MUC1 Protein Expression Research Articles

Abstract 3070: Activated EGFR and PPARδ regulate MUC1 expression in human uterine and pancreatic cancer cell lines

Abstract The high molecular weight, transmembrane mucin, MUC1 is expressed at high levels in many epithelial tumors including endometrial and pancreatic cancers. Aberrant glcosylation and overexpression of MUC1 make tumor cells poorly adherent, protect tumor cells from the host immune system, promote metastases from primary sites and enhance resistance to chemotherapeutic drugs. MUC1 has three distinct domains: an extracellular domain (ECD) composed of a tandem repeat region rich in serine, threonine and proline; a transmembrane domain (TM) and; a short cytoplasmic tail domain (CT) involved in a variety of signal transduction events. Epidermal Growth Factor Receptor (EGFR/erbB) family members regulate cell growth and survival in both normal and cancer cells. MUC1 interacts with erbB receptors and also affects downstream signaling pathways in a EGFR-ligand dependent manner. We previously demonstrated that the Peroxisome Proliferator Activated Receptor ≤ (PPARδ) agonist rosiglitazone severely inhibits progesterone stimulated MUC1 expression (Wang et al. 2010. Mol. Endocrinol. 24: 1368-1379). Two independent approaches were used to determine if rosiglitazone as well as EGFR tyrosine kinase inhibitor, AG1478 reduced MUC1 expression in a high MUC1-expressing cell line, HES; the uterine adenocarcinoma cell line, HEC-1A; and in the human pancreatic adenocarcinoma cell lines, HPAFII and CAPAN2. EGFR activation by Epidermal Growth Factor (EGF) stimulated MUC1 protein expression (1.5-2 fold) in both uterine and pancreatic cancer cells. The PPARδ agonist, rosiglitatzone, markedly (> 90%) reduced EGFR-driven MUC1 expression detected by antibodies directed against the MUC1 ECD and CT. Preliminary studies indicate that rosiglitazone effects on EGFR-driven MUC1 expression is indirect. Furthermore, rosiglitazone treatments enhanced EGFR degradation via the lysosomal degradation pathway in the presence of EGF-family ligands in both uterine and pancreatic cancer cell lines. A combination of biochemical and molecular biological assays have been used to identify the transcriptional and signal transduction pathways activated by EGFR, leading to increased MUC1 expression and the impact of AG1478 and rosiglitazone on these events. Understanding the molecular basis of how these agents reduce MUC1 expression may offer novel therapeutic avenues to improve cancer chemotherapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3070. doi:1538-7445.AM2012-3070

Chronic Anti-inflammatory Drug Therapy Inhibits Gel-Forming Mucin Production in a Murine Xenograft Model of Human Pseudomyxoma Peritonei

Intraperitoneal accumulation of mucinous ascites in pseudomyxoma peritonei (PMP) promotes an inflammatory/fibrotic reaction that progresses to bowel obstruction and eventual patient demise. Cytokines and inflammation-associated transcription factor binding sites, such as glucocorticoid response elements and COX-2, regulate secretory mucin, specifically MUC2, production. We hypothesized that anti-inflammatory drugs targeting inflammation-associated pathways may reduce mucin production and subsequent disease morbidity in PMP. The effects of dexamethasone and Celebrex were assessed in mucin-secreting human colon cancer LS174T cells in vitro and murine xenograft models of LS174T and human appendiceal PMP in vivo by serial parametric measurements, MUC2 transcripts via real-time RT-PCR, and MUC2 protein expression via immunofluorescence assays. Dexamethasone significantly inhibited basal MUC2 mRNA levels in LS174T cells, inhibited mucinous tumor accumulation in an intraperitoneal PMP xenograft model, and prolonged survival in a subcutaneous LS174T xenograft model. Celebrex significantly inhibited sodium butyrate-stimulated MUC2 mRNA levels in LS174T cells and demonstrated a statistically nonsignificant trend toward reduced mucinous tumor growth and prolonged survival in the xenograft models. MUC2 protein analysis by immunofluorescence demonstrated a dual effect of dexamethasone on mucin production and tumor cell count. Inflammatory mediators are known to regulate mucin production and may promote overexpression of MUC2 by neoplastic cells with goblet cell phenotype in PMP. Anti-inflammatory drugs, dexamethasone and Celebrex, could inhibit extracellular mucin production in PMP by targeting inflammatory cascades and, therefore, may decrease compressive symptoms, increase the disease-free interval, and reduce the extent or frequency of morbid cytoreductive surgeries.

PPARγ inhibits airway epithelial cell inflammatory response through a MUC1-dependent mechanism

This study was conducted to examine the relationship between the peroxisome proliferator-associated receptor-γ (PPARγ) and MUC1 mucin, two anti-inflammatory molecules expressed in the airways. Treatment of A549 lung epithelial cells or primary mouse tracheal surface epithelial (MTSE) cells with phorbol 12-myristate 13-acetate (PMA) increased the levels of tumor necrosis factor (TNF)-α in cell culture media compared with cells treated with vehicle alone. Overexpression of MUC1 in A549 cells decreased PMA-stimulated TNF-α levels, whereas deficiency of Muc1 expression in MTSE cells from Muc1 null mice increased PMA-induced TNF-α levels. Treatment of A549 or MTSE cells with the PPARγ agonist troglitazone (TGN) blocked the ability of PMA to stimulate TNF-α levels. However, the effect of TGN required the presence of MUC1/Muc1, since no differences in TNF-α levels were seen between PMA and PMA plus TGN in MUC1/Muc1-deficient cells. Similarly, whereas TGN decreased interleukin-8 (IL-8) levels in culture media of MUC1-expressing A549 cells treated with Pseudomonas aeruginosa strain K (PAK), no differences in IL-8 levels were seen between PAK and PAK plus TGN in MUC1-nonexpressing cells. EMSA confirmed the presence of a PPARγ-binding element in the MUC1 gene promoter. Finally, TGN treatment of A549 cells increased MUC1 promoter activity measured using a MUC1-luciferase reporter gene, augmented MUC1 mRNA levels by quantitative RT-PCR, and enhanced MUC1 protein expression by Western blot analysis. These combined data are consistent with the hypothesis that PPARγ stimulates MUC1/Muc1 expression, thereby blocking PMA/PAK-induced TNF-α/IL-8 production by airway epithelial cells.

MUC2 protein expression status is useful in assessing the effects of hyperthermic intraperitoneal chemotherapy for peritoneal dissemination of colon cancer

We conducted a molecular biological investigation to determine the outcomes of hyperthermic intraperitoneal chemotherapy (HIPEC) treatment, and whether it is effective in all cases for patients with peritoneal dissemination of colon cancer. In the HIPEC group, the 3-year survival rate was 39.2%, whereas in the non-HIPEC group the 3-year survival rate was 15.6%. MUC2 expression was investigated in the HIPEC group, in patients positive for MUC2 expression, and the 3-year survival rate was 0.0%, while in patients negative for MUC2 expression, the 3-year survival rate was 61.1%. In addition, as a result of introducing MUC2-siRNA into a colon cancer cell line with high expression of the MUC2 gene, the cell death rate from heat and anticancer agents increased 40% in comparison with colon cancer cells in which scrambled siRNA had not been introduced. HIPEC therapy is thought to be effective in prolonging survival in patients with peritoneal dissemination of colon cancer, and MUC2 expression is thought to be useful as an indicator to assess its effectiveness in colon cancer cells.

MUC1 Protein Expression in Tumor Cells Regulates Transcription of Proinflammatory Cytokines by Forming a Complex with Nuclear Factor-κB p65 and Binding to Cytokine Promoters: IMPORTANCE OF EXTRACELLULAR DOMAIN

MUC1 is a transmembrane glycoprotein abnormally expressed in all stages of development of human adenocarcinomas. Overexpression and hypoglycosylation of MUC1 in cancer cells compared with normal epithelial cells are likely to alter its function and affect the behavior of cancer cells. The extracellular domain, specifically the highly O-glycosylated VNTR (variable number of tandem repeats) region, plays an important role in cell-cell communication; however, we show here that it also participates intracellularly in activation of the NF-κB pathway. Transfection of MUC1(-) tumor cells with cDNA encoding MUC1 with 22 tandem repeats (MUC1/22TR) or two tandem repeats (MUC1/2TR) or two isoforms that lack the VNTR region (MUC1/Z and MUC1/Y) showed that the highest expression levels of NF-κB family members correlated with the presence of VNTR and the highest number of tandem repeats. Because expression of MUC1 with VNTR on tumors was previously associated with chemotactic activity for cells of the innate immune system, we investigated the influence of MUC1 expression on the NF-κB-dependent transcriptional regulation of proinflammatory cytokines. ChIP and real-time PCR experiments revealed that MUC1/22TR up-regulated IL-6 and TNF-α expression by binding to their promoter regions in a NF-κB p65-dependent manner in both MUC1-transfected and human breast cancer cells that express endogenous MUC1. This newly detected complex of MUC1 and p65 is a novel mechanism that tumors can use to promote inflammation and cancer development.

MUC1 Inhibition Reverses the Poor Immunogenicity of Leukemia Stem Cells Rendering Them Susceptible to Immunotherapy

Abstract 1883 Introduction:Acute myeloid leukemia (AML) arises from a malignant stem cell (LSC) population that is resistant to cytotoxic therapy and represents a reservoir for disease recurrence. A focus of research interest lies in developing cellular immunotherapy for the treatment of AML, in order to immunologically eradicate disease without the toxicity associated with allogeneic transplantation. In this study, we evaluated whether LSCs are effective targets for leukemia specific immunotherapy. To investigate this issue, we have developed a novel class of human T Cell Receptor-Like (TCRL) recombinant antibodies, which target MHC-peptide complexes mimicking their recognition. TCRL can be utilized to quantify the density of antigen presentation by LSCs; however, being able to distinguish LCSs from normal stem cells immunologically has been challenging. The tumor associated complex glycoprotein MUC1 may be used to define such an immature cell population. Methods and Results:We have previously demonstrated that LSCs strongly express the tumor associated antigen, MUC1, in contrast to normal hematopoietic stem cells. TCRL specific for the HLA-A2/MUC1 complex detectable by flow cytometry were constructed in order to quantify MUC1 antigen presentation. CD34+ cells were isolated from HLA-A2.1 AML patients with active disease and were analyzed for expression of MUC1 protein with the DF3 anti-MUC1 antibody and for HLA presentation of MUC1 peptide with the HLA-A2.1/MUC1 TCRL. Remarkably, despite the strong cell surface expression of the MUC1 protein (mean expression 68%, n=8), minimal levels of HLA bound MUC1 peptide was detected (mean expression 7.6%, n=8). CD34+ leukemia cells strongly expressed HLA-A2, indicating that the lack of TCRL recognition of the HLA-MUC1 complex was due to the lack of processing and presentation of the MUC1 peptide. Similarly, we demonstrated that leukemia progenitors minimally express HLA-A2-WT1 peptide complexes (mean expression 5%, n=3). These findings suggest that leukemia progenitors exhibit an immuno-privileged phenotype, potentially shielding them from leukemia specific immunotherapy. We have developed a MUC1 inhibitor peptide, G0-203, that has been shown to induce differentiation and apoptosis of leukemia progenitors. We evaluated whether, in addition to inducing differentiation of leukemia progenitors, exposure to GO-203 would augment antigen presentation. Isolated CD34+ AML cells were treated with 5μM GO-203 for 24 hours. Expression of HLA-A2/MUC1 peptide complexes, as determined by binding of the MUC1 TCRL antibody, was assessed before and after treatment. Exposure to GO-203 markedly upregulated the mean expression of HLA-A2.1/MUC1 peptide complexes by CD34+ leukemia cells (10% to 62%; n=4, p<0.05). Similarly, an upregulation in the mean expression of HLA-A2.1/WT1 peptide complexes was observed on CD34+ progenitors following exposure to GO-203 (mean expression 5% to 44%, n=3, p<0.05). Consistent with these findings, GO-203-treated leukemia progenitors exhibited an increase in potency as antigen presenting cells, as manifested by a 2 fold increase in autologous T cell proliferation in response to stimulation with GO-203 treated compared to untreated progenitors. In addition, an increase in MUC1 pentamer positive T cells was observed following stimulation with GO-203 treated compared to untreated AML progenitors (6.5% versus 3% MUC1 pentamer positive cells respectively, n=2). We subsequently evaluated whether the upregulation of antigen presentation correlated with enhanced targeting by immune effector cells. We have previously demonstrated that DC/AML fusion cells potently stimulate the expansion of leukemia specific T cells. We now show that GO-203 exposure results in an increased susceptibility of leukemia progenitors to targeting by vaccine stimulated T cells, as manifested by a 3-fold increase in cytotoxic T lymphocyte-mediated lysis as measured by Granzyme B activity. Conclusions:The results demonstrate that leukemia progenitors exhibit low levels of HLA presentation of leukemia specific antigens such as MUC1 and WT1, resulting in their poor immunogenicity and potential resistance to tumor specific immunotherapy. Exposure of leukemia progenitors to a MUC1 inhibitor peptide, GO-203, upregulates HLA-based presentation of leukemia associated antigens, rendering these cells more susceptible to immune based targeting. Disclosures:Kufe:genus oncology: Consultancy, Equity Ownership.

Targeting the Intracellular MUC1 C-terminal Domain Inhibits Proliferation and Estrogen Receptor Transcriptional Activity in Lung Adenocarcinoma Cells

Mucin 1 (MUC1) is a diagnostic factor and therapy target in lung adenocarcinoma. MUC1 C-terminal intracellular domain (CD) interacts with estrogen receptor (ER) α and increases gene transcription in breast cancer cells. Because lung adenocarcinoma cells express functional ERα and ERβ, we examined MUC1 expression and MUC1-ER interaction. Because blocking MUC1 CD with an inhibitory peptide (PMIP) inhibited breast tumor growth, we tested whether PMIP would inhibit lung adenocarcinoma cell proliferation. We report that MUC1 interacts with ERα and ERβ within the nucleus of H1793 lung adenocarcinoma cells in accordance with MUC1 expression. PMIP was taken up by H23 and H1793 cells and inhibited the proliferation of H1793, but not H23 cells, concordant with higher MUC1 protein expression in H1793 cells. Lower MUC1 protein expression in H23 does not correspond to microRNAs miR-125b and miR-145 that have been reported to reduce MUC1 expression. PMIP had no effect on the viability of normal human bronchial epithelial cells, which lack MUC1 expression. PMIP inhibited estradiol-activated reporter gene transcription and endogenous cyclin D1 and nuclear respiratory factor-1 gene transcription in H1793 cells. These results indicate MUC1-ER functional interaction in lung adenocarcinoma cells and that inhibiting MUC1 inhibits lung adenocarcinoma cell viability.

Numb modulates intestinal epithelial cells toward goblet cell phenotype by inhibiting the Notch signaling pathway

Numb was originally identified as an important cell fate determinant that is asymmetrically inherited during mitosis and controls the fate of sibling cells by inhibiting the Notch signaling pathway in neural tissue. The small intestinal epithelium originates from the division of stem cells that reside in the crypt, which further differentiate into goblet cells, absorptive cells, paneth cells, and enteroendocrine cells. However, Numb's involvement in the differentiation process of intestinal epithelium is largely unknown. In the present study, we confirm that both the Numb mRNA and protein isoforms are expressed in adult mouse intestinal mucosa. Numb protein is ubiquitously expressed throughout the crypt–villus axis of the small intestinal epithelium and is mainly localized to the cytoplasmic membrane. Down-regulation of endogenous Numb using RNA interference in cultured intestinal LS174T cells increased Notch signaling, leading to the up-regulation of Hes1 and the down-regulation of Hath1. Knockdown of Numb alleviated MUC2 protein expression and led to loss of the goblet cell phenotype in LS174Tl cells. Our results provide the first evidence that Numb, an important cell fate determinant, modulates intestinal epithelial cells towards the goblet cell phenotype by inhibiting the Notch signaling pathway.

Abstract 2032: Regulation of MUC1 expression by activated EGFR and PPARγ in human uterine and pancreatic cancer cell lines

Abstract Many epithelial tumors including endometrial and pancreatic cancers, overexpress the high molecular weight, transmembrane mucin, MUC1, on their cell surface. Overexpression and aberrant glycosylation of MUC1 makes tumor cells poorly adherent, increases drug resistance and promotes cancer cell metastasis. MUC1 has three distinct domains: an extracellular domain (ECD) composed of a tandem repeat region rich in serine, threonine and proline; a transmembrane domain (TM) and; a short cytoplasmic tail domain (CT) involved in a variety of signal transduction events. Previous studies (Wang et al. 2010. Mol. Endocrinol. 24: 1368-1379) demonstrated that the Peroxisome Proliferator Activated Receptor γ (PPARγ) agonist, rosiglitazone severely inhibits progesterone stimulated MUC1 expression. Two independent approaches were used to determine if rosiglitazone as well as inhibitors of Epidermal Growth Factor Receptor (EGFR) tyrosine kinase activity reduced MUC1 expression in the human uterine epithelial cell line, HES, the uterine adenocarcinoma cell line, HEC-1A, and in the human pancreatic adenocarcinoma cell lines, HPAFII and CAPAN2. EGFR activation by Epidermal Growth Factor (EGF) stimulated (1.5-2 fold) MUC1 protein expression 1.5-2.0 fold in both uterine and pancreatic cancer cells, while treatment with EGFR tyrosine kinase inhibitor, AG1478, reduced both basal and stimulated MUC1 protein expression. Furthermore, the PPARγ agonist, rosiglitatzone, markedly (&amp;gt; 90%) reduced EGFR-driven MUC1 expression detected by antibodies directed against the MUC1 ECD and CT. A combination of biochemical and molecular biological assays have been used to identify the transcriptional and signal transduction pathways activated by EGFR leading to increased MUC1 expression and the impact of AG1478 and rosiglitazone on these events. Understanding the molecular basis of how these agents reduce MUC1 expression may offer novel therapeutic avenues to improve cancer chemotherapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2032. doi:10.1158/1538-7445.AM2011-2032

Androgen receptor regulates expression of the MUC1‐C oncoprotein in human prostate cancer cells

The MUC1 heterodimeric oncoprotein is aberrantly overexpressed in human prostate cancers with more aggressive pathologic and clinical features. However, the signals that regulate MUC1 expression in prostate cancer cells are not well understood. MUC1 expression was studied in androgen-dependent and -independent prostate cancer cell lines by quantitative RT-PCR, immunoblotting and assessment of MUC1 promoter activation. Chromatin immunoprecipitation (ChIP) studies were performed to assess androgen receptor (AR) occupancy on the MUC1 promoter. Post-transcriptional regulation of MUC1 expression was assessed by miR-125b-mediated effects on activity of the MUC1 3' untranslated region (3'UTR). The present studies demonstrate that AR occupies a consensus AR element on the MUC1 promoter in androgen-dependent LNCaP, but not in androgen-independent DU145 and PC3, prostate cancer cells. The results further show that AR downregulates MUC1 gene transcription. Stable introduction of exogenous AR in PC3 (PC3/AR) cells and then silencing of AR confirmed AR-mediated repression of the MUC1 promoter. AR signaling has also been shown to drive miR-125b expression. The present studies further demonstrate that miR-125b suppresses MUC1 translation in LNCaP cells and that an anti-sense miR-125b upregulates expression of MUC1 protein. In addition, stable expression of miR-125b in DU145 cells resulted in decreases in MUC1 levels. These findings demonstrate that AR signaling regulates MUC1 expression by transcriptional and posttranscriptional mechanisms in prostate cancer cells.

Blockade of MUC1 Expression by Glycerol Guaiacolate Inhibits Proliferation of Human Breast Cancer Cells

We sought to determine whether administration of glycerol guaiacolate at an optimal biological dose inhibits human breast cancer cell growth. Human breast cancer MCF-7 and ZR-75-1 cells were treated with glycerol guaiacolate and the therapeutic efficacy and biological activity of this drug was investigated on breast cancer cell growth. MCF-7 cells were injected into the mammary fat pad of overectamized female athymic nude mice. Ten days later, animals were treated with daily intraperitoneal injections of glycerol guaiacolate for six weeks. Tumor size and volume was monitored and immunohistochemistry analysis on MUC1, p21 and ki-67 was performed. Glycerol guaiacolate decreased breast cancer cell growth in a dose-dependent manner, decreased cell migration, and caused G1 cell cycle arrest. Our results demonstrate that glycerol guaiacolate inhibits MUC1 protein and mRNA expression levels and significantly increased p21 expression in human breast cancer cells as well as induced PARP cleavage. Similarly, glycerol guaiacolate inhibited breast tumor growth in vivo as well as enhanced p21 expression and decreased breast tumor cell proliferation (ki-67 expression). Collectively, our results demonstrate that glycerol guaiacolate decreased MUC1 expression and enhanced cell growth inhibition by inducing p21 expression in breast cancer cells. These findings suggest that glycerol guaiacolate may provide a novel and effective approach for the treatment of human breast cancer.

Arnebia preventing the expression of Muc1 protein decrease results in anti-implantation in early pregnant mice

Background The study was designed to explore the relationship between the anti-implantation activity of arnebia and the expression of Muc1 protein in the endometrium of early pregnant mice. Study design The aqueous extract of arnebia was administered to mice on Days 1–4 postcoitum, and the mice were sacrificed to asses the implantation rate on Day 8 postcoitum. On the night of Day 4 postcoitum and on the morning of Day 5 postcoitum, the treated mice were sacrificed to study the influence on endometrium. The extract of arnebia was administered to mice on Days 11–14 postcoitum, and the mice were sacrificed to asses abortion on Day 18 postcoitum. The reversible effect of arnebia on mice was also studied. Results The endometrium in the experimental group exhibited morphological changes compared with that in the control group. The expression of Muc1 protein was increased with the increasing doses of arnebia, while in control group, it increased a little. The experiments of pseudopregnancy and normal mice show the identical expression of Muc1. The anti-implantation effect of arnebia was reversible. Conclusion The arnebia may prevent embryo implantation by inhibiting the decrease of the Muc1 protein.

MUC1 oncogene amplification correlates with protein overexpression in invasive breast carcinoma cells

The MUC1 gene is aberrantly overexpressed in approximately 90% of human breast cancers. Several studies have shown that MUC1 overexpression is due to transcriptional regulatory events. However, the importance of gene amplification as a mechanism leading to the increase of MUC1 expression in breast cancer has been poorly characterized. The aim of this study was to evaluate the role of MUC1 gene amplification and protein expression in human breast cancer development. By means of real-time quantitative polymerase chain reaction and immunohistochemical methods, 83 breast tissue samples were analyzed for MUC1 gene amplification and protein expression. This analysis showed MUC1 genomic amplification and a positive association with the histopathological group in 12% (1 out of 8) of benign lesions and 38% (23 out of 60) of primary invasive breast carcinoma samples ( P = 0.004). Array–comparative genomic hybridization meta-analysis of 886 primary invasive breast carcinomas obtained from 22 studies showed MUC1 genomic gain in 43.7% (387 out of 886) of the samples. Moreover, we identified a highly statistical significant association between MUC1 gene amplification and MUC1 protein expression assessed by immunohistochemistry and Western blot test ( P < 0.0001). In conclusion, this study demonstrated that MUC1 copy number increases from normal breast tissue to primary invasive breast carcinomas in correlation with MUC1 protein expression.

Abstract 1561: Guaiacol glyceryl ether, a novel agent to reduce MUC1 expression in breast cancer cells

Abstract Purpose: The overexpression and loss of polarity of Mucin1 (MUC1) has been documented in aggressive breast cancer. We have identified guaifenesin as a novel treatment to reduce or eliminate MUC1 expression on human breast cancer cells. Methods: MCF-7 and ZR-75-1 human breast cancer cells were grown for 48 hours in varying concentrations of guaifenesin to determine if a dose dependent effect on MUC1 protein expression resulted with treatment. In addition, cells were also harvested, RNA isolated, reverse transcribed and PCR was performed using ABI quantitative real time PCR machine. In another set of experiments, MCF-7 cells were plated and grown for 72 hours in the absence or presence of 12.5 mM guaifenesin and the cells were harvested and stained with propidium iodine for analysis by flow cytometry. The Cell-Titer Glo assay was performed on MCF-7 and ZR-75-1 cells incubated for 48 hours in the presence of guaifenesin at varying concentrations and the LD50 was determined. In a final set of experiments, MCF-7 cells, incubated in the presence or absence of guaifenesin, were analyzed for their ability to migrate /proliferate in vitro by using a standard scratch assay. For in vivo studies ovarectomized female nude mice were injected with MCF-7-luciferase tagged cells into the mammary fat pad 24 hours after an estrogen pellet was implanted into the right flank. Treatment (vehicle or 400 mg/kg/daily) was initiated after 10 days and tumor growth was monitored through Kodak imaging system. Six weeks after the treatment tumors were isolated, measured and processed for immunohistochemistry to analyze the expression of MUC1, TUNEL and CD31. Results: Initially, MUC1 gene expression was confirmed in MCF-7 and ZR-75-1 human breast cancer cell lines. Following confirmation, the LD50 was determined to be 12.5 mM. MUC1 gene expression was successfully decreased in a dose-dependent manner in both MCF-7 and ZR-75-1 cell lines when treated with guaifenesin. When MCF-7 cells are treated with 12.5mM guaifenesin, G1 arrest occurs within 24 hours and by 72 hours, 94.2% of cells are in G1 compared to 76.2% in control (drug free) conditions. In addition, there was a significant decrease in the ability of guaifenesin treated MCF-7 cells to migrate into the scratch compared to the control (untreated) cells. In vivo studies revealed that guaifenesin significantly decreased the breast tumor weight and volume which was associated with a low expression of MUC1 in tumor sections as evaluated by immunohistochemistry. Conclusion: These findings suggest that guaifenesin may provide a novel approach to inhibit breast cancer growth. 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 1561.

Abstract 5696: Down-regulation of MUC1 by the herbal formulation, triphala in colon cancer cells

Abstract Natural products from medicinal plants and traditional medicine have been a fertile source for identification of novel molecules with human health-protective effects. Triphala is an Ayurvedic medicine used as a dietary supplement to prevent and cure ailments of the gastro-intestinal tract in India from ancient times. It is a formulation made from dry powder of three fruits, Terminalia chebula, Emblica officinalis and Terminalia bellerica and has been used to treat microbial infections and ulcers of stomach and colon. In order to identify bioactive principles and their molecular targets, colon cancer cells HCT-116 (5×103 cells) were seeded in 96 well plates and treated with the methanolic extract of triphala at varying concentrations for 24, 48 and 72 hours. Cell proliferation assay demonstrated a dose-dependent inhibition of cell growth and the IC50 was determined to be ∼100 μg/ml. RNA isolated from colon cancer cells treated for 6 hours with the methanolic extract at 100 and 200 μg/ml concentrations revealed a dose-dependent down-regulation of MUC1 gene expression as detected by qRT-PCR. Also, MUC1 antibody immunohistochemistry showed down-regulation of MUC1 protein expression, when cells grown in chamber slides were treated with the methanolic extract. In addition, qRT-PCR revealed down-regulation of expression of the inflammatory molecules IL-6, IFNγ, COX-2, NF-κB and the cell cycle-regulatory molecule, cyclin D1. These results suggest that triphala extract exerts its anti-tumorigenic effects by influencing the expression of mucins via modulation of inflammatory molecules that are up-stream of mucin expression, in colon cancer cells. FTICR-MS (Fourier-transform Ion Cyclotron Resonance MS) analysis of the methanolic extract of triphala readily confirmed the presence of previously reported compounds such as gallic acid, ellagic acid, several mucic acid gallates, chebulic acid, ascorbic acid, citric acid, and several tannins. Sub-fractionation of the methanolic extract with ethyl acetate, when tested on colon cancer cells, showed higher efficacy (IC50 = 50 µg/ml) suggesting enrichment of select bioactive principles. FTICR-MS analysis of the ethyl acetate extract revealed enrichment of gallic acid, ellagic acid, and several other tannins, while mucic acid and their gallates were not. HPLC fingerprinting of the methanolic and ethyl acetate extracts confirmed the enrichment of phenolics into ethyl acetate fraction. The fractionation results suggest that enrichment of distinct set of active principles of triphala into different fractions is a viable strategy to help identify their specific molecular targets. More extensive fractionation of the methanolic extract of triphala is currently underway (Supported from Agnes Brown Duggan Endowment). 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 5696.

Risk of gastric cancer is associated with the MUC1 568 A/G polymorphism

Identifying the genetic variants that alter MUC1 protein expression may further our understanding of the risk for development of gastric cancer (GC). We used PCR-SSPs to identify the genotype of MUC1 A/G polymorphism at its 568 site of exon 2 and immunohistochemistry to detect MUC1 protein expression in GC patients and non-cancer subjects and analyzed the association between this polymorphism and MUC1 protein expression. We found that the frequency of AA genotype was significantly high in the GC patients and the risk for GC in AA genotype carriers increased 1.81-fold. Moreover, we found a significant underexpression of MUC1 protein in GC as compared to non-cancer subjects, which was negatively correlated to AA genotype of MUC1 (r=-0.1790, P=0.004). Furthermore, this study provides a possible mechanistic insight that the MUC1 A/G polymorphism at its 568 site disrupts the physiological functions of MUC1 which is important to the physiological protection of gastric mucosa. Thus we have provided evidence that may identify the MUC1 A/G polymorphism at 568 site, as a potential genetic factor which leads to an increase in susceptibility for GC through alteration of MUC1 gene and MUC1 expression in the population that carry the A allele.

MUC1 is a downstream target of STAT3 and regulates lung cancer cell survival and invasion

Signal transducer and activator of transcription 3 (STAT3) is aberrantly activated in human cancer including lung cancer and has been implicated in transformation, tumorigenicity, and metastasis. One putative downstream gene regulated by Stat3 is MUC1 which also has important roles in tumorigenesis. We determined if Stat3 regulates MUC1 in lung cancer cell lines and what function MUC1 plays in lung cancer cell biology. We examined MUC1 expression in non-small cell lung cancer (NSCLC) cell lines and found high levels of MUC1 protein expression associated with higher levels of tyrosine phosphorylated STAT3. STAT3 knockdown downregulated MUC1 expression whereas constitutive STAT3 expression increased MUC1 expression at mRNA and protein levels. MUC1 knockdown induced cellular apoptosis concomitant with reduced Bcl-XL and sensitized cells to cisplatin treatment. MUC1 knockdown inhibited tumor growth and metastasis in an orthotopic mouse model of lung cancer by activating apoptosis and inhibiting cell proliferation in vivo. These results demonstrate that constitutively activated STAT3 regulates expression of MUC1, which mediates lung cancer cell survival and metastasis in vitro and in vivo. MUC1 appears to be a cooperating oncoprotein with multiple oncogenic tyrosine kinase pathways and could be an effective target for the treatment of lung cancer.

The regulation of intestinal mucin MUC2 expression by short-chain fatty acids: implications for epithelial protection

SCFAs (short-chain fatty acids), fermentation products of bacteria, influence epithelial-specific gene expression. We hypothesize that SCFAs affect goblet-cell-specific mucin MUC2 expression and thereby alter epithelial protection. In the present study, our aim was to investigate the mechanisms that regulate butyrate-mediated effects on MUC2 synthesis. Human goblet cell-like LS174T cells were treated with SCFAs, after which MUC2 mRNA levels and stability, and MUC2 protein expression were analysed. SCFA-responsive regions and cis-elements within the MUC2 promoter were identified by transfection and gel-shift assays. The effects of butyrate on histone H3/H4 status at the MUC2 promoter were established by chromatin immunoprecipitation. Butyrate (at 1 mM), as well as propionate, induced an increase in MUC2 mRNA levels. MUC2 mRNA levels returned to basal levels after incubation with 5-15 mM butyrate. Interestingly, this decrease was not due to loss of RNA stability. In contrast, at concentrations of 5-15 mM propionate, MUC2 mRNA levels remained increased. Promoter-regulation studies revealed an active butyrate-responsive region at -947/-371 within the MUC2 promoter. In this region we identified an active AP1 (c-Fos/c-Jun) cis-element at -818/-808 that mediates butyrate-induced activation of the promoter. Finally, MUC2 regulation by butyrate at 10-15 mM was associated with increased acetylation of histone H3 and H4 and methylation of H3 at the MUC2 promoter. In conclusion, 1 mM butyrate and 1-15 mM propionate increase MUC2 expression. The effects of butyrate on MUC2 mRNA are mediated via AP-1 and acetylation/methylation of histones at the MUC2 promoter.

Modulation of MUC1 Protein Expression by PPAR‐gamma in Pancreatic Cancer Cells

The survival rate of pancreatic cancer is approximately four months. Most pancreatic carcinomas overexpress MUC1. MUC1 is a glycoprotein that serves as a barrier to infection in normal cells. However, MUC1 protects cancer cells from immune responses and cell death. Therefore, reducing MUC1 protein expression is expected to increase the sensitivity of tumor cells to killing by cells of the immune system. PPARs are transcription factors of the nuclear receptor family. PPARγ heterodimerizes with retinoid X receptor and binds to PPAR response elements. Recent studies in our laboratory have shown that rosiglitazone reduces cytokine‐stimulated MUC1 expression in various cancer cell lines. Therefore, we hypothesized that rosiglitazone will reduce MUC1 protein expression in pancreatic cancer cells. Pancreatic cancer cell lines were cultured and serum starved for several days. The cell lines were treated with and without rosiglitazone. Western blotting and autoradiography were used to separate and detect the MUC1 proteins synthesized by the cells. Our results indicate that rosiglitazone significantly reduces MUC1 protein expression in Capan2 and HPAF2 cell lines. PPARγ agonists in combination with tumoricidal agents offer a promising avenue to increase pancreatic tumor cell sensitivity to killing by decreasing MUC1 levels. Funding provided by the HHMI Science Education Grant and the McNair Scholars Program.

Hypoxia enhances MUC1 expression in a lung adenocarcinoma cell line

Expression of a transmembrane mucin MUC1 is emphasized in most cases of carcinoma. High expression of MUC1 is closely associated with cancer progression and metastasis, leading to poor prognosis. However, little is known about how MUC1 is overexpressed in malignant tumor. In this study, we demonstrated that: (1) Hypoxia, a typical feature of malignant tumor, enhanced the expression of MUC1 mRNA and protein in a human lung adenocarcinoma cell line; (2) the hypoxia-induced increase in MUC1 mRNA was mediated by the transcriptional activity of MUC1 promoter, but not mRNA stability. Moreover; (3) CoCl 2, an inducer of Hypoxia Inducible Factor (HIF)-1α, increased the expression of MUC1 mRNA; and (4) HIF-1α-targeted siRNA but not its control siRNA decreased hypoxia-induced MUC1 mRNA. These data suggest that hypoxia enhances the expression of MUC1 through the transcriptional regulation by HIF-1α in a human lung epithelial cell line.