A genetic variant located in miR-423 is associated with reduced breast cancer risk.

BACKGROUND/AIM: Since microRNAs (miRNAs) act as translational regulators of multiple genes, single nucleotide polymorphisms (SNP) in them can have potentially wide-ranging effects. Using an association approach, this research examined the effects of the rs6505162 SNP, an A > C polymorphism located in the pre-miRNA region of miR-423, on breast cancer development. MATERIALS AND METHODS: Caucasian Australian women with breast cancer and controls matched for age and ethnicity were genotyped for rs6505162 and their genotypic and allelic frequencies analysed for significant differences. RESULTS: Analysis indicated that there were significant differences between the case and control populations (χ2 = 6.70, p = 0.035), with the CC genotype conferring reduced risk of breast cancer development (odds ratio = 0.50 95% confidence interval = 0.27-0.92, p = 0.03). CONCLUSION: Further functional research is required to determine the mechanism of action of this SNP on miRNA function.

Single nucleotide polymorphism in hsa-mir-196a-2 and breast cancer risk: a case control study.

microRNAs are small, non-coding RNAs that influence gene expression on a post-transcriptional level. They participate in diverse biological pathways and may act as either tumor suppressor genes or oncogenes. As they may have an effect on thousands of target mRNAs, single-nucleotide polymorphisms in microRNA genes might have major functional consequences, because the microRNA's properties and/or maturation may change. miR-196a has been reported to be aberrantly expressed in breast cancer tissue. Additionally, the SNP rs11614913 in hsa-mir-196a-2 has been found to be associated with breast cancer risk in some studies although not in others. This study evaluated the association between rs11614913 and breast cancer risk in a Caucasian case-control cohort in Queensland, Australia. Results do not support an association of the tested hsa-mir-196a-2 polymorphism with breast cancer susceptibility in this cohort. As there is a discrepancy between our results and previous findings, it is important to assess the role of rs11614913 in breast cancer by further larger studies investigating different ethnic groups.

Matrix metalloproteinase localisation by in situ-RT-PCR in archival human breast biopsy material.

Utilising archival human breast cancer biopsy material we examined the stromal/epithelial interactions of several matrix metalloproteinases (MMPs) using in situ-RT-PCR (IS-RT-PCR). In breast cancer, the stromal/epithelial interactions that occur, and the site of production of these proteases, are central to understanding their role in invasive and metastatic processes. We examined MT1-MMP (MMP-14, membrane type-1-MMP), MMP-1 (interstitial collagenase) and MMP-3 (stromelysin-1) for their localisation profile in progressive breast cancer biopsy material (poorly differentiated invasive breast carcinoma (PDIBC), invasive breast carcinomas (IBC) and lymph node metastases (LNM)). Expression of MT1-MMP, MMP-1 and MMP-3 was observed in both the tumour epithelial and surrounding stromal cells in most tissue sections examined. MT1-MMP expression was predominantly localised to the tumour component in the pre-invasive lesions. MMP-1 gene expression was relatively well distributed between both tissue compartments, while MMP-3 demonstrated highest expression levels in the stromal tissue surrounding the epithelial tumour cells. The results demonstrate the ability to distinguish compartmental gene expression profiles using IS-RT-PCR. Further, we suggest a role for MT1-MMP in early tumour progression, expression of MMP-1 during metastasis and focal expression pattern of MMP-3 in areas of expansion. These expression profiles may provide markers for early breast cancer diagnoses and present potential therapeutic targets.

Progesterone, glucocorticoid, but not estrogen receptor mRNA is altered in breast cancer stroma.

Our laboratory has previously found that anti-mitogenic nuclear receptor mRNA is elevated in late stage tumours and this study was performed to scrutinize the possibility of cancer-stroma crosstalk using hormone signaling in these tissues. RNA levels in stromal tissue were examined for the estrogen alpha, estrogen beta, androgen, progesterone and glucocorticoid nuclear receptors by a semi-quantitative PCR. Significant differences in expression between the cancer stroma and control tissue were seen, analyzing for both cancer grade and estrogen receptor status. Stroma and control tissue were significantly different for the progesterone and glucocorticoid nuclear receptors (p=5.908 x 10(-7) and 2.761 x 10(-5), respectively). Glucocorticoid receptor also showed a significant increase to mRNA levels in the stroma of estrogen receptor negative tumours (p=5.85 x 10(-5)). By contrast, the estrogen receptors alpha and beta, those most closely associated with breast tissue growth, showed no significant change in mRNA (p=0.372 and 0.655, respectively). Androgen receptor mRNA also remained unaffected (p=0.174).

Detection of mRNA levels for the estrogen alpha, estrogen beta and androgen nuclear receptor genes in archival breast cancer tissue.

Previous studies in our laboratory have shown association of nuclear receptor expression and histological breast cancer grade. To further investigate these findings, it was the objective of this study to determine if expression levels of the estrogen alpha, estrogen beta and androgen nuclear receptor genes varied in different breast cancer grades. RNA extracted from paraffin embedded archival breast tumour tissue was converted into cDNA and cDNA underwent PCR to enable quantitation of mRNA expression. Expression data was normalised against the 18S ribosomal gene multiplex and analysed using ANOVA. Analysis indicated a significant alteration of expression for the androgen receptor in different cancer grades (P=0.014), as well as in tissues that no longer possess estrogen receptor alpha proteins (P=0.025). However, expression of estrogen receptors alpha and beta did not vary significantly with cancer grade (P=0.057 and 0.622, respectively). Also, the expression of estrogen receptor alpha or beta did not change, regardless of the presence of estrogen receptor alpha protein in the tissue (P=0.794 and 0.716, respectively). Post-hoc tests indicate that the expression of the androgen receptor is increased in estrogen receptor negative tissue as well as in grade 2 and grade 3 tumours, compared to control tissue. This increased expression in late stage breast tumours may have implications to the treatment of breast tumours, particularly those lacking expression of other nuclear receptor genes.

Chromosomal aberrations in squamous cell carcinoma and solar keratoses revealed by comparative genomic hybridization.

OBJECTIVE: To identify chromosomal copy numbers of frequent genetic aberrations within squamous cell carcinomas (SCCs) and solar keratoses (SKs), and provide further evidence to support or challenge current dogma concerning the relationship between these lesions. DESIGN: Retrospective analysis of genetic aberrations in DNA from SK and SCC biopsy specimens by comparative genomic hybridization. SETTING: University-based research laboratory in Queensland, Australia. PATIENTS: Twenty-two biopsy specimens from patients with diagnosed SKs (n = 7), cutaneous SCCs (n = 10), or adjoining lesions (n = 5). MAIN OUTCOME MEASURES: Identification of frequent genetic aberrations both specific to SK and SCC and shared by these lesions to investigate their clonal relationship. RESULTS: Shared genomic imbalances were identified in SK and SCC. Frequent gains were located at chromosome arms 3q, 17q, 4p, 14q, Xq, 5p, 9q, 8q, 17p, and 20q, whereas shared regional losses were observed at 9p, 3p, 13q, 17p, 11p, 8q, and 18p. Significant loss of 18q was observed only in SCC lesions. CONCLUSIONS: Our results demonstrate that numerous chromosomal aberrations are shared by the 2 lesions, suggesting a clonal relationship between SK and SCC. Additionally, the genomic loss of 18q may be a significant event in SK progression to SCC. Finally, the type and frequency of aberrations suggests a common mode of tumorigenesis in SCC-derived tumors.

Expression of glucocorticoid and progesterone nuclear receptor genes in archival breast cancer tissue.

BACKGROUND: Previous studies in our laboratory have shown associations of specific nuclear receptor gene variants with sporadic breast cancer. In order to investigate these findings further, we conducted the present study to determine whether expression levels of the progesterone and glucocorticoid nuclear receptor genes vary in different breast cancer grades. METHODS: RNA was extracted from paraffin-embedded archival breast tumour tissue and converted into cDNA. Sample cDNA underwent PCR using labelled primers to enable quantitation of mRNA expression. Expression data were normalized against the 18S ribosomal gene multiplex and analyzed using analysis of variance. RESULTS: Analysis of variance indicated a variable level of expression of both genes with regard to breast cancer grade (P = 0.00033 for glucocorticoid receptor and P = 0.023 for progesterone receptor). CONCLUSION: Statistical analysis indicated that expression of the progesterone nuclear receptor is elevated in late grade breast cancer tissue.

An assessment of MMP and TIMP gene expression in cell lines and stroma - tumour differences in microdissected breast cancer biopsies.

To examine matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinases (TIMP) mRNA levels in archival breast cancer biopsies, we employed microdissection to separate tumour tissue from the surrounding breast tissue, or stroma and RT-PCR to determine gross qualitative and small quantitative differences in the patterns of expression. In this study, a significant correlation (p < 0.05, by Mann-Whitney U analysis) between TIMP-2 expression and lymph node involvement was identified, while MMP-11 and TIMP-1 expression patterning also significantly (p < 0.05) differed between those tumours showing calcification and those that did not. When compared by Spearmans' rho correlation analysis, a significant association (p < 0.05, rho = 0.404) was identified in the pattern of MMP-2 and MMP-9 gene expression. In this study, the use of microdissection and a systematic strategy of RT-PCR analysis have allowed us to investigate localized MMP and MMP inhibitor expression within breast tumours. We have identified patterns of gene expression that may further reveal aspects of breast carcinogenesis, and a robust method for examining changes in clinically important genes using archival biopsies and across stroma-tumour boundaries.

Pathology in East Timor.

East Timor is the newest nation in Southeast Asia and, with its recent turbulent history, also one of the poorest. Infectious and parasitic diseases such as tuberculosis and malaria are endemic, and the poor infrastructure in the wake of Indonesia's sudden withdrawal has left the country with enormous health challenges. Australia's key role in the emergence of East Timor as an independent country brings with it a long-term interest in the future development of its health services, including pathology.

Polymorphic variants of NFKB1 and its inhibitory protein NFKBIA, and their involvement in sporadic breast cancer.

Nuclear factor kappa-beta (NF-kappaB) is a transcription factor responsible for modulating the expression of many genes involved in cell proliferation, differentiation, apoptosis and metastasis. NF-kappaB interacts with IkappaB inhibitory proteins to regulate gene expression. This study investigated common variants within the genes coding for NF-kappaB and IkappaB, NFKB1 and NFKBIA, for involvement in sporadic breast cancer. Genotypes were determined in a population of breast cancer affected individuals and age-matched controls. Results do not support an involvement of the tested NFKB1 and NFKBIA polymorphisms in susceptibility to sporadic breast cancer, in the tested Caucasian population.

Differential gene expression in breast cancer cell lines and stroma-tumor differences in microdissected breast cancer biopsies revealed by display array analysis.

To examine gene-expression patterning in late-stage breast cancer biopsies, we used a microdissection technique to separate tumor from the surrounding breast tissue or stroma. A DD-PCR protocol was then used to amplify expressed products, which were resolved using PAGE and used as probe to hybridize with representative human arrays and cDNA libraries. The probe derived from the tumor-stroma comparison was hybridized with a gene array and an arrayed cDNA library derived from a GCT of bone; 21 known genes or expressed sequence tags were detected, of which 17 showed differential expression. These included factors associated with epithelial to mesenchymal transition (vimentin), the cargo selection protein (TIP47) and the signal transducer and activator of transcription (STAT3). Northern blot analysis was used to confirm those genes also expressed by representative breast cancer cell lines. Notably, 6 genes of unknown function were restricted to tumor while the majority of stroma-associated genes were known. When applied to transformed breast cancer cell lines (MDA-MB-435 and T47D) that are known to have different metastatic potential, DD array analysis revealed a further 20 genes; 17 of these genes showed differential expression. Use of microdissection and the DD-PCR array protocol allowed us to identify factors whose localized expression within the breast may play a role in abnormal breast development or breast carcinogenesis.