Designer Glycopeptides for Cytotoxic T Cell–based Elimination of Carcinomas

Tumors express embryonic carbohydrate antigens called tumor-associated carbohydrate antigens (TACA). TACA-containing glycopeptides are appealing cytotoxic T cell (CTL)-based vaccines to prevent or treat cancer because the same sugar moieties are expressed in a variety of tumors, rendering a vaccination strategy applicable in a large population. Here we demonstrate that by using glycopeptides with high affinity for the major histocompatibility complex and glycosylated in a position corresponding to a critical T cell receptor (TcR) contact, it is possible to induce anti-TACA CTL in vivo. In the current study we show that designer glycopeptides containing the Thomsen-Freidenreich (TF) antigen (β-Gal-[1→3]-α-GalNAc-O-serine) are immunogenic in vivo and generate TF-specific CTL capable of recognizing a variety of tumor cells in vitro including a MUC1-expressing tumor. The fine specificity of the TF-specific CTL repertoire indicates that the TcR recognize the glycosylated amino acid residue together with TF in a conventional major histocompatibility complex class I–restricted fashion. These results have high potential for immunotherapy against a broad range of tumors

The MUC1 Cytoplasmic Tail and Tandem Repeat Domains Contribute to Mammary Oncogenesis in FVB Mice

Background: Though the importance of the transmembrane mucin MUC1 in mammary oncogenesis has long been recognized, the relative contributions of the cytoplasmic tail and tandem repeat domains are poorly understood.Methods: To address this, mouse models of mammary carcinogenesis were created expressing full-length, cytoplasmic tail-deleted, or tandem repeat-deleted MUC1 constructs.Results: Overexpression of full-length MUC1 resulted in tumor formation in young mice (12 months); however, loss of either the cytoplasmic tail or the tandem repeat domain abrogated this oncogenic capacity. Aged mice in all strains developed late-onset mammary tumors similar to those previously described for the FVB background.Conclusions: This study is the fi rst spontaneous cancer model to address the relative importance of the cytoplasmic tail and tandem repeat domains to MUC1-driven mammary oncogenesis, and suggests that both of these domains are essential for tumor formation

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.Facultad de Ciencias MédicasCentro de Investigaciones Inmunológicas Básicas y Aplicada

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.Facultad de Ciencias MédicasCentro de Investigaciones Inmunológicas Básicas y Aplicada

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.Facultad de Ciencias MédicasCentro de Investigaciones Inmunológicas Básicas y Aplicada

MUC1 alters oncogenic events and transcription in human breast cancer cells

INTRODUCTION: MUC1 is an oncoprotein whose overexpression correlates with aggressiveness of tumors and poor survival of cancer patients. Many of the oncogenic effects of MUC1 are believed to occur through interaction of its cytoplasmic tail with signaling molecules. As expected for a protein with oncogenic functions, MUC1 is linked to regulation of proliferation, apoptosis, invasion, and transcription. METHODS: To clarify the role of MUC1 in cancer, we transfected two breast cancer cell lines (MDA-MB-468 and BT-20) with small interfering (si)RNA directed against MUC1 and analyzed transcriptional responses and oncogenic events (proliferation, apoptosis and invasion). RESULTS: Transcription of several genes was altered after transfection of MUC1 siRNA, including decreased MAP2K1 (MEK1), JUN, PDGFA, CDC25A, VEGF and ITGAV (integrin α(v)), and increased TNF, RAF1, and MMP2. Additional changes were seen at the protein level, such as increased expression of c-Myc, heightened phosphorylation of AKT, and decreased activation of MEK1/2 and ERK1/2. These were correlated with cellular events, as MUC1 siRNA in the MDA-MB-468 line decreased proliferation and invasion, and increased stress-induced apoptosis. Intriguingly, BT-20 cells displayed similar levels of apoptosis regardless of siRNA, and actually increased proliferation after MUC1 siRNA. CONCLUSION: These results further the growing knowledge of the role of MUC1 in transcription, and suggest that the regulation of MUC1 in breast cancer may be more complex than previously appreciated. The differences between these two cell lines emphasize the importance of understanding the context of cell-specific signaling events when analyzing the oncogenic functions of MUC1, and caution against generalizing the results of individual cell lines without adequate confirmation in intact biological systems

Proteomic Interrogation of Human Chromatin

Chromatin proteins provide a scaffold for DNA packaging and a basis for epigenetic regulation and genomic maintenance. Despite understanding its functional roles, mapping the chromatin proteome (i.e. the “Chromatome”) is still a continuing process. Here, we assess the biological specificity and proteomic extent of three distinct chromatin preparations by identifying proteins in selected chromatin-enriched fractions using mass spectrometry-based proteomics. These experiments allowed us to produce a chromatin catalog, including several proteins ranging from highly abundant histone proteins to less abundant members of different chromatin machinery complexes. Using a Normalized Spectral Abundance Factor approach, we quantified relative abundances of the proteins across the chromatin enriched fractions giving a glimpse into their chromosomal abundance. The large-scale data sets also allowed for the discovery of a variety of novel post-translational modifications on the identified chromatin proteins. With these comparisons, we find one of the probed methods to be qualitatively superior in specificity for chromatin proteins, but inferior in proteomic extent, evidencing a compromise that must be made between biological specificity and broadness of characterization. Additionally, we attempt to identify proteins in eu- and heterochromatin, verifying the enrichments by characterizing the post-translational modifications detected on histone proteins from these chromatin regions. In summary, our results provide insights into the value of different methods to extract chromatin-associated proteins and provide starting points to study the factors that may be involved in directing gene expression and other chromatin-related processes

Adenovirus-mediated and targeted expression of the sodium-iodide symporter permits in vivo radioiodide imaging and therapy of pancreatic tumors

Pancreatic cancer is the fourth leading cause of cancer death in the United States. It is highly aggressive with no uniformly effective chemotherapy available for metastatic disease. The sodium–iodide symporter (NIS) is a transmembrane protein responsible for uptake of iodide into cells. The presence of NIS in thyroid cells permits diagnostic imaging and therapy of thyroid tumors, using radioiodide. Previous studies from this laboratory reported mucin-1 (MUC1)-driven expression of NIS in cancer cells. MUC1 overexpression has also been reported in 90% of pancreatic tumors. In this study Ad5/MUC1/NIS was used to infect pancreatic cancer cells both in vitro and in vivo, to investigate the potential for radioiodide imaging and ablation of this disease. In vitro studies revealed a 43-fold increase in iodide uptake in NIS-transduced cells compared with controls. In vivo imaging revealed effective iodide uptake and retention at the site of NIS-transduced tumors, with optimal uptake (13% of injected dose) observed 5 hr after iodide administration. Intravenous delivery was performed to investigate potential hepatotoxicity of the construct in the event of virus leakage. Intravenous injection of Ad5/CMV/NIS resulted in robust iodide uptake throughout mouse liver, whereas no uptake was detected in the liver of animals given Ad5/MUC1/NIS intravenously. Administration of therapeutic doses of 131I resulted in significant regression of NIS-transduced tumors, with a mean 50% reduction in volume within 10 weeks of therapy (p < 0.0001). The ability to target NIS expression to pancreatic cancer, which has such limited treatment options, may be highly beneficial and warrants further investigation.Funding for this work was received from the Prospect Creek Foundation, a Mayo Foundation Prostate Cancer SPORE grant (CA91956), the Mayo Breast Cancer Program, and the Molecular Medicine Program of the Mayo Clinic College of Medicine.peer-reviewe

Expression of Human MUC1 During Early Pregnancy in the Human MUC1 Transgenic Mouse Model1

Embryo implantation involves direct interaction of the blastocyst with the luminal epithelium of the receptive uterus. MUC1, a transmembrane mucin expressed at the apical surface of uterine epithelia, acts as a barrier to microbial infection and enzymatic attack. Loss of MUC1 is believed to be a prerequisite for a functionally receptive uterus across many species. Human and murine MUC1 regulation by steroid hormones displays important differences. Estrogen (E2) stimulates MUC1 expression in mice, and progesterone (P4) antagonizes E2 action in this regard. MUC1 expression is severely reduced during the receptive uterine state in mice. In contrast, human MUC1 expression is maximal at the receptive or midluteal phase, when P4 levels are high. No information is available regarding regulation of human MUC1 in vivo at the site of embryo attachment. Our aim was to better understand regulation of human MUC1 during early pregnancy in vivo. For this purpose, we used a transgenic mouse carrying full-length human MUC1 gene (Tg(MUC1)79.24Gend) as well as endogenous MUC1 as a model system. Human MUC1 was detected by real-time RT-PCR, Western blotting, and immunohistochemistry during early pregnancy. Our data indicate that human MUC1 persists at reduced (20% relative to Day 1 postcoitum) levels in receptive-phase uteri, including the site of embryo attachment. In contrast, mouse MUC1 was much more severely (>98% relative to Day 1 postcoitum) reduced in the same context. These observations are consistent with distinct regulation between the human and mouse genes. Because these genes are expressed in the same transcriptional context (i.e., mouse uterine epithelia), structural differences between human and murine genes must account for these differences in MUC1 regulation