Gendler, Sandra

Abstract MUC1 is overexpressed and aberrantly glycosylated in more than 60% of pancreatic ductal adenocarcinomas. The functional role of MUC1 in pancreatic cancer has yet to be fully elucidated due to a dearth of appropriate models. In this study, we have generated mouse models that spontaneously develop pancreatic ductal adenocarcinoma (KC), which are either Muc1-null (KCKO) or express human MUC1 (KCM). We show that KCKO mice have significantly slower tumor progression and rates of secondary metastasis, compared with both KC and KCM. Cell lines derived from KCKO tumors have significantly less tumorigenic capacity compared with cells from KCM tumors. Therefore, mice with KCKO tumors had a significant survival benefit compared with mice with KCM tumors. In vitro, KCKO cells have reduced proliferation and invasion and failed to respond to epidermal growth factor, platelet-derived growth factor, or matrix metalloproteinase 9. Further, significantly less KCKO cells entered the G2–M phase of the cell cycle compared with the KCM cells. Proteomics and Western blotting analysis revealed a complete loss of cdc-25c expression, phosphorylation of mitogen-activated protein kinase (MAPK), as well as a significant decrease in nestin and tubulin-α2 chain expression in KCKO cells. Treatment with a MEK1/2 inhibitor, U0126, abrogated the enhanced proliferation of the KCM cells but had minimal effect on KCKO cells, suggesting that MUC1 is necessary for MAPK activity and oncogenic signaling. This is the first study to utilize a Muc1-null PDA mouse to fully elucidate the oncogenic role of MUC1, both in vivo and in vitro. Cancer Res; 71(13); 4432–42. ©2011 AACR.

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.

Abstract MUC1 is an excellent target for immunotherapeutic strategies for cancers expressing Mucin 1 (breast, pancreas etc). MUC1 was recently selected by the NCI working group as the number 2 cancer vaccine target antigen, based on its immunogenicity, oncogenicity and wide-spread cell-surface expression. More than 90% of breast cancers over express MUC1, it stimulates both humoral and cellular immunity, its normal apical distribution is lost in cancer cells and aberrant glycosylation exposes peptide epitopes and novel carbohydrate antigens. Clinical trials using unglycosylated MUC1 epitopes have resulted in MUC1 specific cytotoxic T lymphocytes (CTLs) with limited effects on clinical response. Patients with breast tumors have exhibited spontaneous immune responses to MUC1 with the presence of T cells and antibodies specific for glycosylated MUC1. Because these natural immune responses are often not efficient in eradicating the tumor, it is necessary to specifically modulate these immune responses. Hence it is possible that better immunogenicity would be obtained using peptides more representative of the novel (glycosylated) forms of MUC1 as seen in cancer to which individuals may be less tolerant. The presentation of antigenic peptides by MHC class I molecules is crucial for the development of vaccines. MUC1 peptides from the VNTR region, which has five potential glycosylation sites, can be presented by a variety of class I molecules and can generate MUC1 specific CTLs. But these epitopes lack definite anchors and bind with low affinity compared to high affinity peptides. Hence, we developed several peptides from the MUC1 H-2Kb peptide (SAPDTRPAP) epitope with all possible modifications to improve their MHC and TCR binding efficiency. Preclinical studies were performed using the modified H-2Kb-restricted peptides along with the DC stimulants (CpG-ODN and GM-CSF) and foreign helper epitope (hepatitis B virus optimized I-Ab T helper peptide) in incomplete Freund's adjuvant in C57BL/6 mice transgenic for human MUC1. Peptides that were glycosylated at threonine and had methionine or leucine as the terminal amino acid induced a strong CTL response and were able to overcome tolerance in MUC1.Tg.mice. In the breast cancer model with the MUC1- expressing MMT cell line, the onset of tumor was delayed with peptide immunization and there was significant reduction in the tumor weight. Increased lytic activity of CD8+ CTLs and infiltration of T lymphocytes to the tumor site were observed with modified peptides as compared to the effect of MHC class I Kb and Db restricted native MUC1 peptides. Interestingly, the CTLs specific for glycosylated peptides were able to recognize both the Tn (GalNAc) and peptide epitopes. Thus, MUC1 peptides with carbohydrate at the appropriate position may be an optimal vaccine candidate because they can generate CTLs of dual specificity and high affinity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2403.

Abstract Allogeneic hematopoietic stem cell (HSC) transplantation is a promising area of therapy for various hematological malignancies like chronic/acute myeloid leukemia, myelodysplasia and lymphomas. Its effectiveness at inducing the graft versus tumor (GVT) effect is however, compromised by the graft versus host disease (GVHD) frequently observed in recipients post transplant. One of the strategies utilized in minimizing the effect of GVHD while still preserving GVT activity has been to target immunosuppressive cell types like Treg cells or myeloid derived suppressor cells (MDSCs), in an attempt to correct the balance between GVHD and GVT. MDSCs have been shown to be able to inhibit GVHD but not much is known about the ability of MDSCs to preserve GVT. We have previously shown that myeloid progenitors lacking Muc1 from Muc1 knockout (KO) C57BL/6 (H-2b) mice generate more effective MDSCs in vitro and in vivo. In this current study, we study the potential of using MDSCs generated in the absence of Muc1 to alleviate T cell expansion while preserving CTL activity. Our preliminary data shows that MDSCs generated from Muc1 KO C57BL/6 (H-2b) bone marrow are able to inhibit C57BL/6 (H-2b) T cell proliferation in an allogeneic mixed lymphocyte reaction, using Balb/c (H-2d) stimulators, more effectively than wildtype (WT) MDSCs. Interestingly, despite the reduced T cell proliferation in the presence of KO MDSCs, the CTL activity of these C57BL/6 T cells was still preserved, as demonstrated by their ability to lyse Balb/c A-20 leukemia cells. These results inidicate that co transplant of MDSCs generated in the absence of Muc1 can alleviate GVHD across fully mismatched major histocompatibility complex (MHC) barriers while still preserving the GVT ability of the allograft. This would also validate the clinical desirability of down regulating MUC1 in HSCs for more effective generation of MDSCs in a clinical allogeneic HSC transplantation setting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1935.

Abstract It has long been known that immune suppression is induced by the presence of a tumor, resulting in compromised T cell and dendritic cell (DC) functionality. What is not well understood is how soon the immune system recovers normal functionality following surgical removal of tumors and adjuvant therapy. This timing is of critical importance, as the effectiveness of therapeutic immune strategies relies on optimal presentation of antigens and activation of T cells. Blood from breast cancer patients was collected prior to surgery and at 3 and 6 months post adjuvant therapy. Patients were treated with surgery (breast conservation or mastectomy) and chemotherapy (CT) (n=22) or surgery without chemotherapy (n=38). Most patients had radiation therapy (RT). Blood from 22 healthy woman of similar age served as controls. T cell functionality (TCF) was determined following stimulation with plate-bound anti-CD3 (1 µg/ml) and anti-CD28 (0.5 µg/ml) and proliferation was measured by 3H-thymidine uptake. DC functionality (DCF) was determined by ability to present allo-antigens in a mixed lymphocyte reaction. Values for the normal samples determined the normal ranges (NR). Responses were grouped into 4 categories based on their status at 6 months: Group 1 - remained in NR; Group 2 - climbed into NR; Group 3 - remained below NR; Group 4 - fell below NR. Analysis of immunosuppression will be based on lymphocyte subsets, cytokine, COX-2, and PGE2 metabolite levels.T Cell Proliferation AllWith CTNo CT N=60N=22N=38Group 120%14%24%Group 223%23%24%Group 338%41%37%Group 418%23%16% DC Function AllWith CTNo CT N=58N=20N=38Group 143%35%47%Group 219%25%16%Group 317%15%18%Group 421%25%18% Forty-three percent of patients had normal levels (Groups 1 and 2) for TCF and 62% for DCF by 6 months post adjuvant therapy, suggesting that breast cancer patients are potentially amenable to vaccine therapy. 28% of patients had both TCF and DCF in NR; 50% had either TCF or DCF in NR; and 22% had neither in NR. The percentage of patients who had TCF in NR by adjuvant therapy was: 35% CT+RT (6/17); 33% CT (1/3); 52% (17/33) RT; and 20% (1/5) neither. The percentage of patients who had DCF in NR by adjuvant therapy was: 67% (10/15) CT+RT; 33% (1/3) CT; 67% (22/33) RT; and 40% (2/5) neither. Proper understanding of the effects of tumor, chemotherapy and radiation therapy on immune function, especially the effects on T cells and DCs, may enable us to identify the appropriate patients in whom to study immunotherapy approaches in women with early breast cancer and to examine strategies to counteract cancer-related defects in immune function. (supported by P50CA116201) Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4131.

The colonic mucus layer serves as an important barrier and prevents colonic bacteria from invading the mucosa and cause inflammation. The regulation of colonic mucus secretion is poorly understood. The aim of this study was to investigate the role of the mucus barrier in induction of colitis. Furthermore, regulation of mucus secretion by luminal bacterial products was studied. The colon of anesthetized Muc2 −/− , Muc1 −/− , wild-type (wt), and germ-free mice was exteriorized, the mucosal surface was visualized, and mucus thickness was measured with micropipettes. Colitis was induced by DSS (dextran sodium sulfate, 3%, in drinking water), and disease activity index (DAI) was assessed daily. The colonic mucosa of germ-free and conventionally housed mice was exposed to the bacterial products LPS (lipopolysaccharide) and PGN (peptidoglycan). After DSS induction of colitis, the thickness of the firmly adherent mucus layer was significantly thinner after 5 days and onward, which paralleled the increment of DAI. Muc2 −/− mice, which lacked firmly adherent mucus, were predisposed to colitis, whereas Muc1 −/− mice were protected with significantly lower DAI by DSS compared with wt mice. The mucus barrier increased in Muc1 −/− mice in response to DSS, whereas significantly fewer T cells were recruited to the inflamed colon. Mice housed under germ-free conditions had an extremely thin adherent colonic mucus layer, but when exposed to bacterial products (PGN or LPS) the thickness of the adherent mucus layer was quickly restored to levels observed in conventionally housed mice. This study demonstrates a correlation between decreasing mucus barrier and increasing clinical symptoms during onset of colitis. Mice lacking colonic mucus (Muc2 −/− ) were hypersensitive to DSS-induced colitis, whereas Muc1 −/− were protected, probably through the ability to increase the mucus barrier but also by decreased T cell recruitment to the afflicted site. Furthermore, the ability of bacteria to regulate the thickness of the colonic mucus was demonstrated.

Abstract Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of myeloid cells that inhibit T-cell activity and contribute to the immune suppression characteristic of most tumors. We discovered that bone marrow (BM) progenitor cells from the Muc1 knockout (KO) mice differentiated into CD11b+Gr1+ MDSCs in vitro under granulocyte macrophage colony-stimulating factor and interleukin-4 signaling. MUC1 is a tumor-associated mucin and its cytoplasmic tail (MUC1-CT) can regulate β-catenin to promote oncogenesis. Given the importance of β-catenin in hematopoiesis, we hypothesized that the MUC1 regulation of β-catenin is important for MDSC development. Our current study shows that the aberrant development of BM progenitors into CD11b+Gr1+ MDSCs is dependent on the down-regulation of β-catenin levels that occurs in the absence of Muc1. In light of this, KO mice showed enhanced EL4 tumor growth and were able to better tolerate allogeneic BM185 tumor growth, with an accumulation of CD11b+Gr1+ cells in the blood and tumor-draining lymph nodes. WT mice were able to similarly tolerate allogeneic tumor growth when they were injected with CD11b+Gr1+ cells from tumor-bearing KO mice, suggesting that tolerance of allogeneic tumors is dependent on MDSC-mediated immune suppression. This further delineates the ability of Muc1 to control MDSC development, which could directly affect tumorigenesis. Knowledge of the biology by which Muc1 regulates the development of myeloid progenitors into MDSCs would also be very useful in enhancing the efficacy of cancer vaccines in the face of tumor immune suppression. [Cancer Res 2009;69(8):3554–62]

Abstract MUC1 (MUC1 in humans and Muc1 in nonhuman species) is a transmembrane mucin-like glycoprotein expressed in epithelial cells lining various mucosal surfaces as well as hematopoietic cells. Recently, we showed that Muc1−/− mice exhibited greater inflammatory responses to Pseudomonas aeruginosa or its flagellin compared with their wild-type littermates, and our studies with cultured cells revealed that MUC1/Muc1 suppressed the Toll-like receptor (TLR) 5 signaling pathway, suggesting its anti-inflammatory role. Here we demonstrate that other TLR signaling (TLR2, 3, 4, 7, and 9) is also suppressed by MUC1/Muc1. The results from this study suggest that MUC1/Muc1 may play a crucial role during airway infection and inflammation by various pathogenic bacteria and viruses.

Cell surface mucins are large transmembrane glycoproteins involved in diverse functions ranging from shielding the airway epithelium against pathogenic infection to regulating cellular signaling and transcription. Although hampered by the relatively recent characterization of cell surface mucins and the difficulties inherent in working with molecules of their size, numerous studies have placed the tethered mucins in the thick of normal and diseased lung physiology. This review focuses on the three best-characterized cell surface mucins expressed in the respiratory tract: MUC1, MUC4, and MUC16.

Mucins are the major glycoprotein component of respiratory tract secretions. Little is known about their expression in the upper respiratory tract. In order to define this expression, in situ hybridization was performed on 19 normal and 4 vasomotor rhinitis (VMR) inferior turbinates to identify mucin mRNA. MUC1, MUC2, MUC4, MUC5AC, MUC5B, and MUC7 were expressed in both the normal and VMR turbinates. MUC 4 and MUC5AC were the most highly expressed mucins. MUC1, MUC2, MUC4, and MUC5AC were expressed mainly by the epithelial border, whereas MUC5B and MUC7 were expressed by the submucosal glands. MUC1 and MUC4 exhibited a diffuse expression by multiple cell types along the mucosal border, whereas MUC2 and MUC5AC expression appeared to be limited to a subpopulation of epithelial cells, most likely goblet cells. Although MUC1, MUC4, and MUC5AC showed sporadic submucosal glandular expression, MUC5B and MUC7 appeared to be the predominant submucosal gland mucins in the inferior turbinates. MUC3 and MUC6 expression, which have been found primarily in the gastric mucosa, were not seen in any of the inferior turbinate samples examined. The only difference seen between normal and VMR turbinates was a slight decrease in MUC1 expression in the VMR group. The variety of mucins expressed and the diversity of their expression patterns may have significance in terms of the rheologic and particle clearance properties of nasal secretions. Understanding the expression patterns in normal turbinates will serve as the foundation for further study of these mucins in disease states.