Martín Enrique Rabassa

Patterns of MUC1 Tissue Expression Defined by an Anti-MUC1 Cytoplasmic Tail Monoclonal Antibody in Breast Cancer

Our aim was to determine the pattern of expression of MUC1 mucin cytoplasmic tail (MUC1 CT) in breast carcinoma. A total of 98 invasive breast adenocarcinoma tumor samples were assayed by immunohistochemical (IHC) analysis. The pattern of reaction was classified as membrane, cytoplasmic, or mixed. Subcellular fractions were prepared after SDS-PAGE and Western blotting. The antibodies employed were anti-MUC1 CT (CT2 monoclonal antibody, MAb) and C595 MAb against the extracellular MUC1 core protein. With the CT2 MAb, IHC showed a high percentage of positive staining in 93% of specimens, with membrane staining the most common pattern observed. C595 MAb was reactive in 73% of specimens. Similar percentages of membrane and cytoplasmic staining were found, mainly in a mixed pattern. Western blotting showed different bands. With the CT2 MAb, the membrane fraction showed the most intense reaction; a strong band of reaction was detected at approximately <30 kD. With the C595 MAb, in most cases a double band at 200 kD was found. In breast epithelium, the pattern of MUC1 CT expression may constitute an indicator of MUC1 production because it does not depend on glycosylation. The pattern and extension of MUC1 CT positivity do not vary according to the histopathological subtype of the tumor.

Lewis x is highly expressed in normal tissues: a comparative immunohistochemical study and literature revision.

An immunohistochemical analysis was employed to determine the expression of carbohydrate antigens associated to mucins in normal epithelia. Tissue samples were obtained as biopsies from normal breast (18), colon (35) and oral cavity mucosa (8). The following carbohydrate epitopes were studied: sialyl-Lewis x, Lewis x, Lewis y, Tn hapten, sialyl-Tn and Thomsen-Friedenreich antigen. Mucins were also studied employing antibodies against MUC1, MUC2, MUC4, MUC5AC, MUC6 and also normal colonic glycolipid. Statistical analysis was performed and Kendall correlations were obtained. Lewis x showed an apical pattern mainly at plasma membrane, although cytoplasmic staining was also found in most samples. TF, Tn and sTn haptens were detected in few specimens, while sLewis x was found in oral mucosa and breast tissue. Also, normal breast expressed MUC1 at a high percentage, whereas MUC4 was observed in a small number of samples. Colon specimens mainly expressed MUC2 and MUC1, while most oral mucosa samples expressed MUC4 and MUC1. A positive correlation between MUC1VNTR and TF epitope (τ=0.396) was found in breast samples, while in colon specimens MUC2 and colonic glycolipid versus Lewis x were statistically significantly correlated (τ=0.28 and τ=0.29, respectively). As a conclusion, a defined carbohydrate epitope expression is not exclusive of normal tissue or a determined localization, and it is possible to assume that different glycoproteins and glycolipids may be carriers of carbohydrate antigens depending on the tissue localization considered.

MUC1 expression and anti-MUC1 serum immune response in head and neck squamous cell carcinoma (HNSCC): a multivariate analysis

BackgroundHNSCC progression to adjacent tissue and nodes may be mediated by altered glycoproteins and glycolipids such as MUC1 mucin. This report constitutes a detailed statistical study about MUC1 expression and anti-MUC1 immune responses in relation to different clinical and pathological parameters which may be useful to develop new anti HNSCC therapeutic strategies.Patients and methodsFifty three pre treatment HNSCC patients were included: 26 (49.1%) bearing oral cavity tumors, 17 (32.1%) localized in the larynx and 10 (18.8%) in the pharynx. Three patients (5.7%) were at stage I, 5 (9.4%) stage II, 15 (28.3%) stage III and 30 (56.6%) at stage IV. MUC1 tumor expression was studied by immunohistochemistry employing two anti-MUC1 antibodies: CT33, anti cytoplasmic tail MUC1 polyclonal antibody (Ab) and C595 anti-peptidic core MUC1 monoclonal antibody. Serum levels of MUC1 and free anti-MUC1 antibodies were detected by ELISA and circulating immune complexes (CIC) by precipitation in polyethylene glycol (PEG) 3.5%; MUC1 isolation from circulating immune complexes was performed by protein A-sepharose CL-4B affinity chromatography followed by SDS-PAGE and Western blot. Statistical analysis consisted in Multivariate Principal Component Analysis (PCA); ANOVA test (Tukeys test) was employed to find differences among groups; nonparametrical correlations (Kendalls Tau) were applied when necessary. Statistical significance was set to p < 0.05 in all cases.ResultsMUC1 cytoplasmic tail was detected in 40/50 (80%) and MUC1 protein core in 9/50 (18%) samples while serum MUC1 levels were elevated in 8/53 (15%) patients. A significant statistical correlation was found between MUC1 serum levels and anti-MUC1 IgG free antibodies, while a negative correlation between MUC1 serum levels and anti-MUC1 IgM free antibodies was found. Circulating immune complexes were elevated in 16/53 (30%) samples and were also statistically associated with advanced tumor stage. MUC1 was identified as an antigenic component of IgG circulating immune complexes. Moreover, poorly differentiated tumors were inversely correlated with tumor and serum MUC1 detection and positively correlated with node involvement and tumor mass.ConclusionPossibly, tumor cells produce MUC1 mucin which is liberated to the circulation and captured by IgG antibodies forming MUC1-IgG-CIC. Another interesting conclusion is that poorly differentiated tumors are inversely correlated with tumor and serum MUC1 detection.

Glycerol-3-phosphate acyltransferase-2 is expressed in spermatic germ cells and incorporates arachidonic acid into triacylglycerols.

Background De novo glycerolipid synthesis begins with the acylation of glycerol-3 phosphate catalyzed by glycerol-3-phosphate acyltransferase (GPAT). In mammals, at least four GPAT isoforms have been described, differing in their cell and tissue locations and sensitivity to sulfhydryl reagents. In this work we show that mitochondrial GPAT2 overexpression in CHO-K1 cells increased TAG content and both GPAT and AGPAT activities 2-fold with arachidonoyl-CoA as a substrate, indicating specificity for this fatty acid. Methods and Results Incubation of GPAT2-transfected CHO-K1 cells with [1-14C]arachidonate for 3 h increased incorporation of [14C]arachidonate into TAG by 40%. Consistently, arachidonic acid was present in the TAG fraction of cells that overexpressed GPAT2, but not in control cells, corroborating GPAT2s role in synthesizing TAG that is rich in arachidonic acid. In rat and mouse testis, Gpat2 mRNA was expressed only in primary spermatocytes; the protein was also detected in late stages of spermatogenesis. During rat sexual maturation, both the testicular TAG content and the arachidonic acid content in the TAG fraction peaked at 30 d, matching the highest expression of Gpat2 mRNA and protein. Conclusions These results strongly suggest that GPAT2 expression is linked to arachidonoyl-CoA incorporation into TAG in spermatogenic germ cells.

DIFFERENTIAL EXPRESSION OF MUC1 AND CARBOHYDRATE ANTIGENS IN PRIMARY AND SECONDARY HEAD AND NECK SQUAMOUS CELL CARCINOMA

In head and neck squamous cell carcinoma (HNSCC), tumor markers may be helpful to evaluate prognosis accurately as well as to improve therapy selection. Detection of human MUC1 has been widely employed for the evaluation of carcinoma patients. This article aims to study MUC1, Tn, sTn, and Lewis antigenic expression in primary HNSCC, lymph node metastasis, and local recurrences.

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 metabolism—with the associated ontological terms folding, ubiquitination, and proteosomal degradation—cell 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.

High expression of sLex associated with poor survival in Argentinian colorectal cancer patients.

Aim Colorectal cancer (CRC) is one of the most prevalent malignancies in Argentina with 11,043 new cases and 6,596 deaths estimated to have occurred in 2008. The present study was developed to clarify the differential expression of MUC1, MUC2, sLex, and sLea in colorectal cancer patients and their relationship with survival and clinical and histological features. Methods Ninety primary tumor samples and 43 metastatic lymph nodes from CRC patients were studied; follow-up was documented. Twenty-six adenoma and 68 histological normal mucosa specimens were analyzed. An immunohistochemical approach was applied and statistical analysis was performed. Results In tumor samples, MUC1, sLea, and sLex were highly expressed (94%, 67%, and 91%, respectively); also, we found a significantly increased expression of the 3 antigens in primary tumors and metastatic lymph nodes compared with normal mucosa and adenomas. MUC2 was expressed in 52% of both normal mucosa and CRC samples; this reactivity significantly decreased in metastatic lymph nodes (p<0.05). A multiple comparison analysis showed that MUC1 and sLex discriminated among 3 groups: normal, adenoma, and CRC tissues. The increase of sLex expression showed an association with recurrence, and survival analysis showed that a high sLex staining was significantly associated with a poor survival. By multivariate analysis MUC1 inmunoreactivity correlated positively and significantly with tumor size, while MUC2 expression showed the opposite correlation. Conclusions The correlation of sLex overexpression in primary tumors and metastatic lymph nodes, the discrimination among the normal, adenoma, and CRC groups based on sLex expression, as well as its association with recurrence and survival, all suggest a prognostic role of sLex in Argentinian CRC patients.

Nuclear localization of MUC1 extracellular domain in breast, head and neck, and colon cancer.

Background The glycoprotein MUC1 is overexpressed and underglycosylated in cancer cells. MUC1 is translated as a single polypeptide that undergoes autocleavage into 2 subunits (the extracellular domain and the cytoplasmic tail), and forms a stable heterodimer at the apical membrane of normal epithelial cells. The MUC1 cytoplasmic tail localizes to the cytoplasm of transformed cells and is targeted to the nucleus. Aims To study the expression of the MUC1 extracellular subunit in cell nuclei of neoplastic breast, head and neck, and colon samples. Materials and Methods 330 primary tumor samples were analyzed: 166 invasive breast carcinomas, 127 head and neck tumors, and 47 colon tumors; 10 benign breast disease (BBD) and 40 normal specimens were also included. A standard immunohistochemical method with antigen retrieval was performed. Nuclear fractions from tissue homogenates and breast cancer cell lines (ZR-75, MDA-MB-231, MCF7, and T47D) were obtained and analyzed by Western blotting (WB). The anti-MUC1 extracellular subunit monoclonal antibody HMFG1 was used for immunohistochemistry. Results 37/166 breast cancer specimens, 5/127 head and neck cancer specimens, 2/47 colon cancer samples, and 3/10 BBD samples showed immunohistochemical staining at the nuclear level. No nuclear reaction was detected in normal samples. By WB, breast and colon cancer purified nuclear fractions showed reactivity at 200 kDa in 3/30 breast and 3/20 colon cancer samples as well as purified nuclear fractions obtained from breast cancer cell lines. Conclusions This study shows that the MUC1 extracellular domain might be translocated to the cell nucleus in breast, head and neck, and colon cancer as well as BBD.

Factors associated with breast cancer in an Argentine city

In Argentina, breast cancer is the most commonly diagnosed cancer and the first leading cause of cancer deaths; this information is mainly based on estimations since only recently, Argentina has organized a National Registry of Tumors. Argentine studies addressing socioeconomic factors and their possible effect on breast cancer prevalence are scarce, and there is not any systematic action to prevent and control breast cancer as well. The aim of this study was to explore the relationship among socioeconomic factors, breast cancer risk factors, womens adherence to mammography screening, and breast cancer prevalence in La Plata, the capital city of Buenos Aires Province (Argentina), an administrative and University city. We performed a cross‐sectional study of women with low socioeconomic power (low group, LG) and a middle group (MG) from October 2012 to December 2012; 739 women between 45 and 79 years old were personally interviewed, being 360 (MG) and 379 (LG). A structured questionnaire previously validated was employed. Variables included were as follows: socioeconomic group, breast cancer risk and socioeconomic factors, mammographic screening parameters, ever diagnosed breast cancer, Physician Enrollment, and Health System which consists of three sectors: Public (free), Private, and Social/Union Security. Statistical analysis included chi‐squared and Kendalls tau‐b tests, ANOVA or Pearson correlation, principal component analysis (PCA), and Regression procedures, which included Logistic Binary and Ordinal Regressions. Tables 1 and 2 summarize risk and socioeconomic factors and breast cancer mammogram screening rates, respectively. Univariate analysis among variables showed that University women had less number of children, presented a lower rate of breastfeeding, had a high rate of mammographic screening, first mammogram <40 years old, and performed their mammograms every year. They had a high rate of Union Insurance and of Physician Enrollment with a significant difference with respect to women without a University degree. There were 36 women (4.2%) diagnosed with breast cancer; MG had the highest percentage with a significant difference; in MG, a significant association of breast cancer with age, menopausal status, hormonal replacement, and family history was found as well as with a high frequency of mammograms and early age of first mammogram. LG women did not show any of those associations except for a higher number of cases among those who attended to a Physician who had a high frequency of mammograms. The PCA identified two principal components which accounted for 32% of the total variation in the model (Figure S1); this analysis showed that the social groups were clearly separated (Figure S2). Employing the Logistic Regression, age was the only variable statistically related to breast cancer: odds ratio (OR) of 1.067 (1.000‐ 1.137) for each year of age. The Ordinal Regression showed that the educational level, marital status, breast cancer, Health System and Physician Enrollment were statistically associated with the frequency of mammograms. Low educational level was associated with low rate of mammograms; OR for women with primary education: 0.320 (0.178‐0.778) and with secondary level: 0.406 (0.219‐0.753), compared with University women. Married/in couple women showed a high rate of mammograms: OR of 1.950 (1.290‐2.948). Having breast cancer was associated with a high rate of mammograms (OR of 8.846, 1.966‐39.805) while women who attended the Public hospitals had a low rate of mammograms, OR 0.600 (0.369‐0.973) and those who had a Physician had a high frequency of mammograms, OR of 4.697 (3.053‐7.221). Received: 22 August 2017 | Revised: 29 August 2017 | Accepted: 30 August 2017 DOI: 10.1111/tbj.13147