Michael D. Koch

Expression and Functions of Transmembrane Mucin MUC13 in Ovarian Cancer

MUC13, a transmembrane mucin, is normally expressed in gastrointestinal and airway epithelium. Its aberrant expression has been correlated with gastric colon and cancer. However, the expression and functions of MUC13 in ovarian cancer are unknown. In the present study, the expression profile and functions of MUC13 were analyzed to elucidate its potential role in ovarian cancer diagnosis and pathogenesis. A recently generated monoclonal antibody (clone PPZ0020) was used to determine the expression profile of MUC13 by immunohistochemistry using ovarian cancer tissue microarrays and 56 additional epithelial ovarian cancer (EOC) samples. The expression of MUC13 was significantly (P < 0.005) higher in cancer samples compared with the normal ovary/benign tissues. Among all ovarian cancer types, MUC13 expression was specifically present in EOC. For the functional analyses, a full-length MUC13 gene cloned in pcDNA3.1 was expressed in a MUC13 null ovarian cancer cell line, SKOV-3. Here, we show that the exogenous MUC13 expression induced morphologic changes, including scattering of cells. These changes were abrogated through c-Jun NH(2) kinase (JNK) chemical inhibitor (SP600125) or JNK2 siRNA. Additionally, a marked reduction in cell-cell adhesion and significant (P < 0.05) increases in cell motility, proliferation, and tumorigenesis in a xenograft mouse model system were observed upon exogenous MUC13 expression. These cellular characteristics were correlated with up-regulation of HER2, p21-activated kinase 1, and p38 protein expression. Our findings show the aberrant expression of MUC13 in ovarian cancer and that its expression alters the cellular characteristics of SKOV-3 cells. This implies a significant role of MUC13 in ovarian cancer.

MUC13 mucin augments pancreatic tumorigenesis

The high death rate of pancreatic cancer is attributed to the lack of reliable methods for early detection and underlying molecular mechanisms of its aggressive pathogenesis. Although MUC13, a newly identified transmembrane mucin, is known to be aberrantly expressed in ovarian and gastro-intestinal cancers, its role in pancreatic cancer is unknown. Herein, we investigated the expression profile and functions of MUC13 in pancreatic cancer progression. The expression profile of MUC13 in pancreatic cancer was investigated using a recently generated monoclonal antibody (clone PPZ0020) and pancreatic tissue microarrays. The expression of MUC13 was significantly (P < 0.005) higher in cancer samples compared with normal/nonneoplastic pancreatic tissues. For functional analyses, full-length MUC13 was expressed in MUC13 null pancreatic cancer cell lines, MiaPaca and Panc1. MUC13 overexpression caused a significant (P < 0.05) increase in cell motility, invasion, proliferation, and anchorage-dependent or -independent clonogenicity while decreasing cell–cell and cell-substratum adhesion. Exogenous MUC13 expression significantly (P < 0.05) enhanced pancreatic tumor growth and reduced animal survival in a xenograft mouse model. These tumorigenic characteristics correlated with the upregulation/phosphorylation of HER2, p21-activated kinase 1 (PAK1), extracellular signal-regulated kinase (ERK), Akt, and metastasin (S100A4), and the suppression of p53. Conversely, suppression of MUC13 in HPAFII pancreatic cancer cells by short hairpin RNA resulted in suppression of tumorigenic characteristics, repression of HER2, PAK1, ERK, and S100A4, and upregulation of p53. MUC13 suppression also significantly (P < 0.05) reduced tumor growth and increased animal survival. These results imply a role of MUC13 in pancreatic cancer and suggest its potential use as a diagnostic and therapeutic target. Mol Cancer Ther; 11(1); 24–33. ©2011 AACR.

Increased Expression and Aberrant Localization of Mucin 13 in Metastatic Colon Cancer

MUC13 is a newly identified transmembrane mucin. Although MUC13 is known to be overexpressed in ovarian and gastric cancers, limited information is available regarding the expression of MUC13 in metastatic colon cancer. Herein, we investigated the expression profile of MUC13 in colon cancer using a novel anti-MUC13 monoclonal antibody (MAb, clone ppz0020) by immunohistochemical (IHC) analysis. A cohort of colon cancer samples and tissue microarrays containing adjacent normal, non-metastatic colon cancer, metastatic colon cancer, and liver metastasis tissues was used in this study to investigate the expression pattern of MUC13. IHC analysis revealed significantly higher (p<0.001) MUC13 expression in non-metastatic colon cancer samples compared with faint or very low expression in adjacent normal tissues. Interestingly, metastatic colon cancer and liver metastasis tissue samples demonstrated significantly (p<0.05) higher cytoplasmic and nuclear MUC13 expression compared with non-metastatic colon cancer and adjacent normal colon samples. Moreover, cytoplasmic and nuclear MUC13 expression correlated with larger and poorly differentiated tumors. Four of six tested colon cancer cell lines also expressed MUC13 at RNA and protein levels. These studies demonstrate a significant increase in MUC13 expression in metastatic colon cancer and suggest a correlation between aberrant MUC13 localization (cytoplasmic and nuclear expression) and metastatic colon cancer.

Combined Staining of TAG-72, MUC1, and CA125 Improves Labeling Sensitivity in Ovarian Cancer: Antigens for Multi-targeted Antibody-guided Therapy

Single antigen-targeted intraperitoneal radioimmunotherapy for ovarian cancer has shown limited success. Due to the heterogeneous expression of tumor antigens on cancer cells, a multi-antigen targeting approach appears logical to augment the therapeutic efficacy of antibody-guided therapy. In the interest of developing this novel approach, ovarian cancer tissue microarray slides containing cancer and benign/non-neoplastic tissue samples (n = 92) were processed for single-, double-, and triple-antigen labeling using antibodies for the tumor-associated antigens TAG-72, MUC1, and CA125. Among all ovarian cancer types, 72%, 61%, and 50% of the samples showed immunolabeling for TAG-72, MUC1, and CA125, respectively. Expression level of these antigens was significantly (p<0.005) higher in advanced stage carcinomas compared with early stage. Of the 48 epithelial ovarian cancer samples, individual anti-TAG-72, MUC1, and CA125 antibody probing showed labeling in 89.5%, 87.5%, and 73.0% of the cases, respectively. In the majority of the cancer samples (>70%), a heterogeneous labeling pattern was observed (only 30–40% of the cancer cells within the sample were labeled). However, upon combining the three antigens (triple-antigen labeling), 98% of the epithelial ovarian cancer samples were labeled and >95% of the cancer cells within each sample were labeled. Our data indicate that the heterogeneous expression of cancer antigens appears to be a major obstacle in antibody-guided therapy, and this can be overcome by multiple antigen targeting. Therapeutic efficacy of antibody-guided therapy for ovarian cancer treatment will be enhanced by the combined targeting of TAG-72, MUC1, and CA125. (J Histochem Cytochem 55: 867–875, 2007)

Evaluation and comparison of staining patterns of factor XIIIa (AC-1A1), adipophilin, and GATA3 in sebaceous neoplasia

Reliable nuclear immunohistochemical stains for sebaceous neoplasms have not been readily available. Positive nuclear staining has been reported for GATA3 and factor XIIIa (AC‐1A1). We sought to determine the diagnostic utility of these nuclear stains by comparing their staining pattern to adipophilin, a consistently positive cytoplasmic stain.

Status of HER-2 gene amplification in breast cancers from Native American women

In the United States, one in eight women will be diagnosed with breast cancer during her lifetime. The American Cancer Society estimates that in 2008, approximately 182,460 new female breast cancer cases will be diagnosed and 40,480 women will die from this disease [1]. However, breast cancer incidence and mortality rates in females vary across ethnic and racial groups. The National Cancer Institute’s Surveillance, Epidemiology, and End Results (SEER) program indicates that incidence rates for breast cancer among Native American women are significantly lower than the Caucasian population [2]. Yet, Native American women tend to present to their physician with larger, more advanced-stage tumors that lead to lower survival rates, when compared to their Caucasian counterparts [3][sn1]. Moreover, mortality rates for Native American women are not decreasing as they are for the Caucasian population [1,4]. It has been particularly difficult to study the molecular basis of breast cancer in the Native American people because the population is small and has limited access to healthcare. Therefore, this report describes a small cohort study of 60 breast cancer samples from Native American patients and determines the status of HER-2 amplification.

Abstract 3589: Comparative expression profile of transmembrane mucin MUC1 in breast cancer from American Indian and Caucasian women

Introduction: Considerable disparity exists in breast cancer incidence and mortality among different racial/ethnic populations and geographic locations. For unknown reasons, American Indian women of the Northern Plains have a greater incidence and higher death rate due to breast cancer than Caucasian women living in the same geographical region. Additionally, American Indian women living in the Northern Plains also have a much higher incidence and death rate due to breast cancer than American Indian women living elsewhere in the United States. To investigate the potential molecular basis for this cancer health disparity, we have compared the expression profile of transmembrane mucin, MUC1 on breast cancer cases from American Indian and Caucasian women living on the Northern Plains. MUC1 is known to be overexpressed in breast cancer and nuclear localization of MUC1 can have downstream effects on cell signaling and increase oncogenic phenotypes. We are also exploring the link between smoking, exposure to HPV infection, and the development of breast cancer in American Indian women. Methods: We have examined the expression profile of MUC1 in breast cancer tissue from American Indian (n=40) and Caucasian (n=60) women. The staining was analyzed by experienced pathologists and scored according to intensity (0-4) and percent of cancer cells stained positive (0-4). A composite score was calculated by multiplying the scores of intensity and percent of cells stained. Tissues were categorized by modified Bloom-Richardson9s histological grade and the mean composite scores were compared amongst American Indian and Caucasian groups. Results and Conclusions: We have observed that grade 3 breast cancer samples from American Indian women had a higher percent of nuclear MUC1 staining and a lower amount of p53 expression. This result correlates with previous data showing that MUC1 inhibits the expression of p53. In conclusion, breast cancer tissue from American Indian women has a different expression profile of molecular markers and this finding may help to explain the cancer health disparity. Further investigation of these differences may promote understanding of the etiological differences and may identify effective treatment strategies for breast cancer in American Indian women. 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 3589. doi:1538-7445.AM2012-3589

Abstract B60: Role of MUC1 mucin in American Indian breast cancer health disparity.

Introduction: Considerable breast cancer health disparity exists among different racial/ethnic populations and geographic locations. For unknown reasons, American Indian women of the Northern Plains have a greater incidence and higher death rate due to breast cancer than Caucasian women living in the same geographical region. Additionally, American Indian women living in the Northern Plains also have a much higher incidence and death rate due to breast cancer than American Indian women living elsewhere in the United States. To investigate the potential molecular basis for breast cancer health disparity, we have initiated a study to compare the expression profiles of cancer associated proteins on breast cancer cases from American Indian and Caucasian women living on the Northern Plains. One protein of interest, MUC1, a transmembrane mucin, is known to be overexpressed in breast cancer and nuclear localization of MUC1 can have downstream effects on cell signaling and increase oncogenic phenotypes. American Indian women are exposed to cigarette smoke at rates higher than Caucasian women; therefore, we are also assessing the prevalence of aryl hydrocarbon receptor (AhR) activation in breast cancer tissues. Cigarette compounds can activate the AhR pathway and induce the expression of a variety of genes, some of which are known to be oncogenic. Methods: We have examined the expression profiles of MUC1 in breast cancer tissue collected from American Indian (n=40) and Caucasian (n=60) women by immunohistochemical analysis. The staining was analyzed by experienced pathologists and scored according to intensity (0-4) and percent of cancer cells stained positive (0-4). A composite score was calculated by multiplying the scores of intensity and percent of cells stained. Tissues were categorized by modified Bloom-Richardsons histological grade and the mean composite scores were compared amongst American Indian and Caucasian groups. In vitro experiments have also been conducted to examine a potential link between the activation of AhR by cigarette smoke compounds and an increase in MUC1 expression or change in cellular localization. Results and Conclusions: A differential expression/localization pattern of MUC1 was observed in American Indian breast cancer samples compared to Caucasian samples. Grade 3 breast cancer samples obtained from American Indian women showed higher nuclear MUC1 staining compared to Caucasian samples. We have also observed that breast cancer cells aberrantly express AhR and have increased AhR activation as shown by nuclear localization. We also observed a lower amount of nuclear p53 and membrane β-catenin expression. This result correlates with previous data showing that MUC1 inhibits the expression of p53 and decreases membrane β-catenin. In vitro data suggests that exposure to BaP increases the nuclear localization of MUC1. In conclusion, breast cancer tissue from American Indian women has a different expression profile of MUC1 and this finding may help to explain the cancer health disparity. Further investigation of these differences may promote understanding of the etiological differences and may identify effective treatment strategies for breast cancer in American Indian women. Citation Format: Diane M. Maher, Emily Gaster, Phillip Stephenson, Michael Koch, Susan Eliason, Meena Jaggi, Subhash C. Chauhan. Role of MUC1 mucin in American Indian breast cancer health disparity. [abstract]. In: Proceedings of the Fifth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2012 Oct 27-30; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(10 Suppl):Abstract nr B60.

Abstract 1481: MUC13 expression enhances colon cancer progression

Background: Mucin, a family of high molecular weight glycoprotein has been implicated in a variety of cancers. MUC13 is a newly identified transmembrane mucin which has shown deregulated expression in gastric and ovarian cancers. However, limited information is available about the role of MUC13 in colon cancer progression. The present study investigated the expression profile, clinical relevance and functional significance of MUC13 expression in colon cancer progression. Materials and Methods: The MUC13 expression profile was determined by immunohistochemical (IHC) analysis using a novel MUC13 monoclonal antibody (PPZ0020) on a panel of colon cancer tissue microarrays containing non-malignant, colon cancer and liver metastasis tissue samples. Colon cancer cell lines which showed faint (SW480) and high (SW620) MUC13 expression were selected for over-expression and knockdown experiments, respectively, to determine the functional role of MUC13 in colon cancer progression. Functional studies, such as cell proliferation, colony formation, cell migration and cell invasion assays were performed in stable clones. The levels of Sonic hedehog (Shh), B cell moloney murine leukemia virus integration site 1 (Bmi-1) and Matrix metalloproteinase 1 (MMP1) were determined using real time PCR, Western blot and flow cytometry analyses. Results: Differential expression and sub-cellular localization of MUC13 was observed in cancerous and non-malignant tissues. MUC13 IHC analysis showed significantly higher membranous and cytoplasmic MUC13 expression in colon cancer and liver metastasis tissues compared to non-malignant tissue samples. Interestingly, liver metastasis tissue samples showed significantly higher nuclear MUC13 expression in addition to high membranous and cytoplasmic expression. The over-expression of MUC13 enhanced cell proliferation, colony formation, cell migration and cell invasion. In contrast, knockdown of MUC13 resulted in reduced cell proliferation, colony formation, cell migration and cell invasion. Additionally, over-expression of MUC13 increased the expression level of metastatic associated proteins such as Shh, Bmi-1 and MMP1 while knockdown of MUC13 decreased the expression level of these proteins. Conclusions: The present study suggests two important outcomes pertaining to MUC13 in colon cancer: (1) The aberrant expression of MUC13 could serve as a potential diagnostic/prognostic molecular tool for clinical use, and (2) MUC13 over-expression may enhance colon cancer carcinogenesis via modulation of metastatic proteins Bmi-1, Shh and MMP1. These data suggest a potential role of MUC13 in colon cancer progression and metastasis. 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 1481. doi:10.1158/1538-7445.AM2011-1481

Abstract 2358: MUC13 mucin augments pancreatic tumorigenesis

Pancreatic cancer is one of the most lethal malignancies with extremely poor prognosis. The high death rate of pancreatic cancer is attributed to a lack of reliable methods of early diagnosis and underlying molecular mechanisms associated with aggressive pathogenesis. MUC13, a newly identified transmembrane mucin, is known to be aberrantly expressed in ovarian, gastric and colon cancer. However, the expression and functions of MUC13 in pancreatic cancer are unknown. Herein, we have investigated the expression and functions of MUC13 mucin, in pancreatic cancer to determine its potential for early cancer diagnosis and its role in pancreatic cancer pathogenesis. The expression profile of MUC13 in pancreatic cancer was investigated using a recently generated monoclonal antibody (MAb, clone PPZ0020) and pancreatic tissue microarrays. The expression of MUC13 was significantly (p Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 2358.