Rhomboid family gene expression profiling in breast normal tissue and tumor samples.

Rhomboid is an evolutionary conserved and functionally diversified group of proteins composed of proteolytically active and inactive members that are involved in the modulation of multiple biological processes such as epidermal growth factor receptor signaling pathway, endoplasmic reticulum-associated degradation, cell death, and proliferation. Recently, several human rhomboid genes have been associated with the development of chronic myeloid leukemia and pituitary, colorectal, ovarian, and breast cancers. In this study, we evaluated the mRNA and protein expression profiles of rhomboid genes in cancer cell lines and breast tissue/tumor samples. In silico analysis of publicly available gene expression datasets showed that different rhomboid genes are specifically expressed according to the breast cancer intrinsic subtypes. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis showed a significant RHBDD2 mRNA overexpression in advanced breast cancer compared with normal tissue samples (p = 0.012). In addition, we found that RHBDL2 and PARL mRNA expression was associated with a low/intermediate histologic tumor grade (p = 0.024 and p = 0.015, respectively). Immunohistochemistry analysis showed a significant increase of RHBDD2 protein expression in association with breast cancer samples negative for progesterone receptor (p = 0.015). Moreover, protein expression analysis corroborated the quantitative RT-PCR results, indicating that breast primary tumors belonging to patients with a more disseminated disease expressed significantly increased levels of RHBDD2 protein compared with less disseminated tumors (p = 0.01).

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.

Rhomboid domain containing 2 (RHBDD2): a novel cancer-related gene over-expressed in breast cancer.

In the course of breast cancer global gene expression studies, we identified an uncharacterized gene known as RHBDD2 (Rhomboid domain containing 2) to be markedly over-expressed in primary tumors from patients with recurrent disease. In this study, we identified RHBDD2 mRNA and protein expression significantly elevated in breast carcinomas compared with normal breast samples as analyzed by SAGE (n=46) and immunohistochemistry (n=213). Interestingly, specimens displaying RHBDD2 over-expression were predominantly advanced stage III breast carcinomas (p=0.001). Western-blot, RT-PCR and cDNA sequencing analyses allowed us to identify two RHBDD2 alternatively spliced mRNA isoforms expressed in breast cancer cell lines. We further investigated the occurrence and frequency of gene amplification and over-expression affecting RHBDD2 in 131 breast samples. RHBDD2 gene amplification was detected in 21% of 98 invasive breast carcinomas analyzed. However, no RHBDD2 amplification was detected in normal breast tissues (n=17) or breast benign lesions (n=16) (p=0.014). Interestingly, siRNA-mediated silencing of RHBDD2 expression results in a decrease of MCF7 breast cancer cells proliferation compared with the corresponding controls (p=0.001). In addition, analysis of publicly available gene expression data showed a strong association between high RHBDD2 expression and decreased overall survival (p=0.0023), relapse-free survival (p=0.0013), and metastasis-free interval (p=0.006) in patients with primary ER-negative breast carcinomas. In conclusion, our findings suggest that RHBDD2 over-expression behaves as an indicator of poor prognosis and may play a role facilitating 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.

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.

Detection of circulating mammary mucin (Muc1) and MUC1 immune complexes (Muc1-CIC) in healthy women.

There is convincing epidemiological evidence that multiparity provides protection against the development of breast cancer. In the present study we evaluated the levels of MUC1 and MUC1 circulating immune complexes (MUC1-CIC) in 135 serum samples obtained from healthy women. The study population included 13 women who had never been pregnant, 31 primiparous pregnant women, 36 multiparous pregnant women who had lactated, 5 multiparous pregnant women who had never lactated, 24 multiparous non-pregnant women who were lactating at the time of the study, 24 multiparous non-pregnant women who had lactated, and 2 multiparous non-pregnant women who had never lactated. The purpose of this work was to detect MUC1 variations during pregnancy and lactation as well as to study the possible induction of a humoral immune response against MUC1 in these conditions. We employed ELISA techniques to measure MUC1 (CASA test) and MUC1-CIC (IgM and IgG) using two anti-MUC1 monoclonal antibodies (MAbs): C595 and SM3. Statistical analysis was performed using the ANOVA test. The pooled results pertaining to pregnant versus non-pregnant women were compared and significant differences were observed in MUC1 and MUC1-CIC-lgM levels detected with both MAbs; the MUC1-CIC-lgG levels detected with C595 were increased in the pregnant group while the MUC1-CIC-lgG levels detected with SM3 did not show any significant differences. When the results were compared between lactating and non-lactating women, no significant differences were found. In conclusion, MUC1 and MUC1-CIC-lgM, detected with both MAbs, and MUC1-CIC-4gG levels detected with the MAb C595 are apparently induced by pregnancy.

MUC1 cytoplasmic tail detection using CT33 polyclonal and CT2 monoclonal antibodies in breast and colorectal tissue.

UNLABELLED: The immunohistochemical detection (IHC) of MUC1-CT employing a polyclonal antibody (CT33) in relation to CT2 monoclonal antibody (MAb) was analyzed. Western blot (WB) was used to determine the molecular mass of CT. MATERIALS AND METHODS: We studied 163 breast and 89 colorectal cancer specimens, 10 breast and 14 colorectal benign conditions, and 12 breast and 20 colorectal normal samples. From each tumor sample, subcellular fractions were obtained and analyzed by SDS-PAGE and WB. A nonparametric statistical analysis was employed; data were standardized and a Kendall-Tau correlation was applied. RESULTS: By IHC, 146/163 (90%) and 151/163 (93%) of breast cancer were positive with CT33 and CT2, respectively; a statistically significant correlation was obtained (t=0.5199). Seven out of ten (70%) benign breast specimens were positive with CT33 while all samples stained with CT2; in normal breast sample tissues, all were positive with both Abs. In colorectal cancer samples, both antibodies stained 47/89 (53%) samples; CT2 reacted in 13/14 (93%) of benign samples while CT33 showed a positive reaction in 9/14 (64%) of benign specimens. In normal samples, CT2 showed staining in 17/20 (85%) of samples and CT33 was reactive in 12/20 (60%). By WB, in breast and colorectal cancer samples, similar results were obtained with both antibodies: a main band at about 30kDa which represents the smaller subunit. CONCLUSION: CT33 polyclonal antibody has demonstrated its efficacy to detect MUC1 in breast and colorectal cancer tissues with similar reactivity to CT2. It is worthwhile to affirm that CT33 is a good indicator of MUC1 expression.

Humoral immune response induced by the protein core of MUC1 mucin in pregnant and healthy women.

UNLABELLED: Serum levels of MUC1 and antibodies (Abs) against MUC1 (IgG and IgM-MUC1) were evaluated in healthy women related to pregnancy and lactation status. A total of 149 serum samples were obtained from: nulliparous, primiparous pregnant, multiparous pregnant that have lactated, multiparous pregnant without lactation, multiparous non-pregnant actual lactating, multiparous non-pregnant that have lactated and finally, multiparous non-pregnant women without lactation. In all assays, we included pre- and post-serum samples belonging to a breast cancer patient vaccinated with a MUC1 derived peptide. CASA test was employed to measure MUC1 while IgG- and IgM-MUC1 serum Abs were evaluated with an ELISA using a 100 mer peptide as catcher. In all groups, mean IgM levels were higher than IgG mean values; when samples were grouped in pregnants versus non-pregnants, a significant difference was detected with both Abs, being raised in non-pregnants. When samples were grouped in lactating versus non-lactating a significant difference was detected with IgG-MUC1, being raised in lactating women while no significant difference was found with IgM-MUC1. The evaluation of serum MUC1 levels confirmed previous results since a significant difference between pregnant versus non-pregnant groups was found while lactating versus non-lactating samples did not. CONCLUSIONS: (i) Increased MUC1 serum levels are apparently associated with pregnancy but not with lactation; (ii) MUC1 Abs are mainly associated with lactation and with non-pregnant status. These results may be considered a contribution on studies about protection against breast cancer induced by pregnancy and lactation.