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Ovarian cancer typically presents at an advanced stage, and although the majority of cases initially respond well to platinum-based therapies, chemoresistance almost always occurs leading to a poor long-term prognosis. While various cellular autonomous mechanisms contribute to intrinsic or acquired platinum resistance, the tumour microenvironment (TME) plays a central role in resistance to therapy and disease progression by providing cancer stem cell niches, promoting tumour cell metabolic reprogramming, reducing chemotherapy drug perfusion and promoting an immunosuppressive environment. As such, the TME is an attractive therapeutic target which has been the focus of intense research in recent years. This review provides an overview of the unique ovarian cancer TME and its role in disease progression and therapy resistance, highlighting some of the latest preclinical and clinical data on TME-targeted therapies. In particular, it focuses on strategies targeting cancer-associated fibroblasts, tumour-associated macrophages, cancer stem cells and cancer cell metabolic vulnerabilities.

Genomic technologies are increasingly used clinically for both diagnosis and guiding cancer therapy. However, formalin fixation can compromise DNA quality. This study aimed to optimise tissue fixation using normal colon, liver and uterus (n=8 each) by varying neutral buffered formalin (NBF) concentration (1%–5% w/v) and fixation time (24–48 hours). Fixation using 4% NBF improved DNA quality (assessed by qPCR) compared with routine (4% unbuffered formal saline-fixed) specimens (p

Eicosanoids comprise a diverse group of bioactive lipids which orchestrate inflammation, immunity, and tissue homeostasis, and whose dysregulation has been implicated in carcinogenesis. Among the various eicosanoid metabolic pathways, studies of their role in endometrial cancer (EC) have very much been confined to the COX-2 pathway. This study aimed to determine changes in epithelial eicosanoid metabolic gene expression in endometrial carcinogenesis; to integrate these with eicosanoid profiles in matched clinical specimens; and, finally, to investigate the prognostic value of candidate eicosanoid metabolic enzymes. Eicosanoids and related mediators were profiled using liquid chromatography-tandem mass spectrometry in fresh frozen normal, hyperplastic, and cancerous (types I and II) endometrial specimens (n = 192). Sample-matched epithelia were isolated by laser capture microdissection and whole genome expression analysis was performed using microarrays. Integration of eicosanoid and gene expression data showed that the accepted paradigm of increased COX-2-mediated prostaglandin production does not apply in EC carcinogenesis. Instead, there was evidence for decreased PGE

Laser capture microdissection (LCM) allows expression profiling of specific cell populations within tissues. However, isolation of high-quality RNA from laser capture microdissected frozen tissue is beset by problems arising from intrinsic tissue RNase activity. Herein, we describe an optimized staining/LCM/RNA extraction protocol developed for the isolation of epithelial RNA from frozen tissue sections using human endometrial cancer as a model tissue. This method combines excellent, reproducible visualization of tissue morphology with the isolation of high-integrity RNA suitable for downstream applications such as expression microarray analysis. We present quantitative and qualitative RNA data obtained from200 endometrial epithelial samples (normal, hyperplastic, and cancerous), where 92% of samples had RIN values of 7 and above and highlight common pitfalls faced by investigators. This method should also be broadly applicable to a range of other tissue types.

Background: Variations in systemic inflammatory response biomarker levels have been associated with adverse clinical outcome in various malignancies. This study determined the prognostic significance of preoperative neutrophil:lymphocyte (NLR), platelet:lymphocyte (PLR) and monocyte:lymphocyte (MLR) ratios in endometrial cancer. Methods: Clinicopathological and 5-year follow-up data were obtained for a retrospective series of surgically treated endometrial cancer patients (n=605). Prognostic significance was determined for overall (OS) and cancer-specific survival (CSS) using Cox proportional hazards models and Kaplan–Meier analysis. Receiver–operator characteristic and log-rank functions were used to optimise cut-offs. NLR, PLR and MLR associations with clinicopathological variables were determined using non-parametric tests. Results: Applying cut-offs of ⩾2.4 (NLR), ⩾240 (PLR) and ⩾0.19 (MLR), NLR and PLR (but not MLR) had independent prognostic significance. Combining NLR and PLR scores stratified patients into low (NLR-low and PLR-low), intermediate (NLR-high or PLR-high) and high risk (NLR-high and PLR-high) groups: multivariable hazard ratio (HR) 2.51; P

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has long been used as a default reference gene in quantitative mRNA profiling experiments. However, its expression reportedly varies in response to a range of pathophysiological variables (inflammation, oxidative stress, hyperinsulinaemia, hypoxia) which feature prominently in sepsis. We therefore assessed the applicability of using GAPDH as a reference gene for expression studies in sepsis compared to other housekeeping genes (succinate dehydrogenase complex subunit A (SDHA), hypoxanthine phosphoribosyltransferase (HPRT)-1). Severe sepsis resulted in a 42.4-fold increase in median GAPDH expression (P

Estrogen receptor β1 (ERβ1) downregulation occurs in many breast cancers, but the responsible molecular mechanisms remain unclear. Here, we report that levels of ERβ1 expression are negatively regulated by the microRNA miR-92. Expression analysis in a cohort of primary breast tumors confirmed a significant negative correlation between miR-92 and both ERβ1 mRNA and protein. Inhibition of miR-92 in MCF-7 cells increased ERβ1 expression in a dose-dependent manner, whereas miR-92 overexpression led to ERβ1 downregulation. Reporter constructs containing candidate miR-92 binding sites in the 3′-untranslated region (UTR) of ERβ1 suggested by bioinformatics analysis confirmed that miR-92 downregulated ERβ1 via direct targeting of its 3′-UTR. Our results define a potentially important mechanism for downregulation of ERβ1 expression in breast cancer. Cancer Res; 70(11); 4778–84. ©2010 AACR.

Purpose: Tamoxifen remains therapy of choice for premenopausal estrogen receptor α–positive breast cancer. However, resistance and recurrence are serious problems. Our previous work indicated that carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) was significantly up-regulated in tamoxifen-resistant (TAMr) MCF-7 derivatives. The aim of this study was to determine the functional role of CEACAM6 in endocrine-resistant breast cancer and to retrospectively test whether it was predictive of resistance in a large cohort of breast cancers with long-term follow-up. Experimental Design: siRNA silencing of CEACAM6 was done in TAMr cells and effects on clonogenicity and endocrine sensitivity were determined. CEACAM6 immunohistochemistry was done on a tissue microarray comprising 108 relapsed primary human breast cancers and 243 tamoxifen-sensitive controls. Results: siRNA-mediated silencing of CEACAM6 reduced both clonogenicity and anchorage-dependent and anchorage-independent growth of TAMr cells. Importantly, CEACAM6 silencing restored sensitivity of TAMr cells to 4-hydroxytamoxifen and proliferative response to 17β-estradiol. Immunohistochemistry showed significantly more CEACAM expression in the relapsed group compared with nonrelapsed controls [35 of 108 (33.3%) and 32 of 243 (13.2%), respectively; odds ratio, 3.16 (95% confidence interval, 1.83-5.47); P < 0.0001]. Additionally, we derived an outcome predictor model based on CEACAM expression that restratified patients in the Nottingham prognostic index intermediate-risk group into either higher-risk or lower-risk group. Conclusions: Our data support an important role for CEACAM6 in endocrine resistance, which can serve as a powerful predictor of future recurrence.

Laser capture microdissection of frozen tissue sections allows homogeneous cell populations to be isolated for expression profiling. However, this requires striking a balance between retaining adequate morphology for accurate microdissection and maintaining RNA integrity. Various staining protocols were applied to frozen endometrial carcinoma tissue sections. Although alcohol-based methods were superior to aqueous stains for maintaining RNA integrity, they suffered from irreproducible staining intensity. We developed a modified alcohol-based, buffered cresyl violet staining protocol that provides reproducible staining with minimal RNA degradation suitable for tissues with moderate to high levels of intrinsic RNase activity.

Genomic representational difference analysis (RDA) was carried out on a total of nine Wilms tumors and one cystic partially differentiated nephroblastoma (CPDN; a sub-type of Wilms) to look for novel genetic deletions involving tumor suppressor genes. Genomic DNA from either short-term cultured Wilms tumor cells or a WT xenograft was used to create driver representations, and genomic DNA from matched normal kidney or normal kidney cells grown in short-term culture was used to create the tester. Genuine difference products were obtained from only one of the tumors. However, none of these fragments were found to be deleted in the original tumor biopsy, microdissected tumor or in the lung metastasis from this patient. It is, therefore, likely that the deletions were due to random losses associated with the genetic instability of the cultured cells from this particular tumor. We did not isolate difference products from any of the other tumors, showing that they did not have chromosomal losses, homozygous deletions or regions of LOH that were detectable by RDA.

Germ cell tumors (GCTs) are rare but clinically and pathologically diverse tumors that occur in an extensive range of age groups, from children to older adults and which include both seminomatous and nonseminomatous tumors. Current clinical management for both male and female teenagers and young adults (TYAs) with GCTs remains inconsistent, alternating between pediatric and adult multidisciplinary oncology teams, based on locally defined age cutoffs. Therefore, we reviewed available literature to determine the biological similarities and differences between GCTs in young children (0-12 years), TYAs (13-24 years), and older adults (24 years). GCTs arising in pediatric and adult populations in general showed marked molecular biological differences within identical histological subtypes, whereas there was a distinct paucity of available data for GCTs in the TYA population. These findings highlight that clinical management based simply on chronological age may be inappropriate for TYA and suggests that the optimal future management of GCTs should consider specific molecular biological factors in addition to clinical parameters in the context of patient-specific age group rather than medical specialty.

Cytokines are key regulators of ovarian physiology, particularly in relation to folliculogenesis and ovulation, where they contribute to creating an environment supporting follicle selection and growth. Their manifold functions include regulating cellular proliferation/differentiation, follicular survival/atresia, and oocyte maturation. Several cytokines, such as TGF-β-superfamily members, are involved at all stages of folliculogenesis while the production of others is stage-dependent. This review draws upon evidence from both human and animal models to highlight the species-specific roles at each milestone of follicular development. Given these pivotal roles and their ease of detection in follicular fluid, cytokines have been considered as attractive biomarkers of oocyte maturational status and of successful assisted reproductive outcome. Despite this, our understanding of cytokines and their interactions remains incomplete, and is still frequently limited to overly simplistic descriptions of their interrelationships. Given our increased appreciation of cytokine activity in complex and highly regulated networks, we put forward the case for using Bayesian modelling approaches to describe their hierarchical relationships in order to predict causal physiological interactions in vivo.

ERβ1 is often down-regulated in breast cancer compared to normal breast but mechanisms surrounding this are unclear. We examined whether loss of heterozygosity (LOH) or methylation at ERβ promoters (0N, 0K) and/or untranslated exon 0N were involved in ERβ down-regulation in breast cancer tissues and cell lines and if treatment with the de-methylating agent 5-aza-deoxycytidine and/or the histone deacetylase inhibitor Trichostatin A could influence expression in vitro. We found no evidence of correlation between LOH at 14q22-24 (genomic locus containing ERβ/ESR2), and ERβ1 expression in primary breast cancers. A negative correlation between ERβ1 mRNA expression and methylation status was observed for promoter 0N in BT-20, MDA-MB-453 and T47D cells. Promoter 0K was consistently unmethylated. In primary breast tumours, methylation of the untranslated exon 0N, downstream of promoter 0N, but not of promoter 0N itself, correlated with down-regulation of ERβ. In MDA-MB-453 cells, treatment with 5-aza-deoxycytidine was sufficient to induce ERβ1 expression from the 0N promoter while in BT-20 both agents were required. Examination of various sites on ESR2 highlighted epigenetic but not genetic regulation of ERβ1. In particular methylation adjacent to promoter 0N was a key regulatory event for ERβ1 silencing. A combination of de-methylating agents and histone deacetylase inhibitors fully restored ERβ1 expression which may offer a novel therapeutic angle for breast cancer management.

Estrogen receptor (ER) action is modulated by posttranslational modifications. Although ERalpha phosphorylation correlates with patient outcome, ERbeta is similarly phosphorylated but its significance in breast cancer has not been addressed. We investigated whether ERbeta that is phosphorylated at serine 105 (S105-ERbeta) is expressed in breast cancer and assessed potential clinical implications of this phosphorylation. Following antibody validation, S105-ERbeta expression was studied in tissue microarrays comprising 108 tamoxifen-resistant and 351 tamoxifen-sensitive cases and analyzed against clinical data. S105-ERbeta regulation in vitro was assessed by Western blot, flow cytometry, and immunofluorescence. Nuclear S105-ERbeta was observed in breast carcinoma and was associated with better survival (Allred scoreor =3), even in tamoxifen-resistant cases, and additionally correlated with ERbeta1 and ERbeta2 expression. Distinct S105-ERbeta nuclear speckles were seen in some higher grade tumors. S105-ERbeta levels increased in MCF-7 cells in response to 17beta-estradiol, the ERbeta-specific agonist diarylpropionitrile, and the partial ERbeta-agonist genistein. S105-ERbeta nuclear speckles were also seen in MCF-7 cells and markedly increased in size and number at 24 hours following 17beta-estradiol and, in particular diarylpropionitrile, treatment. These speckles were coexpressed with ERbeta1 and ERbeta2. Presence of S105-ERbeta in breast cancer and association with improved survival, even in endocrine resistant breast tumors suggest S105-ERbeta might be a useful additional prognostic marker in this disease.

Although ERs (oestrogen receptors) mediate breast tumour behaviour, the precise role of ERbeta remains unclear. This is mainly because analyses have been complicated by the presence in breast tissue of three ERbeta protein variants (ERbeta1, ERbeta2 and ERbeta5) that derive from differential 3' splicing. We have recently identified the first known mechanisms responsible for the differential control of isoform expression, involving regulation of translation via 5'-UTRs (untranslated regions). In the present study, we have uncovered further complexity involving the influence of multiple promoters and cross-talk between 5'- and 3'-UTRs. We demonstrate that full-length ERbeta mRNAs are transcribed from three separate promoters; two promoters are well-established within the literature, whereas the third represents a novel finding. Each promoter produces transcripts with distinct 5'-UTRs. The differential 3' splicing that produces transcripts coding for the ERbeta isoforms also defines isoform-specific 3'-UTRs. We identified exact 3'-UTR sequences for each isoform, and have shown that alternative polyadenylation sites are used in a cell-type specific manner to produce transcripts with 3'-UTRs of different lengths. Critically, we show that 5'- and 3'-UTRs combine to specify the efficiencies with which individual transcripts are translated, with 3'-UTR length having a key influence. In addition, we demonstrate how 17beta-oestradiol, a key driver of breast cancer development, affects the regulation of ERbeta expression at both transcriptional and translational levels.

Background: Despite many published studies on ERβ, progress towards understanding its role in breast cancer remains slow. This is largely due to discordant data between mRNA and protein studies as well as failure to take into account the biologically distinct ERβ isoforms and their heterogeneous expression profile. Methods: We compared expression of ERβ, -2 and -5 genes in HB2 and MCF-7 breast cell lines, primary breast fibroblasts (n=5) and whole tissue and laser microdissected epithelial and stromal cells obtained from 25 human breast tumours. Results: Our study shows that the level of gene expression of ERβ isoforms depends on the cell population within a given tumour and varies dramatically in different cellular compartments. This has implications for gene expression analyses and could explain some of the contradictory data published to date, rendering “grind and bind” analyses of ERβ uninformative. Conclusions: With the technology now available, we suggest a more refined approach be adopted to help resolve some of the controversy surrounding ERβ.

Tamoxifen resistance (TAMr) in breast cancer is a serious clinical dilemma, with no satisfactory explanation. We hypothesised that changes in the expression of steroid hormone receptors (ERalpha, ERbeta), their downstream target genes (PR, pS2) and their associated co-regulators (AIB-1, SRC-1, SRA, NCoR-1, SMRT and REA) could be related to the acquisition of TAMr. To test this hypothesis, we developed in vitro TAMr cell line models by continuous exposure of MCF-7 cells to 4-hydroxytamoxifen (4-HT) over 12 (MCF-7MMU1) and 21 (MCF-7MMU2) months, respectively and examined the expression of the above by Western blotting and immunohistochemistry. In addition, we further examined the changes in global gene expression in TAMr cells in comparison with TAM-sensitive cells by microarray analysis. We report here that acquisition of TAMr is associated with changes in the expression of PR, pS2 and several co-activators, but not ERs. In addition, genes associated with cell cycle, cell adhesion and extracellular matrix, were up-regulated while those associated with apoptosis or growth factors/hormones were down-regulated. Based on our results, it appears that increased co-activator expression, in concert with alterations in genes associated with controlling cell proliferation and survival contribute to TAMr in breast cancer.

Nucleotide excision repair is the principal mechanism for the removal of bulky DNA adducts caused by a range of chemotherapeutic drugs, and contributes to cisplatin resistance. In this study, we used synthetic siRNAs targeted to XPA and ERCC1 and compared their effectiveness in sensitising mismatch repair deficient prostate cancer cell lines to cisplatin and mitomycin C. Downregulation of ERCC1 sensitised DU145 and PC3 cells to cisplatin and mitomycin C. In contrast, XPA downregulation did not sensitise either cell line to mitomycin C, and only sensitised DU145 cells to cisplatin. The effects of ERCC1 downregulation may be due to its role in homologous recombination repair. Excision repair of cisplatin adducts in PC3 cells was attenuated to a similar extent by XPA and ERCC1 downregulation. Downregulation of XPA but not ERCC1 caused an increase in the number of cisplatin-induced RAD51 foci in PC3 cells, suggesting that HRR is able to substitute for NER in these cells. We observed co-localisation of ERCC1 and RAD51 in cisplatin treated PC3 cells by immunofluorescence and co-immunoprecipitation, which may represent recruitment of ERCC1/XPF to sites of recombination repair. These results indicate that ERCC1 is a broader therapeutic target than XPA with which to sensitise cancer cells to chemotherapy because of its additional role in recombination repair.

Although most advanced cancers are incurable, the majority of testicular germ cell tumors can be cured using cisplatin-based combination chemotherapy. The nucleotide excision repair (NER) pathway removes most DNA adducts produced by cisplatin, and the low levels of NER in testis tumor cells may explain why these cancers are curable. Three NER proteins: ERCC1, XPF, and XPA, are present at low levels in testis tumor cell lines, and addition of these proteins to protein extracts of testis tumor cells increases their in vitro DNA repair capacity to normal levels. The aim of this study was to identify the mechanism responsible for the low levels of these DNA repair proteins. The levels of the mRNA transcripts for ERCC1, XPF, and XPA were measured in a panel of 14 different human cancer cell lines, using real-time PCR. Three ERCC1 splice variants were identified and quantitated. Three alternative transcription start points (TSPs) were identified for ERCC1 but none were testis-specific. The significantly lower levels of ERCC1, XPF, and XPA protein in testis tumor cell lines cannot be explained solely by differences in transcriptional efficiency or mRNA stability. For ERCC1, post-transcriptional control by alternative splicing does not account for the testis-specific low levels of protein expression. Pulse-chase experiments showed that the half-life of ERCC1 protein in a testis tumor cell line was not significantly different to that in a prostate cancer cell line. Taken together, these results suggest that constitutive levels of these DNA repair proteins are controlled at the level of translation.

Transfection of full-length antisense cDNA is used frequently to achieve stable downregulation of gene expression. However, screening for clones that express the antisense mRNA is complicated by the presence of endogenous sense mRNA. Thus, clones usually are screened for downregulation of the target protein by Western blotting, which can be time consuming. Here, we used strand-specific RT-PCR to identify antisense-expressing clones, which can then be screened for protein downregulation. This approach allows earlier identification of potentially useful clones and cuts down on the number of clones to be screened by Western blotting.

BCR-ABL confers apoptotic resistance to a range of genotoxic agents, and this protection is mediated in part by prolonging the G2 checkpoint. The p53 tumour suppressor protein regulates the transcription of regulatory genes involved in cell cycle arrest and apoptosis. To investigate the effect of p53 on the BCR-ABL-mediated G2M checkpoint response, we transiently transfected the BCR-ABL-positive, p53-negative cell line K562 with wild-type human p53. The p53-transfected cells showed a decreased ability to arrest in G2 and an increase in apoptosis in response to etoposide treatment, relative to the control mock-transfected cells. p53-transfected and control cells were treated with etoposide and trapped at mitosis with nocodazole. The mitotic index of p53-transfected cells was higher than that of the control cells, which suggests that p53 abrogates the G2 checkpoint response to etoposide treatment in K562 cells. We found that the expression of the cell cycle checkpoint protein Chk1 was reduced in the etoposide-treated p53-transfected cells by 24 h, and this correlated with a reduction in the extent of etoposide-induced phosphorylation of CDK1 at tyrosine 15 (Y15). We conclude, therefore, that p53 overrides the strong G2 checkpoint response to etoposide in K562 cells, by directly or indirectly downregulating Chk1 expression, which, in turn, contributes to the proapoptotic effect of p53.