Iron cycle disruption by heme oxygenase-1 activation leads to a reduced breast cancer cell survival.

Heme oxygenase-1 (HO-1), which catalyzes heme degradation releasing iron, regulates several processes related to breast cancer. Iron metabolism deregulation is also connected with several tumor processes. However the regulatory relationship between HO-1 and iron proteins in breast cancer remains unclear. Using human breast cancer biopsies, we found that high HO-1 levels significantly correlated with low DMT1 levels. Contrariwise, high HO-1 levels significantly correlated with high ZIP14 and prohepcidin expression, as well as hemosiderin storage. At mRNA level, we found that high HO-1 expression significantly correlated with low DMT1 expression but high ZIP14, L-ferritin and hepcidin expression. In in vivo experiments in mice with genetic overexpression or pharmacological activation of HO-1, we detected the same expression pattern observed in human biopsies. In in vitro experiments, HO-1 activation induced changes in iron proteins expression leading to an increase of hemosiderin, ROS levels, lipid peroxidation and a decrease of the growth rate. Such low growth rate induced by HO-1 activation was reversed when iron levels or ROS levels were reduced. Our findings demonstrate an important role of HO-1 on iron homeostasis in breast cancer. The changes in iron proteins expression when HO-1 is modulated led to the iron accumulation deregulating the iron cell cycle, and consequently, generating oxidative stress and low viability, all contributing to impair breast cancer progression.

Breast cancer cutaneous metastases are associated to uMUC1 and sialyl Lewis x and to highly malignant primary tumors.

Breast cancer spreading to different organs have been related to different molecules and mechanisms, but cutaneous metastasis remains unexplored. Increasing evidence showed that MUC1 and some of its carbohydrate associated antigens may be implicated in breast cancer metastasis. In this study we analyzed these tumor markers in order to identify breast cancer cutaneous metastatic profiles. A cohort of 26 primary tumors from breast cancer patients with cutaneous metastases were included; also, cutaneous and lymphatic node metastatic samples and primary tumors from breast cancer patients without metastases were analysed. Immunohistochemical (IHC) studies demonstrated that both underglycosylated MUC1 (uMUC1) and sialyl Lewis x (sLex) to be positively associated with cutaneous metastatic primary tumors (p < 0.05). Notably, a high percentage of tumors with cutaneous metastases were characterized as triple negative and Her2+ tumors (37.5 % and 29 %, respectively). Some discordant results were found between primary tumors and their matched cutaneous metastases. To determine if MUC1 variants may be carriers of carbohydrate antigens, subcellular fractions from a cutaneous metastatic lesion were obtained, immunoprecipitated and analyzed by Western blot. We found that the isolated uMUC1 with a molecular weight of>200 kDa was also the site for binding of anti-sLex MAb; in coincidence, a high correlation of positive IHC expression of both markers was observed. Our findings confirm that breast cancer cutaneous metastases were associated to highly malignant primary tumors and sustain the hypothesis that u-MUC1 and sLe x may drive breast cancer cutaneous metastases.

IDO is highly expressed in breast cancer and breast cancer-derived circulating microvesicles and associated to aggressive types of tumors by in silico analysis.

Indoleamine-2,3-dioxygenase (IDO) has been established as a normal mechanism of peripheral tolerance and immunosuppression. Besides, malignant tumors release microvesicles (MV) related with tumor dissemination. The aims of this study were to determine the expression of IDO in breast cancer and circulating microvesicles from breast cancer patients and to perform an in silico analysis to find genes co-expressed to IDO. One hundred and twenty-two tissue and serum breast samples (91 malignant, 21 benign, and 10 normal), and MCF7, MDA-MB-231, and T47D breast cancer cell lines were included. Standard immunohistochemistry (IHC), immunocytochemistry (ICC), Western blot (WB), and RT-PCR were employed. Microvesicle isolation from plasma samples was obtained by serial centrifugation and ultracentrifugation. By IHC, 60 % breast cancer, 43 % benign, and 20 % normal samples were positive. Significant differences were found among normal, benign, and malignant samples. Breast cancer stages I, II, and III expressed IDO in 42, 66, and 71 % of samples, respectively, while breast cancer cell lines also reacted; by WB, 9/25 microvesicles fractions showed bands at 42 kD. In silico analysis of IDO 1 gene expression in breast cancer showed its association with several genes related to immune response and apoptosis. Moreover, IDO and co-expressed genes were found predominately in basal and erbB2 subtypes. The cumulative data indicate a high expression of IDO in breast cancer which increased with higher stages. Furthermore, IDO was found in association with circulating breast cancer MV, while experimental and in silico gene expression revealed that IDO was mainly expressed in a triple-negative subgroup.

Identification of signaling pathways modulated by RHBDD2 in breast cancer cells: a link to the unfolded protein response.

Rhomboid domain containing 2 (RHBDD2) was previously observed overexpressed and amplified in breast cancer samples. In order to identify biological pathways modulated by RHBDD2, gene expression profiles of RHBDD2 silenced breast cancer cells were analyzed using whole genome human microarray. Among the statistically significant overrepresented biological processes, we found protein metabolism­with the associated ontological terms folding , ubiquitination, and proteosomal degradation­cell death, cell cycle, and oxidative phosphorylation. In addition, we performed an in silico analysis searching for RHBDD2 co-expressed genes in several human tissues. Interestingly, the functional analysis of these genes showed similar results to those obtained with the microarray data, with negative regulation of protein metabolism and oxidative phosphorylation as the most enriched gene ontology terms. These data led us to hypothesize that RHBDD2 might be involved in endoplasmic reticulum (ER) stress response. Thus, we specifically analyzed the unfolding protein response (UPR) of the ER stress process. We used a lentivirus-based approach for stable silencing of RHBDD2 mRNA in the T47D breast cancer cell line, and we examined the transcriptional consequences on UPR genes as well as the phenotypic effects on migration and proliferation processes. By employing dithiothreitol as an UPR inducer, we observed that cells with silenced RHBDD2 showed increased expression of ATF6, IRE1, PERK, CRT, BiP, ATF4, and CHOP (p <0.01). We also observed that RHBDD2 silencing inhibited colony formation and decreased cell migration. Based on these studies, we hypothesize that RHBDD2 overexpression in breast cancer could represent an adaptive phenotype to the stressful tumor microenvironment by modulating the ER stress response.

RHBDD2: a 5-fluorouracil responsive gene overexpressed in the advanced stages of colorectal cancer.

In previous studies, we identified rhomboid domain containing 2 (RHBDD2) gene to be markedly overexpressed in breast cancer patients that developed recurrence of the disease. In this study, we evaluated for the first time RHBDD2 gene expression in colorectal cancer (CRC). Five public available DNA microarray studies were compiled in a homogeneous dataset of 906 colorectal samples. The statistical analysis of these data showed a significant increase of RHBDD2 expression in the advanced stages of CRC (p < 0.01). We validated these findings by immunohistochemistry on 130 colorectal tissue samples; RHBDD2 protein overexpression was also observed in the advanced stages of the disease (p < 0.001). In addition, we investigated RHBDD2 expression in response to the chemotherapy agent 5-fluorouracile (5FU). We detected a significant increase of RHBDD2 mRNA and protein after 5FU treatment (20-40 µM; p < 0.001). Overall, these results showed that RHBDD2 overexpression might play a role in colorectal cancer progression.

Antigenic differences between metastatic cells in bone marrow and primary tumours and the anti-MUC1 humoral immune response induced in breast cancer patients.

The dissemination of a malignant neoplasia is a complex process, which requires a set of molecules that remains unknown. It has been suggested that mucins and their carbohydrate-associated antigens may be implicated in tumour spreading which may be also influenced by an anti-MUC1 immune response. In this pilot study, we report the pattern of carbohydrate and peptidic MUC1-associated epitopes on carcinoma cells isolated from bone marrow (BM), taking into account primary tumour histopathologic features. We also bring information about the anti-MUC1 humoral response in these patients. Seventeen patients with invasive breast carcinoma were included. A sample of the primary tumour, a serum sample and a BM aspirate were obtained from each patient. Clinical features studied were tumour size, number of metastatic nodes, histological type and disease stage. Standard immunohistochemistry was performed with antigenic retrieval using different monoclonal antibodies (MAbs): anti carbohydrate antigens: Lewis x (KM380), sLewis x (KM93), Lewis y (C14) and Tn, anti-MUC1 peptide core MAbs: C595, HMFG2 and SM3, anti-cytokeratins, anti-protoncogenes ErbB2 and ErbB3 (IgG) MAbs and also anti-CD34 and anti-CD45 MAbs. ELISA techniques were employed to study circulating MUC1 as well as free and complexed anti-MUC1 antibodies. Immunohistochemical results showed that carbohydrate antigenic expression increases in BM neoplastic cells compared to the original tumours. However, we were not able to demonstrate that a humoral immune response to MUC1 has been induced in these patients. Finally, the employed procedures allow the selective immortalisation of micrometastatic carcinoma cells since short-term cell lines were established.

MUC1 mucin and carbohydrate associated antigens as tumor markers in head and neck squamous cell carcinoma.

UNLABELLED: An immunological analysis to study MUC1 mucin core protein and carbohydrate associated antigens as tissue tumor markers in head and neck carcinoma was performed. Twenty nine patients with the following tumor localizations were included: tongue (n=10), larynx (n=8), oral cavity (n=4), maxillary sinus (n=3), tonsillar ring (n=3) and pharynx (n=1); seven samples of epithelium obtained from normal organs at the same localizations were studied as controls. Immunohistochemical analysis was performed following standard procedures and reaction was graded according to staining intensity and distribution. From each tissue section, membrane, cytoplasmic and nuclear moieties were obtained by differential centrifugation with subsequent fractionation by density gradient centrifugation (6M guanidium chloride-CsCl); subcellular moieties and CsCl derived fractions were analyzed by immunoblotting. Monoclonal antibodies (MAbs) reacting with the core protein of MUCI (C595) and associated carbohydrate antigens were: Tn, 83D4 MAb; Lewis y antigen (Le y), C14 MAb; Lewis x antigen (Le x), KM380 MAb and sialyl Lewis x (sLe x), KM93 MAb. Statistical analysis was undertaken by Spearman rank correlation. In tumor samples, the immunohistochemical identification of MUCl core protein and associated antigens was extended; differences were found in the pattern and intensity of expression; results were corroborated by immunoblotting although in a few samples there was not coincidence between both methods. Localization, tumor mass or node involvement did not show significant differences for any of the antigens studied. CONCLUSIONS: 1) head and neck carcinoma expressed MUCI and associated carbohydrate antigens in high levels; 2) no relationship between antigenic expression and tumor status was found.