M.A.A.K. Folgueira

Antiproliferative effects of 1,25-dihydroxyvitamin D3 on breast cells: a mini review

The hormone 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), the active form of vitamin D3, is an important regulator of calcium homeostasis, exerts antiproliferative effects on various cell systems and can induce differentiation in some kinds of hematopoietic cells. These effects are triggered by its receptor, vitamin D receptor (VDR), a phosphoprotein member of the nuclear receptor superfamily, which functions as a transcriptional factor. VDR binds as a heterodimer with retinoid X receptor (R X R) to hexameric repeats, characterized as vitamin D-responsive elements present in the regulatory region of target genes such as osteocalcin, osteopontin, calbindin-D28K, calbindin-D9K, p21WAF1/CIP1, TGF-beta2 and vitamin D 24-hydroxylase. Many factors such as glucocorticoids, estrogens, retinoids, proliferation rate and cell transformation can modulate VDR levels. VDR is expressed in mammary tissue and breast cancer cells, which are potential targets to hormone action. Besides having antiproliferative properties, vitamin D might also reduce the invasiveness of cancer cells and act as an anti-angiogenesis agent. All of these antitumoral features suggest that the properties of vitamin D could be explored for chemopreventive and therapeutic purposes in cancer. However, hypercalcemia is an undesirable side effect associated with pharmacological doses of 1,25-(OH)2D3. Some promising 1,25-(OH)2D3 analogs have been developed, which are less hypercalcemic in spite of being potent antiproliferative agents. They represent a new field of investigation.

Gene expression profiling of clinical stages II and III breast cancer

Clinical stage (CS) is an established indicator of breast cancer outcome. In the present study, a cDNA microarray platform containing 692 genes was used to identify molecular differences between CSII and CSIII disease. Tumor samples were collected from patients with CSII or CSIII breast cancer, and normal breast tissue was collected from women without invasive cancer. Seventy-eight genes were deregulated in CSIII tumors and 22 in CSII tumors when compared to normal tissue, and 20 of them were differentially expressed in both CSII and CSIII tumors. In addition, 58 genes were specifically altered in CSIII and expression of 6 of them was tested by real time RT-PCR in another cohort of patients with CSII or CSIII breast cancer and in women without cancer. Among these genes, MAX, KRT15 and S100A14, but not APOBEC3G or KRT19, were differentially expressed on both CSIII and CSII tumors as compared to normal tissue. Increased HMOX1 levels were detected only in CSIII tumors and may represent a molecular marker of this stage. A clear difference in gene expression pattern occurs at the normal-to-cancer transition; however, most of the differentially expressed genes are deregulated in tumors of both CS (II and III) compared to normal breast tissue.

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.

Gene trio signatures as molecular markers to predict response to doxorubicin cyclophosphamide neoadjuvant chemotherapy in breast cancer patients

In breast cancer patients submitted to neoadjuvant chemotherapy (4 cycles of doxorubicin and cyclophosphamide, AC), expression of groups of three genes (gene trio signatures) could distinguish responsive from non-responsive tumors, as demonstrated by cDNA microarray profiling in a previous study by our group. In the current study, we determined if the expression of the same genes would retain the predictive strength, when analyzed by a more accessible technique (real-time RT-PCR). We evaluated 28 samples already analyzed by cDNA microarray, as a technical validation procedure, and 14 tumors, as an independent biological validation set. All patients received neoadjuvant chemotherapy (4 AC). Among five trio combinations previously identified, defined by nine genes individually investigated (BZRP, CLPTM1, MTSS1, NOTCH1, NUP210, PRSS11, RPL37A, SMYD2, and XLHSRF-1), the most accurate were established by RPL37A, XLHSRF-1 based trios, with NOTCH1 or NUP210. Both trios correctly separated 86% of tumors (87% sensitivity and 80% specificity for predicting response), according to their response to chemotherapy (82% in a leave-one-out cross-validation method). Using the pre-established features obtained by linear discriminant analysis, 71% samples from the biological validation set were also correctly classified by both trios (72% sensitivity; 66% specificity). Furthermore, we explored other gene combinations to achieve a higher accuracy in the technical validation group (as a training set). A new trio, MTSS1, RPL37 and SMYD2, correctly classified 93% of samples from the technical validation group (95% sensitivity and 80% specificity; 86% accuracy by the cross-validation method) and 79% from the biological validation group (72% sensitivity and 100% specificity). Therefore, the combined expression of MTSS1, RPL37 and SMYD2, as evaluated by real-time RT-PCR, is a potential candidate to predict response to neoadjuvant doxorubicin and cyclophosphamide in breast cancer patients.

Differential regulation of vitamin D receptor expression in distinct leukemic cell lines upon phorbol ester-induced growth arrest

A close correlation between vitamin D receptor (VDR) abundance and cell proliferation rate has been shown in NIH-3T3 fibroblasts, MCF-7 breast cancer and in HL-60 myeloblastic cells. We have now determined if this association occurs in other leukemic cell lines, U937 and K562, and if VDR content is related to c-myc expression, which is also linked to cell growth state. Upon phorbol myristate acetate (PMA) treatment, cells from the three lineages (HL-60, U937 and K562) differentiated and expressed specific surface antigens. All cell lines analyzed were growth inhibited by PMA and the doubling time was increased, mainly due to an increased fraction of cells in the G0/G1 phase, as determined by flow cytometry measurements of incorporated bromodeoxyuridine and cell DNA content. C-myc mRNA expression was down-regulated and closely correlated to cell growth arrest. However, VDR expression in leukemic cell lines, as determined by immunofluorescence and Northern blot assays, was not consistently changed upon inhibition of cell proliferation since VDR levels were down-regulated only in HL-60 cells. Our data suggest that VDR expression cannot be explained simply as a reflection of the leukemic cell growth state.

Expression of Gene Trios as Predictive Markers of Response to Doxorubicin Based Primary Chemotherapy in Breast Cancer Patients.

Some studies have been carried out to identify a gene expression profile predictive of drug response in breast cancer patients and assessed the reproducibility of the model in an independent group of patients. In common, these studies have used the same technique already used to identify the panel of genes in a training set, to verify accuracy of the model in a validation set. We have previously identified through cDNA microarray technology gene trios whose expression was capable of predicting response to neoadjuvant chemotherapy based in doxorubicin and cyclophosphamide in breast cancer patients. We have now evaluated whether expression of these genes analyzed by real time RT-PCR, which represents a more accessible technique, would reproduce cDNA microarray results in separating responsive from non-responsive patients. Twenty eight samples, already studied by cDNA microarray, were analyzed as a technical validation group and subsequently, another 14 samples were evaluated, as a biological validation group. Expression of nine genes (defining five trio combinations, previously identified) was evaluated by RT-PCR in samples from the technical validation group, to define the separating plane, using linear discriminant analysis. Among the five trios, the best separation of tumors was conferred by RPL37A, XLHSRF-1 based trios (with NOTCH1 or NUP210, as third genes), which correctly classified 86% of samples from the technical validation group and 82% in a cross-validation analysis (leave-one-out). Next, samples from the biological validation group were spatially distributed using the pre-established features from these two trios, resulting in 71% correct classification. Additionally, other gene combinations were searched for a higher accuracy in discriminating response and expression of a new trio, RPL37A, SMYD2 and MTSS1, correctly classified 86% and 79% of samples from the technical validation (leave-one-out cross-validation) and the biological validation groups, respectively. Expression values evaluated by cDNA microarray and RT-PCR are reproducible, however they are not exactly the same as these methods are based on different technical principles and normalization approaches. Our data indicate that care should be taken when substituting techniques in an attempt to reproduce a model. In conclusion, expression of the gene trio RPL37A, SMYD2, MTSS1, as evaluated by RT-PCR, is a potential candidate as predictive marker of response to neoadjuvant chemotherapy in breast cancer patients. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2040.

Orthotopic tumorgrafts in nude mice as a model to evaluate calcitriol effects in breast cancer

OBJECTIVES Calcitriol antiproliferative effects were observed in xenografts of breast cancer cell lines, however they were not yet investigated in tumorgrafts, consisting of freshly collected breast cancer samples xenografted into animals. To establish a tumorgraft model, from freshly collected breast cancer samples, which were directly implanted in nude mice, to study calcitriol effects. METHODS Breast cancer samples collected from 12 patients were orthotopically implanted into nude mice. Animals were treated with weekly intratumoral injections of calcitriol 3 μg/Kg, which was previously shown to induce peak serum calcitriol levels in the predicted therapeutic range. RESULTS Success engraftment rate was 25%. Tumorgrafts were established from aggressive (HER2 positive or histological grade 3) highly proliferative samples and original tumor characteristics were preserved. Calcitriol highly induced its target gene, CYP24A1, indicating that the genomic vitamin D pathway is active in tumorgrafts. However, no differences in the expression of proliferation and apoptosis markers (BrdU incorporation, Ki67, CDKN1A, CDKN1B, BCL2 expression) were observed in these highly proliferative tumor samples. CONCLUSIONS Tumorgrafts seem a promising model to explore other calcitriol doses and regimens, considering the heterogeneity of the disease and microenvironment interactions.

Abstract 186: RHOA, RAC1 and PAK1 evaluation in paired stromal fibroblasts of breast cancer primary and of lymph node metastasis: Importance of these biomarkers in lymph node invasion

The importance of tumor-stromal cells interactions in breast tumor progression and invasion has been recognized; therefore modifications in the stromal fibroblasts can play a significant role in overall cancer development and progression, the presence of fibroblasts in the breast cancer microenvironment metastatic lymph nodes was described, suggesting its role in invasion process. Here after having evaluated the differential genomic profile of carcinoma associated fibroblasts (CAFs) when compared to fibroblasts derived from tissues adjacent to fibroadenomas (NAFs), we depicted focal adhesion as an altered prominent pathway in microarray studies; we validated these data through confocal assays. In order to verify possible role of fibroblasts in lymph node invasion we constructed a tissue microarray consisting of primary BC samples and corresponding lymph node metastasis and compared the expression of some adhesion markers in fibroblasts of the two locations. For the in vitro part of this work stromal fibroblasts were isolated from four invasive breast carcinoma samples and from four women with benign breast disorders. RNA was extracted from low-passage cultures and analyzed with the CodeLink Human Whole Genome cDNA Microarray platform to assess the genomic differential expression. For evaluation of Rho-GTPases: RhoA and Rac1 and a Rho-GTPase effector PAK1 (P21-activated kinase 1), two distinct TMAs were constructed from stromal component of 43 primary tumors and from matched lymph node samples, respectively, fibroblasts were characterized by the expression of α-SMA and vimentin. Our immunohistochemistry revealed 83,7% RhoA positivity in fibroblasts of primary tumors and 71,4% of respective metastatic lymph nodes (p=0,271); 71,8% of fibroblasts of primaries presented Rac1 positive staining, whereas the frequency of positivity in same cells of lymph nodes was 55,3% (p=0,16). With respect to Pak1 50% of patients were positive in the primary tumor against 73,7% in lymph nodal fibroblasts (p=0,039). Our data point to the importance of focal adhesion factors in stromal cells. Since we did not found significant changes between primary and metastatic lymph nodes, we highlight fibroblasts as an active participant in the invasion to lymph nodes, supporting the idea that metastatic tumor cells continue to be dependent of their microenvironment. Supported by Fapesp Citation Format: Patricia Bortman Rozenchan, Fiorita GL Mundim, Rosimeire A. Roela, Maria LH Katayama, Fatima S. Pasini, Helena Brentani, Eduardo C. Lyra, Maria AAK Folgueira, Maria M. Brentani. RHOA, RAC1 and PAK1 evaluation in paired stromal fibroblasts of breast cancer primary and of lymph node metastasis: Importance of these biomarkers in lymph node invasion. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 186. doi:10.1158/1538-7445.AM2014-186

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.

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.