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-1based 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.

Epithelial intercellular cohesion, mainly mediated by E-cadherin (CDH1) expression and function, may be deregulated during cancer cell invasion of adjacent tissues and lymphatic and vascular channels. CDH1 expression is down-modulated in invasive lobular breast carcinomas but its regulation in invasive ductal carcinomas (IDC) is less clear. CDH1 expression is repressed by transcription factors such as Snail (SNAI1) and its product is degraded after Hakai ubiquitination. We compared CDH1, SNAI1 and HAKAI mRNA expression in IDC and paired adjacent normal breast tissue and evaluated its relation with node metastasis and circulating tumor cells. Matched tumor/peritumoral and blood samples were collected from 30 patients with early IDC. Epithelial cells from each compartment (tumor/peritumoral) were recovered by an immunomagnetic method and gene expression was determined by real time RT-PCR. There were no differences in CDH1, SNAI1 and HAKAI mRNA expression between tumor and corresponding peritumoral samples and no differential tumoral gene expression according to nodal involvement. Another 30 patients with a long-term follow-up (at least 5 years) and a differential prognosis (good or poor, as defined by breast cancer death) had E-cadherin and Snail protein detected by immunohistochemistry in tumor samples. In this group, E-cadherin-positive expression, but not Snail, may be associated with a better prognosis. This is the first report simultaneously analyzing CDH1, SNAI1 and HAKAI mRNA expression in matched tumor and peritumoral samples from patients with IDC. However, no clear pattern of their expression could distinguish the invasive tumor compartment from its adjacent normal tissue.

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.

The objective of the present study was to determine the effects of retinoic acid on the growth of the mouse mammary cells HC11 and HC11ras, which are a model for in vitro breast cancer progression. The expression of the two classes (RARs and RXRs) of retinoic acid receptor mRNAs was determined by Northern blot analysis. Receptor functional integrity was determined by testing whether RAR ß mRNA could be induced by retinoic acid. The effects of a 72-h exposure to 50 µM 13-cis retinoic acid on HC11 and HC11ras cell proliferation and HC11 cell differentiation were investigated by flow cytometric cell cycle analysis, and by determination of ß-casein mRNA expression, respectively. The possibility that retinoic acid would induce the expression of the vitamin D receptor and synergize with vitamin D, a known inhibitor of HC11 cell growth, was also investigated. HC11 cells expressed higher mRNA levels of both RAR a and RAR g when compared to HC11ras cells. In contrast, RAR ß, as well as RXR a, ß and g expression was low in both HC11 and HC11ras cells. In addition, RAR ß mRNA was induced by retinoic acid treatment in both cells. In spite of these observations, no effects were seen on cell proliferation or differentiation upon exposure to retinoic acid. Neither vitamin D receptor induction nor synergy with vitamin D on growth inhibition was observed. We conclude that the RAR expression profile could be related to the transformed state in HC11ras cells and that the retinoic acid resistance observed merits further investigation.

Cells usually lose adhesion and increase proliferation and migration during malignant transformation. Here, we studied how proliferation can affect the other two characteristics, which ultimately lead to invasion and metastasis. We determined the expression of ß1 integrins, as well as adhesion and migration towards laminin-1, fibronectin, collagens type I and type IV presented by LISP-1 colorectal cancer cells exposed to 2.5% dimethyl sulfoxide (DMSO), an agent capable of decreasing proliferation in this poorly differentiated colorectal cell line. Untreated cells (control), as shown by flow cytometry and monoclonal antibodies, expressed alpha2 (63.8 ± 11.3% positive cells), alpha3 (93.3 ± 7.0%), alpha5 (50.4 ± 12.0%) and alpha6 (34.1 ± 4.9%) integrins but not alpha1, alpha4, alphav or ß4. Cells adhered well to laminin-1 (73.4 ± 6.0%) and fibronectin (40.0 ± 2.0%) substrates but very little to collagens. By using blocking monoclonal antibodies, we showed that alpha2, alpha3 and alpha6 mediated laminin-1 adhesion, but neither alpha3 nor alpha5 contributed to fibronectin adherence. DMSO arrested cells at G0/G1 (control: 55.0 ± 2.4% vs DMSO: 70.7 ± 2.5%) while simultaneously reducing alpha5 (24.2 ± 19%) and alpha6 (14.3 ± 10.8%) expression as well as c-myc mRNA (7-fold), the latter shown by Northern blotting. Although the adhesion rate did not change after exposure to DMSO, alpha3 and alpha5 played a major role in laminin-1 and fibronectin adhesion, respectively. Migration towards laminin-1, which was clearly increased upon exposure to DMSO (control: 6 ± 2 cells vs DMSO: 64 ± 6 cells), was blocked by an antibody against alpha6. We conclude that the effects of DMSO on LISP-1 proliferation were accompanied by concurrent changes in the expression and function of integrins, consequently modulating adhesion/migration, and revealing a complex interplay between function/expression and the proliferative state of cells.

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-ß2 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.

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.