Identification of vitamin D3 target genes in human breast cancer tissue.

Multiple epidemiological studies have shown that high vitamin D3 status is strongly associated with improved breast cancer survival. To determine the molecular pathways influenced by 1 alpha, 25-dihydroxyvitamin D3 (1,25D) in breast epithelial cells we isolated RNA from normal human breast and cancer tissues treated with 1,25D in an ex vivo explant system. RNA-Seq revealed 523 genes that were differentially expressed in breast cancer tissues in response to 1,25D treatment, and 127 genes with altered expression in normal breast tissues. GoSeq KEGG pathway analysis revealed 1,25D down-regulated cellular metabolic pathways and enriched pathways involved with intercellular adhesion. The highly 1,25D up-regulated target genes CLMN, SERPINB1, EFTUD1, and KLK6were selected for further analysis and up-regulation by 1,25D was confirmed by qRT-PCR analysis in breast cancer cell lines and in a subset of human clinical samples from normal and cancer breast tissues. Ketoconazole potentiated 1,25D-mediated induction of CLMN, SERPINB1, and KLK6 mRNA through inhibition of 24-hydroxylase (CYP24A1) activity. Elevated expression levels of CLMN, SERPINB1, and KLK6 are associated with prolonged relapse-free survival for breast cancer patients. The major finding of the present study is that exposure of both normal and malignant breast tissue to 1,25D results in changes in cellular adhesion, metabolic pathways and tumor suppressor-like pathways, which support epidemiological data suggesting that adequate vitamin D3 levels may improve breast cancer outcome.

The unique transcriptional response produced by concurrent estrogen and progesterone treatment in breast cancer cells results in upregulation of growth factor pathways and switching from a Luminal A to a Basal-like subtype.

BACKGROUND: In breast cancer, progesterone receptor (PR) positivity or abundance is positively associated with survival and treatment response. It was initially believed that PR was a useful diagnostic marker of estrogen receptor activity, but increasingly PR has been recognised to play an important biological role in breast homeostasis, carcinogenesis and metastasis. Although PR expression is almost exclusively observed in estrogen receptor positive tumors, few studies have investigated the cellular mechanisms of PR action in the context of ongoing estrogen signalling. METHODS: In this study, we contrast PR function in estrogen pretreated ZR-75-1 breast cancer cells with vehicle treated ZR-75-1 and T-47D breast cancer cells using expression microarrays and chromatin immunoprecipitation-sequencing. RESULTS: Estrogen cotreatment caused a dramatic increase in the number of genes regulated by progesterone in ZR-75-1 cells. In T-47D cells that have naturally high levels of PR, estrogen and progesterone cotreatment resulted in a reduction in the number of regulated genes in comparison to treatment with either hormone alone. At a genome level, estrogen pretreatment of ZR-75-1 cells led to a 10-fold increase in the number of PR DNA binding sites detected using ChIP-sequencing. Time course assessment of progesterone regulated genes in the context of estrogen pretreatment highlighted a series of important regulatory pathways, including those driven by epithelial growth factor receptor (EGFR). Importantly, progesterone applied to cells pretreated with estradiol resulted in switching of the PAM50-determined intrinsic breast cancer subtype from Luminal A to Basal-like, and increased the Oncotype DX® Unscaled Recurrence Score. CONCLUSION: Estrogen pretreatment of breast cancer cells increases PR steady state levels, resulting in an unequivocal progesterone response that upregulates key members of growth factor pathways. The transformative changes progesterone exerts on the breast cancer subtype suggest that these subtyping tools should be used with caution in premenopausal women.

Identification of the sentinel lymph node in the SNAC-1 trial.

BACKGROUND: A combination of scintigraphy and a lymphotropic dye (patent blue dye (BD)) is the recommended technique to detect the sentinel lymph node (SLN) in early breast cancer. This study determined the effect of clinical factors on SLN identification in the sentinel node biopsy versus axillary clearance (SNAC) trial. METHODS: A total of 1088 women were registered. Lymphatic mapping was performed using preoperative lymphoscintigraphy (LSG) and gamma probe (GP) combined with peritumoural injection of patent BD (971 patients) or BD alone (106 patients). RESULTS: SLNs were identified in 1024 women (94%), localized with LSG in 779 (81.4%), and were identified by GP in 879 (91.8%). The BD identified SLNs in 890 of 1073 (82%) women. Three patients had allergic reactions. BD detected the SLNs in 141 of 178 women with negative LSG mapping and in 44 of 79 women with no hot SLNs detected intraoperatively. Age, body mass index (BMI) and tumour presentation (screen detected versus symptomatic) were significantly related to the identification of the SLN. For BD, the primary tumour location was significantly related to identification rate. The detection of blue SLN was significantly lower in women with inner quadrant tumours. CONCLUSION: The combined technique resulted in a high identification rate. BD contributed to the identification of the SLNs in patients where LSG and GP failed to identify the sentinel node. Special attention to these techniques is needed in particular groups of patients such as those with high BMI, screen-detected primary tumours and tumour located in the inner quadrants.

Specific-site methylation of tumour suppressor ANKRD11 in breast cancer.

ANKRD11 is a putative tumour suppressor gene in breast cancer, which has been shown in our laboratory to be a co-activator of p53. Our data suggest that down-regulation of ANKRD11 is associated with breast tumourigenesis. Breast cancer cell lines treated with DNA demethylating agents resulted in up-regulation of ANKRD11 expression suggesting that promoter DNA methylation may be responsible for its down-regulation. The transcriptional activity of a CpG-rich region 2kb upstream of the transcription initiation site of ANKRD11 was investigated using dual-luciferase reporter assays. The constructs carrying -661 to -571 bp promoter sequence showed significant transcriptional activity. Using the SEQUENOM Epityper Platform, the region between -770 and +399 bp was analysed in 25 breast tumours, four normal breast tissues and five normal blood samples. The region between -770 and -323 bp was shown to be frequently methylated in breast tumours. The methylation patterns of all analysed CpGs in this region were identical in the normal and tumour samples, except for a 19 bp region containing three CpG sites. These sites had significantly higher levels of methylation in tumours (40%) compared to normal samples (6%). Our findings support the role of ANKRD11 as a tumour suppressor gene and suggest that aberrant DNA methylation of three CpGs in a 19 bp region within the ANKRD11 promoter may be responsible for its down-regulation in breast cancer.

Comparison of volume displacement versus circumferential arm measurements for lymphoedema: implications for the SNAC trial.

BACKGROUND: The Royal Australasian College of Surgeons Sentinel Node versus Axillary Clearance trial is a randomized controlled trial comparing sentinel node biopsy with axillary clearance in breast cancer patients. Primary study end-points include arm volume differences with time, which may indicate the development of lymphoedema. The RACS SNAC trial uses circumferential arm measurements in the estimation of arm volume. This study aimed to assess the accuracy of circumferential volume estimation in comparison with water displacement. METHODS: Eighty-seven women attending the breast clinic at the Women's Health Centre, Royal Adelaide Hospital, were assessed by volumetric and circumferential arm measurements. Correlations between volume estimations and measurements were made, taking into account the width of measuring tape and body mass index. RESULTS: There was a highly significant correlation between circumferential and volumetric arm measurements (Pearson's correlation coefficient = 0.92, P < 0.0001), especially when using the narrow measuring tape. Correlation was best in the overweight BMI group (Pearson's correlation coefficient = 0.94. P < 0.0001) and worst in the obese group (Pearson's correlation coefficient = 0.79, P < 0.0001) but all relationships were statistically significant. CONCLUSION: Using a narrow tape, circumferential arm measurement is an appropriate method for assessing arm volume in the SNAC trial.

Clinical impact of oncoplastic surgery in a specialist breast practice.

BACKGROUND: Oncoplastic breast surgery is an integral and fundamental component of the clinical management of breast cancer. The aim of this study was to determine the proportion of oncoplastic and reconstructive breast cancer procedures undertaken within a specialist breast practice. METHODS: An audit of breast-related cancer procedures was undertaken for patients with early breast cancer between 1 January 2001 and 31 December 2005, treated at the Royal Adelaide Hospital and in private practice. The proportion of oncoplastic and breast reconstructive procedures was calculated to determine the clinical effects on a specialist breast-surgical practice. RESULTS: Breast cancer resection procedures accounted for 1514 of 2113 of operations (72%). Most of these (897 of 1514, 59.2%) were wide local excision or re-excision procedures. Total breast reconstruction operations (i.e. autogenous tissue flaps, tissue expander/implant reconstructions) accounted for 251 procedures. Of these, 67 (26.7%) were carried out at the time of simple mastectomy. Contralateral breast procedures (i.e. reduction mammaplasty, mastopexy and augmentation) accounted for 138 procedures and nipple-areola reconstruction/tattoo accounted for 153 procedures. Oncoplastic procedures, such as skin-sparing mastectomy, latissimus dorsi miniflap and therapeutic mammaplasty accounted for 57 of 599 procedures (9.5%). Breast reconstruction and oncoplastic operations accounted for 599 of 2113 procedures (28%). CONCLUSION: Specialist breast surgeons trained in breast reconstruction and oncoplastic techniques can expect a substantial proportion of their breast practice to include such operative procedures (28% in this series). Subspecialist training in breast surgery should incorporate experience in breast reconstructive and aesthetic surgery for trainees who wish to practise as specialist breast surgeons in the future.