Cintia Milani

Differences in transcriptional effects of 1α,25 dihydroxyvitamin D3 on fibroblasts associated to breast carcinomas and from paired normal breast tissues.

The effects of 1α,25 dihydroxyvitamin D3 (1,25D) on breast carcinoma associated fibroblasts (CAFs) are still unknown. This study aimed to identify genes whose expression was altered after 1,25D treatment in CAFs and matched adjacent normal mammary associated fibroblasts (NAFs). CAFs and NAFs (from 5 patients) were cultured with or without (control) 1,25D 100 nM. Both CAF and NAF expressed vitamin D receptor (VDR) and 1,25D induction of the genomic pathway was detected through up-regulation of the target gene CYP24A1. Microarray analysis showed that despite presenting 50% of overlapping genes, CAFs and NAFs exhibited distinct transcriptional profiles after 1,25D treatment (FDR<0.05). Functional analysis revealed that in CAFs, genes associated with proliferation (NRG1, WNT5A, PDGFC) were down regulated and those involved in immune modulation (NFKBIA, TREM-1) were up regulated, consistent with anti tumor activities of 1,25D in breast cancer. In NAFs, a distinct subset of genes was induced by 1,25D, involved in anti apoptosis, detoxification, antibacterial defense system and protection against oxidative stress, which may limit carcinogenesis. Co-expression network and interactome analysis of genes commonly regulated by 1,25D in NAFs and CAFs revealed differences in their co-expression values, suggesting that 1,25D effects in NAFs are distinct from those triggered in CAFs.

Transcriptional effects of 1,25 dihydroxyvitamin D 3 physiological and supra-physiological concentrations in breast cancer organotypic culture

BackgroundVitamin D transcriptional effects were linked to tumor growth control, however, the hormone targets were determined in cell cultures exposed to supra physiological concentrations of 1,25(OH)2D3 (50-100nM). Our aim was to evaluate the transcriptional effects of 1,25(OH)2D3 in a more physiological model of breast cancer, consisting of fresh tumor slices exposed to 1,25(OH)2D3 at concentrations that can be attained in vivo.MethodsTumor samples from post-menopausal breast cancer patients were sliced and cultured for 24 hours with or without 1,25(OH)2D3 0.5nM or 100nM. Gene expression was analyzed by microarray (SAM paired analysis, FDR≤0.1) or RT-qPCR (p≤0.05, Friedman/Wilcoxon test). Expression of candidate genes was then evaluated in mammary epithelial/breast cancer lineages and cancer associated fibroblasts (CAFs), exposed or not to 1,25(OH)2D3 0.5nM, using RT-qPCR, western blot or immunocytochemistry.Results1,25(OH)2D3 0.5nM or 100nM effects were evaluated in five tumor samples by microarray and seven and 136 genes, respectively, were up-regulated. There was an enrichment of genes containing transcription factor binding sites for the vitamin D receptor (VDR) in samples exposed to 1,25(OH)2D3 near physiological concentration. Genes up-modulated by both 1,25(OH)2D3 concentrations were CYP24A1, DPP4, CA2, EFTUD1, TKTL1, KCNK3. Expression of candidate genes was subsequently evaluated in another 16 samples by RT-qPCR and up-regulation of CYP24A1, DPP4 and CA2 by 1,25(OH)2D3 was confirmed. To evaluate whether the transcripitonal targets of 1,25(OH)2D3 0.5nM were restricted to the epithelial or stromal compartments, gene expression was examined in HB4A, C5.4, SKBR3, MDA-MB231, MCF-7 lineages and CAFs, using RT-qPCR. In epithelial cells, there was a clear induction of CYP24A1, CA2, CD14 and IL1RL1. In fibroblasts, in addition to CYP24A1 induction, there was a trend towards up-regulation of CA2, IL1RL1, and DPP4. A higher protein expression of CD14 in epithelial cells and CA2 and DPP4 in CAFs exposed to 1,25(OH)2D3 0.5nM was detected.ConclusionsIn breast cancer specimens a short period of 1,25(OH)2D3 exposure at near physiological concentration modestly activates the hormone transcriptional pathway. Induction of CYP24A1, CA2, DPP4, IL1RL1 expression appears to reflect 1,25(OH)2D3 effects in epithelial as well as stromal cells, however, induction of CD14 expression is likely restricted to the epithelial compartment.

Human breast tumor slices: A model for identification of vitamin D regulated genes in the tumor microenvironment

While many studies have addressed the direct effects of 1alpha,25(OH)2D3 on breast cancer (BC) cells, stromal-epithelial interactions, which are important for the tumor development, have been largely ignored. In addition, high concentrations of the hormone, which cannot be attained in vivo, have been used. Our aim was to establish a more physiological breast cancer model, represented by BC tissue slices, which maintain epithelial-mesenchymal interactions, cultured with a relatively low 1alpha,25(OH)2D3 concentration, in order to evaluate the vitamin D pathway. Freshly excised human BC samples were sliced and cultured in complete culture media containing vehicle, 0.5 nM or 100 nM 1alpha,25(OH)2D3 for 24 h. BC slices remained viable for at least 24 h, as evaluated by preserved tissue morphology in hematoxylin and eosin (HE) stained sections and bromodeoxyuridine (BrdU) incorporation by 10% of tumor cells. VDR mRNA expression was detected in all samples and CYP24A1 mRNA expression was induced by 1alpha,25(OH)2D3 in both concentrations (but mainly with 100 nM). Our results indicate that the vitamin D signaling pathway is functional in BC slices, a model which preserves stromal-epithelial interactions and mimics in vivo conditions.

Transcriptional profile of fibroblasts obtained from the primary site, lymph node and bone marrow of breast cancer patients

Cancer-associated fibroblasts (CAF) influence tumor development at primary as well as in metastatic sites, but there have been no direct comparisons of the transcriptional profiles of stromal cells from different tumor sites. In this study, we used customized cDNA microarrays to compare the gene expression profile of stromal cells from primary tumor (CAF, n = 4), lymph node metastasis (N+, n = 3) and bone marrow (BM, n = 4) obtained from breast cancer patients. Biological validation was done in another 16 samples by RT-qPCR. Differences between CAF vs N+, CAF vs BM and N+ vs BM were represented by 20, 235 and 245 genes, respectively (SAM test, FDR < 0.01). Functional analysis revealed that genes related to development and morphogenesis were overrepresented. In a biological validation set, NOTCH2 was confirmed to be more expressed in N+ (vs CAF) and ADCY2, HECTD1, HNMT, LOX, MACF1, SLC1A3 and USP16 more expressed in BM (vs CAF). Only small differences were observed in the transcriptional profiles of fibroblasts from the primary tumor and lymph node of breast cancer patients, whereas greater differences were observed between bone marrow stromal cells and the other two sites. These differences may reflect the activities of distinct differentiation programs.

Post-mortem cytogenomic investigations in patients with congenital malformations.

Congenital anomalies are the second highest cause of infant deaths, and, in most cases, diagnosis is a challenge. In this study, we characterize patterns of DNA copy number aberrations in different samples of post-mortem tissues from patients with congenital malformations. Twenty-eight patients undergoing autopsy were cytogenomically evaluated using several methods, specifically, Multiplex Ligation-dependent Probe Amplification (MLPA), microsatellite marker analysis with a MiniFiler kit, FISH, a cytogenomic array technique and bidirectional Sanger sequencing, which were performed on samples of different tissues (brain, heart, liver, skin and diaphragm) preserved in RNAlater, in formaldehyde or by paraffin-embedding. The results identified 13 patients with pathogenic copy number variations (CNVs). Of these, eight presented aneuploidies involving chromosomes 13, 18, 21, X and Y (two presented inter- and intra-tissue mosaicism). In addition, other abnormalities were found, including duplication of the TYMS gene (18p11.32); deletion of the CHL1 gene (3p26.3); deletion of the HIC1 gene (17p13.3); and deletion of the TOM1L2 gene (17p11.2). One patient had a pathogenic missense mutation of g.8535C>G (c.746C>G) in exon 7 of the FGFR3 gene consistent with Thanatophoric Dysplasia type I. Cytogenomic techniques were reliable for the analysis of autopsy material and allowed the identification of inter- and intra-tissue mosaicism and a better understanding of the pathogenesis of congenital malformations.

Expression of heterochromatin protein 1 in the primary tumor of breast cancer patients in the presence or absence of occult metastatic cells in the bone marrow

Gene silencing may occur in breast cancer samples from patients presenting with occult metastatic cells in the bone marrow and one mechanism regulating gene suppression is heterochromatin formation. We have studied whether members of the heterochromatin protein 1 family (HP1Hs alpha, HP1Hs beta and HP1Hs gamma), which take part in chromatin packaging and gene expression regulation, were differentially expressed in tumors from patients with and without occult metastatic cells in their bone marrow. Tumor samples and bone marrow aspirates were obtained from 37 breast cancer patients. Median age was 63 years and 68% of the patients presented with clinical stage I/II disease. Presence of occult metastatic cells in bone marrow was detected through keratin-19 expression by nested RT-PCR in samples from 20 patients (54.1%). The presence of occult metastatic cells in bone marrow was not associated with node involvement, histological grade, estrogen receptor and ERBB2 immunoexpression. Relative gene expression of HP1Hs alpha, HP1Hs beta and HP1Hs gamma was determined by realtime RT-PCR and did not vary according to the presence of occult metastatic cells in bone marrow. In addition, the combined expression of these three transcripts could not be used to classify samples according to the presence of bone marrow micrometastasis. Our work indicates that regulation of heterochromatin formation through HP1 family members may not be the sole mechanism implicated in the metastatic process to the bone marrow.

Gene expression profile of stromal cells from potential metastatic sites in breast cancer patients.

Methods Fibroblasts primary culture was established from 11 breast cancer patients. Expression analysis was evaluated in PT (n=4), N+ (n=3) and BM (n=4) through a customized cDNA microarray platform (4,800 ORESTES) analyzed by SAM (TMEV; FDR 0%) and functional analysis was performed using DAVID. Technical validation was performed in 6 samples and biological validation was performed in fibroblasts obtained from others 25 patients as evaluated by RT-qPCR.

Abstract 4267: Gene expression profile of fibroblasts from different topographical origins in breast cancer patients

Background: In breast cancer, there is increasing evidence that stromal cells, including fibroblasts, may influence tumor development in the primary site, as well as in regional and distant metastatic sites. Objectives: To compare fibroblasts from the primary tumor (PT), lymph nodes and bone marrow (BM) from breast cancer patients, by means of their expression profile. Methods: Fibroblasts primary culture was established from 14 breast cancer patients: PT (n=4), axillary nodes (3 from positive and 3 from negative nodes) and BM (2 with and 2 without micrometastasis, identified by RT-PCR for KRT19). Expression analysis was performed through a customized cDNA microarray platform (4,800 ORESTES) analyzed by SAM multiclass (TMEV; FDR 0%), followed by LIMMA test (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4267. doi:1538-7445.AM2012-4267

Breast Cancer Gene Expression Profile in Post-Menopausal Patients Supplemented with Vitamin D.

Vitamin D supplementation is indicated for post-menopausal women to prevent osteoporosis and lower 25(OH)2D3 or 1,25(OH)2D3 serum levels have been associated with breast cancer incidence or prognosis (metastasis). The antiproliferative effects of vitamin D are observed in breast cancer cell lines exposed to phamacological doses of calcitriol (1,25(OH)2D3, 100nM) but whether physiological doses are sufficient to produce growth inhibition in vivo is not known. The aim of our study was to investigate gene expression profile changes of breast cancer samples from patients supplemented with calcitriol, presenting an anti-proliferative effect on the tumor. Post-menopausal women diagnosed with breast cancer were instructed to take one (0.25ug/day, n=8) or two (0.50ug/day, n=8) tablets of calcitriol after tumor biopsy. Median time of supplementation was 30 days. Sixteen tumor samples were collected during biopsy (before supplementation) and breast surgery (after supplementation). Proliferation index was evaluated by tumor Ki-67 immunohistochemistry (IHC) expression in breast cancer samples before and after calcitirol supplementation and 1000 cells were counted by three observers (p in vitro with a low concentration of calcitriol, 0.5nM (that can be attained with subcutaneous administration of doses of 8ug calcitriol, without hypercalcemia) for 24h, were included in the analysis. All samples had RNA hybridized to the same gene chips. Results were normalized and analyzed using RMA and Mev.TM4 softwares. CYP24A1 , a target gene of vitamin D, presented a positive regulation after calcitriol supplementation in all samples analyzed. Differentially expressed genes were involved in the regulation of cell cycle [ SMAD2 , cyclin E, YWHAQ (14-3-3 family] and calcium signalling ( HTR7, PTGER1 and PTGER2 ). Our results indicate that the tumor proliferation index is reduced upon calcitriol supplementation. Moreover, potentially regulated pathways in breast cancer specimens after administration of low doses of calcitriol are regulation of cell cycle and calcium signaling.Supported by FAPESP 2007/01111-0 – 2007/04799-2 Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6128.