Humanin Promotes Tumor Progression in Experimental Triple Negative Breast Cancer.

Humanin (HN) is a mitochondrial-derived peptide with cytoprotective effect in many tissues. Administration of HN analogs has been proposed as therapeutic approach for degenerative diseases. Although HN has been shown to protect normal tissues from chemotherapy, its role in tumor pathogenesis is poorly understood. Here, we evaluated the effect of HN on the progression of experimental triple negative breast cancer (TNBC). The meta-analysis of transcriptomic data from The Cancer Genome Atlas indicated that HN and its receptors are expressed in breast cancer specimens. By immunohistochemistry we observed up-regulation of HN in TNBC biopsies when compared to mammary gland sections from healthy donors. Addition of exogenous HN protected TNBC cells from apoptotic stimuli whereas shRNA-mediated HN silencing reduced their viability and enhanced their chemo-sensitivity. Systemic administration of HN in TNBC-bearing mice reduced tumor apoptotic rate, impaired the antitumor and anti-metastatic effect of chemotherapy and stimulated tumor progression, accelerating tumor growth and development of spontaneous lung metastases. These findings suggest that HN may exert pro-tumoral effects and thus, caution should be taken when using exogenous HN to treat degenerative diseases. In addition, our study suggests that HN blockade could constitute a therapeutic strategy to improve the efficacy of chemotherapy in breast cancer.

Alternative splicing variant of RHBDD2 is associated with cell stress response and breast cancer progression.

RHBDD2 is an intramembrane pseudoprotease member of the Rhomboid superfamily. Our previous studies in breast and colorectal cancer indicate an association between RHBDD2 overexpression and advanced tumor stages. Two alternative transcriptional variants have been described for RHBDD2, which would be encoding for different RHBDD2 protein isoforms. The expression of these RHBDD2 variants/isoforms and its association with breast cancer was the focus of this study. First, expression of RHBDD2 splicing variants was evaluated in normal and breast tumor samples. RHBDD2 variant 2 overexpression was detected in tumors in respect to normal breast tissues at the mRNA and protein levels (P<0.05). Moreover, RHBDD2 variant 2 expression was associated with poor prognostic factors such as basal­like intrinsic subtype (P<0.05), high proliferation (P<0.01) and long­term risk­of­recurrence (P<0.01) scores. Second, the expression of both variants was evaluated under nutritional­deprived conditions in breast cancer cell lines. Results demonstrated that RHBDD2 splicing was switched from mRNA variant 1 to variant 2 in association with a significant increment of protein isoform B in response to glucose starvation treatment. Therefore, we propose that the switch from the RHBDD2 variant 1, expressed in normal epithelial cells, to variant 2 occurs as an adaptive phenotype to bypass the stressful tumor microenvironment and promote tumor progression. Finally, the RHBDD2 subcellular localization was corroborated at the Golgi apparatus and their associated v­SNARE transport vesicles, suggesting a putative new role for RHBDD2 in the protein trafficking of human breast cancer cells.

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 (r=0.396) was found in breast samples, while in colon specimens MUC2 and colonic glycolipid versus Lewis x were statistically significantly correlated (r=0.28 and r=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.

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.

Humoral immune response against tumoral mucin 1 (MUC1) in breast cancer patients.

The aim of this study was to elucidate whether the IgG humoral immune response to breast cancer cells is directed to the aberrant mucin-1 (MUC1) associated to this type of cancer. To this aim, an adaptation of immunohistochemistry (IHC) was performed on samples of 45 breast cancer tissues, 12 benign disease tissues, and 31 normal tissues, incubated with matched serum samples from the same patients. Each serum sample was also incubated, with a modified immunocytochemistry (ICC), with MCF7 cells. In both techniques, serum was employed instead of the primary antibody. In the case of IHC, the reactivity with sera diminished when added after previous incubation of the tumor/tissue with an anti-MUC1 mAb; the reduction in reactivity was: from 93% to 44% in breast cancer tissues, and from 100% to 67% in benign disease tissues. The reactivity of normal samples (36%) remained unchanged. In the case of ICC, the reactivity with sera decreased after incubation with anti-MUC1 mAb from 71% to 16% in breast cancer tissues, from 83% to 0% in benign disease tissues, and from 52% to 10% in normal serum samples. These results were confirmed employing siRNA MUC1 transient gene knockdown. By Western blot analysis -after immunoprecipitation (IP) of the circulating MUC1- and ELISA, the TF antigen was detected in circulating MUC1 in all breast cancer and benign samples while Tn was detected in 38% of the samples.
The existence of IgG autoantibodies against aberrantly glycosylated MUC1 may have a protective role and may contribute to a better prognosis in some patients. Enhancement of this natural immune response may constitute an alternative therapeutic strategy.

Invasive breast cancer in Argentine women: association between risk and prognostic factors with antigens of a peptidic and carbohydrate nature.

OBJECTIVE: In breast cancer, several tumor markers have been identified. The marker most extensively associated with breast cancer is MUC1. The objective of the study was to analyze prognostic and risk factors in relation to tumor markers in order to clarify breast cancer biology. A total of 349 primary tumor samples and lymph nodes from breast cancer patients were studied. Risk and prognostic factors were considered. An immunohistochemical approach was applied and an extensive statistical analysis was performed, including frequency analysis and analysis of variance. Correlation among variables was performed with principal component analysis. RESULTS: All the antigens showed an increased expression according to tumor size increment; moreover, sialyl Lewis x expression showed a significant increase in relation to disease stage, whereas Tn and TF presented a positive tendency. Vascular invasion was related to sialyl Lewis x expression and number of metastatic lymph nodes. Taking into account risk factors, when a patient had at least one child, Lewis antigens diminished their expression. In relation to breastfeeding, sialyl Lewis x expression diminished, although its apical expression increased. CONCLUSION: Associations between MUC1 and carbohydrate antigens and risk and prognostic factors show the complexity of the cellular biological behavior that these antigens modulate in breast cancer.

Comparative immunohistochemical study of MUC1 and carbohydrate antigens in breast benign disease and normal mammary gland.

The aim was to compare the expression of MUC1 and carbohydrate antigens in 124 tissue samples; 42 fibroadenoma (FA), 23 nonproliferative benign diseases (NPF), 25 usual epithelial hyperplasia (UEH), 7 atypical ductal hyperplasia (ADH), and 27 breast normal tissues. An immunohistochemical approach was adopted, using the following antibodies: reactive with MUC1 variable number of tandem repeats (C595, HMFG2, and SM3 monoclonal antibodies), anti-MUC1-cytoplasmic tail polyclonal antibody (CT33), and anti-carbohydrate antigens (sialyl Lewis x, Lewis x, Lewis y, Tn, and Thomsen-Friedenreich epitopes). Positive area of reaction, intensity, and pattern of expression were considered. A reactivity index was calculated as intensity (I) x 100+percentage of positive area (A). Statistical analysis comprised frequency analysis, P < 0.05, analysis of variance, and multiple correlation with principal component analysis. All samples expressed MUC1, detected by at least one anti-MUC1 antibody whereas Lewis x was the carbohydrate antigen most frequently found in all groups whereas variable number of tandem repeats MUC1 and Lewis x showed the highest correlation: 93% of normal samples, 62.5% of NPF, 87% of FA, 85% of UEH, and finally 80% of ADH. Although principal component analysis using reactivity indexes explained only 39% of data variability, normal samples appeared grouped and separated from benign breast diseases, which remained spread. Thomsen-Friedenreich was the only antigen that showed an increased tendency for positive expression and intensity from NPF through FA, UEH to ADH, whereas it was not detected in normals. With respect to the pattern of expression, an apical pattern was predominantly found in all the groups.

Breast cancer humoral immune response: involvement of Lewis y through the detection of circulating immune complexes and association with Mucin 1 (MUC1).

BACKGROUND: In cancer patients, MUC1 glycoprotein may carry Lewis y which could be involved in immune response. PURPOSES: 1- to evaluate the presence of Lewis y and MUC1 in circulating immune complexes (Lewis y/CIC and MUC1/CIC, respectively) and their correlation; 2- to analyze the possible presence of Lewis y in carbohydrate chains of tumoral MUC1 glycoprotein and 3- to correlate serum and tissue parameters considered. METHODS: Pretreatment serum and tissue breast samples from 76 adenocarcinoma, 34 benign and 36 normal specimens were analyzed. Anti-MUC1 and anti-Lewis y MAbs were employed. To detect Lewis y/CIC and MUC1/CIC, ELISA tests were developed; serum samples containing MUC1 were previously selected by Cancer Associated Serum Antigen (CASA). Immunoprecipitation (IP) was performed in 9 malignant, benign and normal samples and analyzed by SDS-PAGE and Western blot. Lewis y and MUC1 expression was studied by immunohistochemistry (IHC). Statistical analysis was performed employing principal component analysis (PCA), ANOVA, Tukey HSD, Chi square test and classical correlation (p < 0.05). RESULTS: By ELISA, Lewis y/IgM/CIC levels showed statistically significant differences between breast cancer versus benign and normal samples; mean +/- SD values expressed in OD units were: 0.525 +/- 0.304; 0.968 +/- 0.482 and 0.928 +/- 0.447, for breast cancer, benign disease and normal samples, respectively, p < 0.05. Lewis y/IgG/CIC did not show any statistically significant difference. MUC1/IgM/CIC correlated with Lewis y/IgM/CIC. By CASA, 9 samples with MUC1 values above the cut off were selected and IP was performed, followed by SDS-PAGE and Western blot; bands at 200 kDa were obtained with each MAb in all the samples. By IHC, with C14 MAb, 47.5%, 31% and 35% of malignant, benign and normal samples, respectively, showed positive reaction while all the samples were positive with anti-MUC1 MAb; in both cases, with a different pattern of expression between malignant and non malignant samples. CONCLUSION: Our findings support that in breast cancer there was a limited humoral immune response through Lewis y/IgM/CIC levels detection which correlated with MUC1/IgM/CIC. We also found that Lewis y might be part of circulating MUC1 glycoform structure and also that Lewis y/CIC did not correlate with Lewis y expression.

Tissue and serum MUC1 mucin detection in breast cancer patients.

Tumor MUC1 expression as well as levels of MUC1, MUC1 circulating immune complexes (MUC1-CIC) and free antibodies against MUC1 (IgG and IgM-MUC1) were evaluated in 70 breast cancer patients with different stages of disease. Controls included: 135 serum samples from healthy women, normal mammary tissue samples (n = 7) and benign breast disease specimens (n = 6). In all assays, pre- and post-vaccination serum samples from breast cancer patients belonging to a vaccination protocol developed at the Memorial Sloan Kettering Cancer Center (New York, USA) were included as controls. Serum MUC1 was measured through Cancer Associated Serum Antigen test and CA15-3 test. Employing ELISA, MUC1-CIC-IgG/M were measured with either C595 or SM3 monoclonal antibodies (MAb) as catchers and also free antibodies against MUC1 (IgG and IgM) using 100mer peptide as catcher. Employing multivariate statistical analysis, results were correlated with age, tumor type, stage of disease and grade of differentiation. By quantitative immunohistochemistry using three anti-MUC1 core protein MAbs (C595, HMFG2 and SM3), tumor MUC1 was detected in 60/70 (86%) breast cancer specimens which reacted with at least one of these MAbs. High MUCI serum levels were detected in 14/67 (21%); IgG and IgM anti-MUC1 antibodies were found elevated in 32 and 14%, respectively, while IgG-MUC1-CIC-measured with C595 in 42% and IgM-MUC1-CIC in 54%; finally, SM3 was positive in 43 and 18%, respectively. Results of these studies demonstrate that in a group of breast cancer patients, MUC1 was detected both in tissue specimens as well as free in serum samples; furthermore, MUC1 can also circulate complexed with IgG and IgM antibodies; thus an accurate measurement should include free and complexed forms. On the other hand, immunohistochemical studies on breast cancer tissues may contribute to reveal different MUC1 glycoforms.

Investigación de la mucina de tipo 1 (MUC1) circulante y tisular en el cáncer de mama / Investigation of the circulating and tissue type 1 mucin (MUC 1) in the breast cancer

Las mucinas (MUC) son glicoproteínas que se localizan y expresan en la mayoría de los epitelios y han sido recientemente relacionadas con el cáncer de mama. entre ellas, la MUC1 constituye una mólecula de elevado peso molecular que en la célula maligna puede presentar una glicosilación aberrante o incompleta que la convierte en inmunogénica. Los objetivos del presente trabajo de investigación han sido: 1- estudiar la expresión celular de MUC1 en el cáncer de mama y en neoplasias mamarias benignas; 2- estudiar los niveles de MUC1 sérica como marcador tumoral; 3- investigar la relación entre la expresión sérica y tisular y 4- detectar anticuerpos séricos anti MUC1 libres y acomplejados. Se incluyeron 70 pacientes con cáncer de mama de las que se obtuvieron 70 muestras de tejido tumoral y 65 sueros y los controles fueron 5 muestras de tejido normal, 8 displasias y 135 sueros de mujeres sanas. además, se emplearon sueros de pacientes con cáncer de mama pre y post vacunación con un péptido derivado de MUC1. Por otra parte, se utilizaron 3 monoclonales (MAbs) que reaccionan con el centro proteico de la molécula de MUC1: C595, SM3 y HMFG2. Los métodos empleados fueron: inmunocito e inmunohistoquímica con procesamiento de imágenes y análisis cuantitativo, preparación de membranas subcelulares; SDS-PAGE y Western blot, cultivo primario de tumores malignos, ensayos de tumorigenicidad, detección de MUC1 circulante, determinación de complejos inmunes anti-MUC1 y detección de anticuerpos libres anti-MUC1 mediante ELISA (enzimoinmunoensayo en fase líquida); los datos fueron analizados estadísticamente. Los resultados fueron los siguientes: la inmunohistoquímica reveló que las muestras de cáncer de mama fueron positivas en el 73 por ciento con un patrón de expresión fundamentalmente en la membrana plasmática, o bien mixto tanto en la membrana plasmática como en el citoplasma. En los cultivos primarios se observó expresión de MUC1 principalmente citoplásmica en todos los casos con los MAbs empleados. En los controles, el patrón de reactividad fue apical o en la luz de las glándulas siendo negativo el SM3 para las neoplasias benignas. mediante Western-blot fue posible observar que las fracciones subcelulares de neoplasias malignas fueron positivas dando una banda de reacción de >200kD con los tres MAbs ensayados... (TRUNCADO) (AU)

Investigación de la mucina de tipo 1 (MUC1) circulante y tisular en el cáncer de mama / Investigation of the circulating and tissue type 1 mucin (MUC 1) in the breast cancer

Las mucinas (MUC) son glicoproteínas que se localizan y expresan en la mayoría de los epitelios y han sido recientemente relacionadas con el cáncer de mama. entre ellas, la MUC1 constituye una mólecula de elevado peso molecular que en la célula maligna puede presentar una glicosilación aberrante o incompleta que la convierte en inmunogénica. Los objetivos del presente trabajo de investigación han sido: 1- estudiar la expresión celular de MUC1 en el cáncer de mama y en neoplasias mamarias benignas; 2- estudiar los niveles de MUC1 sérica como marcador tumoral; 3- investigar la relación entre la expresión sérica y tisular y 4- detectar anticuerpos séricos anti MUC1 libres y acomplejados. Se incluyeron 70 pacientes con cáncer de mama de las que se obtuvieron 70 muestras de tejido tumoral y 65 sueros y los controles fueron 5 muestras de tejido normal, 8 displasias y 135 sueros de mujeres sanas. además, se emplearon sueros de pacientes con cáncer de mama pre y post vacunación con un péptido derivado de MUC1. Por otra parte, se utilizaron 3 monoclonales (MAbs) que reaccionan con el centro proteico de la molécula de MUC1: C595, SM3 y HMFG2. Los métodos empleados fueron: inmunocito e inmunohistoquímica con procesamiento de imágenes y análisis cuantitativo, preparación de membranas subcelulares; SDS-PAGE y Western blot, cultivo primario de tumores malignos, ensayos de tumorigenicidad, detección de MUC1 circulante, determinación de complejos inmunes anti-MUC1 y detección de anticuerpos libres anti-MUC1 mediante ELISA (enzimoinmunoensayo en fase líquida); los datos fueron analizados estadísticamente. Los resultados fueron los siguientes: la inmunohistoquímica reveló que las muestras de cáncer de mama fueron positivas en el 73 por ciento con un patrón de expresión fundamentalmente en la membrana plasmática, o bien mixto tanto en la membrana plasmática como en el citoplasma. En los cultivos primarios se observó expresión de MUC1 principalmente citoplásmica en todos los casos con los MAbs empleados. En los controles, el patrón de reactividad fue apical o en la luz de las glándulas siendo negativo el SM3 para las neoplasias benignas. mediante Western-blot fue posible observar que las fracciones subcelulares de neoplasias malignas fueron positivas dando una banda de reacción de >200kD con los tres MAbs ensayados... (TRUNCADO)