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1.  The expression of Dicer and Drosha in matched normal tissues, tumours and lymph node metastases in triple negative breast cancer 
BMC Cancer  2014;14:253.
Background
Breast cancer is the most common malignancy in women world-wide. Triple negative breast cancer (TNBC) is a highly aggressive subtype that lacks expression of hormone receptors for estrogen, progesterone and human epidermal growth factor 2; and is associated with a high propensity for metastatic spread. Several studies have identified critical roles for microRNAs in breast cancer, but the role of two critical enzymes involved in microRNA biogenesis, Dicer and Drosha, is not well understood, particularly with respect to metastatic progression in this subtype.
Methods
We examined the expression of Dicer and Drosha in a series of invasive 35 TNBCs with matched normal adjacent tissues (n = 18) and lymph node metastases (n = 15) using semi-quantitative real time RT-PCR. The relationship of their expression with clinical features including age at diagnosis, lymph node positivity and tumour size was analysed.
Results
We report that Dicer was significantly decreased while Drosha was significantly increased in tumours when compared to normal adjacent tissues. While there was no difference in Drosha expression in lymph node metastases when compared to the primary tumour, Dicer was significantly increased. There was no correlation between the expression of either Dicer or Drosha to age at diagnosis, lymph node positivity and tumour size.
Conclusions
In conclusion, Dicer and Drosha are dysregulated in TNBC and matched lymph node metastases however, the clinical relevance of this is still not known. The altered expression of Dicer and Drosha may serve as markers for disrupted miRNA biogenesis in TNBC.
doi:10.1186/1471-2407-14-253
PMCID: PMC4021460  PMID: 24725360
Dicer; Drosha; Breast cancer; Metastasis; Triple negative
2.  Decreased expression of key tumour suppressor microRNAs is associated with lymph node metastases in triple negative breast cancer 
BMC Cancer  2014;14:51.
Background
Breast cancer is the most common malignancy that develops in women, responsible for the highest cancer-associated death rates. Triple negative breast cancers represent an important subtype that have an aggressive clinical phenotype, are associated with a higher likelihood of metastasis and are not responsive to current targeted therapies. miRNAs have emerged as an attractive candidate for molecular biomarkers and treatment targets in breast cancer, but their role in the progression of triple negative breast cancer remains largely unexplored.
Methods
This study has investigated miRNA expression profiles in 31 primary triple negative breast cancer cases and in 13 matched lymph node metastases compared with 23 matched normal breast tissues to determine miRNAs associated with the initiation of this disease subtype and those associated with its metastasis.
Results
71 miRNAs were differentially expressed in triple negative breast cancer, the majority of which have previously been associated with breast cancer, including members of the miR-200 family and the miR-17-92 oncogenic cluster, suggesting that the majority of miRNAs involved in the initiation of triple negative breast cancer are not subtype specific. However, the repertoire of miRNAs expressed in lymph node negative and lymph node positive triple negative breast cancers were largely distinct from one another. In particular, miRNA profiles associated with lymph node negative disease tended to be up-regulated, while those associated with lymph node positive disease were down-regulated and largely overlapped with the profiles of their matched lymph node metastases. From this, 27 miRNAs were identified that are associated with metastatic capability in the triple negative breast cancer subtype.
Conclusions
These results provide novel insight into the repertoire of miRNAs that contribute to the initiation of and progression to lymph node metastasis in triple negative breast cancer and have important implications for the treatment of this breast cancer subtype.
doi:10.1186/1471-2407-14-51
PMCID: PMC3912930  PMID: 24479446
miRNA; Breast cancer; Triple negative; Metastases; Tumour suppressor
3.  A high-throughput protocol for mutation scanning of the BRCA1 and BRCA2 genes 
BMC Cancer  2011;11:265.
Background
Detection of mutations by DNA sequencing can be facilitated by scanning methods to identify amplicons which may have mutations. Current scanning methods used for the detection of germline sequence variants are laborious as they require post-PCR manipulation. High resolution melting (HRM) is a cost-effective rapid screening strategy, which readily detects heterozygous variants by melting curve analysis of PCR products. It is well suited to screening genes such as BRCA1 and BRCA2 as germline pathogenic mutations in these genes are always heterozygous.
Methods
Assays for the analysis of all coding regions and intron-exon boundaries of BRCA1 and BRCA2 were designed, and optimised. A final set of 94 assays which ran under identical amplification conditions were chosen for BRCA1 (36) and BRCA2 (58). Significant attention was placed on primer design to enable reproducible detection of mutations within the amplicon while minimising unnecessary detection of polymorphisms. Deoxyinosine residues were incorporated into primers that overlay intronic polymorphisms. Multiple 384 well plates were used to facilitate high throughput.
Results
169 BRCA1 and 239 BRCA2 known sequence variants were used to test the amplicons. We also performed an extensive blinded validation of the protocol with 384 separate patient DNAs. All heterozygous variants were detected with the optimised assays.
Conclusions
This is the first HRM approach to screen the entire coding region of the BRCA1 and BRCA2 genes using one set of reaction conditions in a multi plate 384 well format using specifically designed primers. The parallel screening of a relatively large number of samples enables better detection of sequence variants. HRM has the advantages of decreasing the necessary sequencing by more than 90%. This markedly reduced cost of sequencing will result in BRCA1 and BRCA2 mutation testing becoming accessible to individuals who currently do not undergo mutation testing because of the significant costs involved.
doi:10.1186/1471-2407-11-265
PMCID: PMC3146935  PMID: 21702907
4.  P53 in human melanoma fails to regulate target genes associated with apoptosis and the cell cycle and may contribute to proliferation 
BMC Cancer  2011;11:203.
Background
Metastatic melanoma represents a major clinical problem. Its incidence continues to rise in western countries and there are currently no curative treatments. While mutation of the P53 tumour suppressor gene is a common feature of many types of cancer, mutational inactivation of P53 in melanoma is uncommon; however, its function often appears abnormal.
Methods
In this study whole genome bead arrays were used to examine the transcript expression of P53 target genes in extracts from 82 melanoma metastases and 6 melanoma cell lines, to provide a global assessment of aberrant P53 function. The expression of these genes was also examined in extracts derived from diploid human melanocytes and fibroblasts.
Results
The results indicated that P53 target transcripts involved in apoptosis were under-expressed in melanoma metastases and melanoma cell lines, while those involved in the cell cycle were over-expressed in melanoma cell lines. There was little difference in the transcript expression of P53 target genes between cell lines with null/mutant P53 compared to those with wild-type P53, suggesting that altered expression in melanoma was not related to P53 status. Similarly, down-regulation of P53 by short-hairpin RNA (shRNA) had limited effect on P53 target gene expression in melanoma cells, whereas there were a large number of P53 target genes whose mRNA expression was significantly altered by P53 inhibition in melanocytes. Analysis of whole genome gene expression profiles indicated that the ability of P53 to regulate genes involved in the cell cycle was significantly reduced in melanoma cells. Moreover, inhibition of P53 in melanocytes induced changes in gene expression profiles that were characteristic of melanoma cells and resulted in increased proliferation. Conversely, knockdown of P53 in melanoma cells resulted in decreased proliferation.
Conclusions
These results indicate that P53 target genes involved in apoptosis and cell cycle regulation are aberrantly expressed in melanoma and that this aberrant functional activity of P53 may contribute to the proliferation of melanoma.
doi:10.1186/1471-2407-11-203
PMCID: PMC3120805  PMID: 21615965
5.  Variant alleles of the CYP1B1 gene are associated with colorectal cancer susceptibility 
BMC Cancer  2010;10:420.
Background
CYP1B1 is a P450 enzyme which is involved in the activation of pro-carcinogens to carcinogens as well as sex hormone metabolism. Because differences in the activity of the enzyme have been correlated with variant alleles of single nucleotide polymorphisms (SNPs), it represents an attractive candidate gene for studies into colorectal cancer susceptibility.
Methods
We genotyped 597 cancer patients and 597controls for three CYP1B1 SNPs, which have previously been shown to be associated with altered enzymatic activity. Using the three SNPs, eight different haplotypes were constructed. The haplotype frequencies were estimated in cases and controls and then compared. The odds ratio for each tumour type, associated with each haplotype was estimated, with reference to the most common haplotype observed in the controls.
Results
The three SNPs rs10012, rs1056827 and rs1056836 alone did not provide any significant evidence of association with colorectal cancer risk. Haplotypes of rs1056827 and rs10012 or rs1056827 and rs1056836 revealed an association with colorectal cancer which was significantly stronger in the homozygous carriers. One haplotype was under represented in the colorectal cancer patient group compared to the control population suggesting a protective effect.
Conclusion
Genetic variants within the CYP1B1 that are associated with altered function appear to influence susceptibility to a colorectal cancer in Poland. Three haplotypes were associated with altered cancer risk; one conferred protection and two were associated with an increased risk of disease. These observations should be confirmed in other populations.
doi:10.1186/1471-2407-10-420
PMCID: PMC2929240  PMID: 20701755
6.  Toll-Like Receptor (TLR) and Nucleosome-binding Oligomerization Domain (NOD) gene polymorphisms and endometrial cancer risk 
BMC Cancer  2010;10:382.
Background
Endometrial cancer is the most common gynaecological malignancy in women of developed countries. Many risk factors implicated in endometrial cancer trigger inflammatory events; therefore, alterations in immune response may predispose an individual to disease. Toll-like receptors (TLRs) and nucleosome-binding oligomerization domain (NOD) genes are integral to the recognition of pathogens and are highly polymorphic. For these reasons, the aim of the study was to assess the frequency of polymorphic variants in TLR and NOD genes in an Australian endometrial cancer population.
Methods
Ten polymorphisms were genotyped in 191 endometrial cancer cases and 291 controls using real-time PCR: NOD1 (rs2075822, rs2907749, rs2907748), NOD2 (rs5743260, rs2066844, rs2066845), TLR2 (rs5743708), TLR4 (rs4986790) and TLR9 (rs5743836, rs187084).
Results
Haplotype analysis revealed that the combination of the variant alleles of the two TLR9 polymorphisms, rs5743836 and rs187084, were protective for endometrial cancer risk: OR 0.11, 95% CI (0.03-0.44), p = 0.002. This result remained highly significant after adjustment for endometrial cancer risk factors and Bonferroni correction for multiple testing. There were no other associations observed for the other polymorphisms in TLR2, TLR4, NOD1 and NOD2.
Conclusions
The variant 'C' allele of rs5743836 causes greater TLR9 transcriptional activity compared to the 'T' allele, therefore, higher TLR9 activity may be related to efficient removal of microbial pathogens within the endometrium. Clearly, the association of these TLR9 polymorphisms and endometrial cancer risk must be further examined in an independent population. The results point towards the importance of examining immune response in endometrial tumourigenesis to understand new pathways that may be implicated in disease.
doi:10.1186/1471-2407-10-382
PMCID: PMC2918576  PMID: 20646321
7.  Demethylation by 5-aza-2'-deoxycytidine in colorectal cancer cells targets genomic DNA whilst promoter CpG island methylation persists 
BMC Cancer  2010;10:366.
Background
DNA methylation and histone acetylation are epigenetic modifications that act as regulators of gene expression. Aberrant epigenetic gene silencing in tumours is a frequent event, yet the factors which dictate which genes are targeted for inactivation are unknown. DNA methylation and histone acetylation can be modified with the chemical agents 5-aza-2'-deoxycytidine (5-aza-dC) and Trichostatin A (TSA) respectively. The aim of this study was to analyse de-methylation and re-methylation and its affect on gene expression in colorectal cancer cell lines treated with 5-aza-dC alone and in combination with TSA. We also sought to identify methylation patterns associated with long term reactivation of previously silenced genes.
Method
Colorectal cancer cell lines were treated with 5-aza-dC, with and without TSA, to analyse global methylation decreases by High Performance Liquid Chromatography (HPLC). Re-methylation was observed with removal of drug treatments. Expression arrays identified silenced genes with differing patterns of expression after treatment, such as short term reactivation or long term reactivation. Sodium bisulfite sequencing was performed on the CpG island associated with these genes and expression was verified with real time PCR.
Results
Treatment with 5-aza-dC was found to affect genomic methylation and to a lesser extent gene specific methylation. Reactivated genes which remained expressed 10 days post 5-aza-dC treatment featured hypomethylated CpG sites adjacent to the transcription start site (TSS). In contrast, genes with uniformly hypermethylated CpG islands were only temporarily reactivated.
Conclusion
These results imply that 5-aza-dC induces strong de-methylation of the genome and initiates reactivation of transcriptionally inactive genes, but this does not require gene associated CpG island de-methylation to occur. In addition, for three of our selected genes, hypomethylation at the TSS of an epigenetically silenced gene is associated with the long term reversion of gene expression level brought about by alterations in the epigenetic status following 5-aza-dC treatment.
doi:10.1186/1471-2407-10-366
PMCID: PMC2912869  PMID: 20618997
8.  The influence of the Cyclin D1 870 G>A polymorphism as an endometrial cancer risk factor 
BMC Cancer  2008;8:272.
Background
Cyclin D1 is integral for the G1 to S phase of the cell cycle as it regulates cellular proliferation. A polymorphism in cyclin D1, 870 G>A, causes overexpression and supports uncontrollable cellular growth. This polymorphism has been associated with an increased risk of developing many cancers, including endometrial cancer.
Methods
The 870 G>A polymorphisms (rs605965) in the cyclin D1 gene was genotyped in an Australian endometrial cancer case-control population including 191 cases and 291 controls using real-time PCR analysis. Genotype analysis was performed using chi-squared (χ2) statistics and odds ratios were calculated using unconditional logistic regression, adjusting for potential endometrial cancer risk factors.
Results
Women homozygous for the variant cyclin D1 870 AA genotype showed a trend for an increased risk of developing endometrial cancer compared to those with the wild-type GG genotype, however this result was not statistically significant (OR 1.692 95% CI (0.939–3.049), p = 0.080). Moreover, the 870 G>A polymorphism was significantly associated with family history of colorectal cancer. Endometrial cancer patients with the homozygous variant AA genotype had a higher frequency of family members with colorectal cancer in comparison to endometrial cancer patients with the GG and combination of GG and GA genotypes (GG versus AA; OR 2.951, 95% CI (1.026–8.491), p = 0.045, and GG+GA versus AA; OR 2.265, 95% CI (1.048–4.894), p = 0.038, respectively).
Conclusion
These results suggest that the cyclin D1 870 G>A polymorphism is possibly involved in the development of endometrial cancer. A more complex relationship was observed between this polymorphism and familial colorectal cancer.
doi:10.1186/1471-2407-8-272
PMCID: PMC2567988  PMID: 18822177
9.  Inflammatory response gene polymorphisms and their relationship with colorectal cancer risk 
BMC Cancer  2008;8:112.
Backgroud
Patients with chronic inflammatory bowel disease (IBD) are at an increased risk of colorectal cancer (CRC) and it is estimated that one in six persons diagnosed with IBD will develop CRC. This fact suggests that genetic variations in inflammatory response genes may act as CRC disease risk modifiers.
Methods
In order to test this hypothesis we investigated a series of polymorphisms in 6 genes (NOD2, DLG5, OCTN1, OCTN2, IL4, TNFα) associated with the inflammatory response on a group of 607 consecutive newly diagnosed colorectal cancer patients and compared the results to controls (350 consecutive newborns and 607 age, sex and geographically matched controls).
Results
Of the six genes only one polymorphism in TNFα(-1031T/T) showed any tendency to be associated with disease risk (64.9% for controls and 71.4% for CRC) which we further characterized on a larger cohort of CRC patients and found a more profound relationship between the TNFα -1031T/T genotype and disease (64.5% for controls vs 74.7% for CRC cases above 70 yrs). Then, we investigated this result and identified a suggestive tendency, linking the TNFα -1031T/T genotype and a previously identified change in the CARD15/NOD2 gene (OR = 1.87; p = 0,02 for CRC cases above 60 yrs).
Conclusion
The association of polymorphisms in genes involved in the inflammatory response and CRC onset suggest that there are genetic changes capable of influencing disease risk in older persons.
doi:10.1186/1471-2407-8-112
PMCID: PMC2386482  PMID: 18433468
10.  Ovarian cancer risk in Polish BRCA1 mutation carriers is not associated with the prohibitin 3' untranslated region polymorphism 
BMC Cancer  2008;8:90.
Background
The variable penetrance of ovarian cancer in BRCA1 mutation carriers suggests that other genetic or environmental factors modify disease risk. The C to T transition in the 3' untranslated region of the prohibitin (PHB) gene alters mRNA function and has recently been shown to be associated with hereditary breast cancer risk in Polish women harbouring BRCA1 mutations.
Methods
To investigate whether the PHB 3'UTR polymorphism also modifies hereditary ovarian cancer risk, we performed a case-control study among Polish women carrying one of the three common founder mutations (5382insC, 300 T > G, 4154delA) including 127 ovarian cases and 127 unaffected controls who had both breasts and ovaries intact. Controls were matched to cases by year of birth and BRCA1 mutation. Genotyping analysis was performed using PCR-based restriction fragment length polymorphism analysis. Odds ratios (OR) were calculated using conditional and penalized univariable and multivariable logistic regression.
Results
A comparison of the genotype frequencies between cases and controls revealed no association of the PHB 3'UTR _CT+TT genotypes with ovarian cancer risk (ORadj 1.34; 95% CI, 0.59–3.11).
Conclusion
Our data suggest that the PHB 3'UTR polymorphism does not modify ovarian cancer risk in women carrying one of the three Polish BRCA1 founder mutations.
doi:10.1186/1471-2407-8-90
PMCID: PMC2364631  PMID: 18397521

Results 1-10 (10)