a cells response to microtubule inhibitory (MI) chemotherapy exemplified by paclitaxel.Methods:Using an siRNA approach, the impact of MAD2 down-regulation on cellular senescence and paclitaxel responsiveness was investigated. The endpoints of senescence, cell viability, migration, cytokine expression, cell cycle analysis and anaphase bridge scoring were carried out using standard approaches.Results:We show that MAD2 down-regulation induces premature senescence in the MCF7 breast epithelial cancer cell line. These MAD2-depleted (MAD2↓) cells are also significantly replicative incompetent but retain viability. Moreover, they show significantly higher levels of anaphase bridges and polyploidy compared to controls. In addition, these cells secrete higher levels of IL-6 and IL-8 representing key components of the senescence-associated secretory phenotype (SASP) with the ability to impact on neighbouring cells. In support of this, MAD2↓ cells show enhanced migratory ability. At 72 h after paclitaxel, MAD2↓ cells show a significant further induction of senescence compared with paclitaxel naive controls. In addition, there are significantly more viable cells in the MAD2↓ MCF7 cell line after paclitaxel reflecting the observed increase in senescence.Conclusion:Considering that paclitaxel targets actively dividing cells, these senescent cells will evade cytotoxic kill. In conclusion, compromised MAD2 levels induce a population of senescent cells resistant to paclitaxel.

The role of transcription factors in prostate cancer and potential for future RNA interference therapy

Introduction: Prostate cancer is a leading cause of cancer-related death in men and current treatments offer only a modest survival benefit in advanced stages of the disease. RNA interference (RNAi) is a therapeutic option that has received great attention in recent years with the potential to treat a variety of disorders, including prostate cancer. Transcription factors are cellular proteins that can up-regulate or down-regulate the transcription of genes and offer promising therapeutic targets. Areas covered: This review will focus on transcription factors that have been identified as key molecules in drug resistance, disease progression and metastases in prostate cancer, which may offer potential as therapeutic targets for RNAi in the future. Expert opinion: By identifying therapeutically viable transcription factor targets in prostate cancer, it is hoped that treatment strategies using RNAi will augment the effect of current chemotherapy regimens, slow disease progression and reduce metastases in prostate cancer, resulting in disease regression.

The role of epithelial–mesenchymal transition drivers ZEB1 and ZEB2 in mediating docetaxel‐resistant prostate cancer

Docetaxel is the main treatment for advanced castration‐resistant prostate cancer; however, resistance eventually occurs. The development of intratumoral drug‐resistant subpopulations possessing a cancer stem cell (CSC) morphology is an emerging mechanism of docetaxel resistance, a process driven by epithelial–mesenchymal transition (EMT). This study characterised EMT in docetaxel‐resistant sublines through increased invasion, MMP‐1 production and ZEB1 and ZEB2 expression. We also present evidence for differential EMT across PC‐3 and DU145 in vitro resistance models as characterised by differential migration, cell colony scattering and susceptibility to the CSC inhibitor salinomycin. siRNA manipulation of ZEB1 and ZEB2 in PC‐3 and DU145 docetaxel‐resistant sublines identified ZEB1, through its transcriptional repression of E‐cadherin, to be a driver of both EMT and docetaxel resistance. The clinical relevance of ZEB1 was also determined through immunohistochemical tissue microarray assessment, revealing significantly increased ZEB1 expression in prostate tumours following docetaxel treatment. This study presents evidence for a role of ZEB1, through its transcriptional repression of E‐cadherin to be a driver of both EMT and docetaxel resistance in docetaxel‐resistant prostate cancer. In addition, this study highlights the heterogeneity of prostate cancer and in turn emphasises the complexity of the clinical management of docetaxel‐resistant prostate cancer.

The use of LC‐MS to identify differentially expressed proteins in docetaxel‐resistant prostate cancer cell lines

Docetaxel is a taxane‐derived chemotherapy drug that has been approved for treatment of prostate cancer. While docetaxel is frequently used as a treatment for hormone‐refractory prostate cancer, a subset of patients either do not respond to this treatment or those that do respond eventually become resistant to the drug over time. Resistance to docetaxel is complex and multi‐factoral and further understanding of the cellular biochemistry underlying resistance is vital to improve treatment efficacy. To identify proteins altered in the resistant phenotype, three parental cell lines DU145, 22RV1 and PC‐3, as well as their docetaxel resistant sub‐lines, were subjected to quantitative label‐free LC‐MS proteomic profiling. A total of 189 significant (p < 0.05) protein abundance changes were identified in the DU145 resistant sub‐lines, 254 in the 22RV1 sub‐lines, and 51 and 72 in the 8 and 12 nM resistant PC‐3 sub‐lines, respectively. From these, 29 proteins demonstrated a significant (p < 0.05) fold change across two or more resistant variants. These included proteins indicative of an epithelial‐to‐mesenchemyl transition as well as altered heat shock response elements.

Identification of transcription factors associated with castration-resistance: Is the serum responsive factor a potential therapeutic target?

Advanced prostate cancer is treated by hormone ablation therapy. However, despite an initial response, the majority of men relapse to develop castration‐resistant disease for which there are no effective treatments.