Normal Glandular Epithelial Cells Research Articles

Abstract 2052: A novel antibody blocks anti-apoptotic activity of MUC1 in pancreatic cancer cell lines

Abstract Introduction: The third leading cause of cancer-related deaths in the United States is pancreatic cancer, >95% of which is Pancreatic Ductal Adenocarcinoma or PDAC. MUC1 is a transmembrane glycoprotein expressed on normal glandular epithelial cells. In PDAC, MUC1 is overexpressed and aberrantly glycosylated and is designated tumor associated MUC1 (tMUC1). Over 80% of human PDAC expresses tMUC1. In a NCI initiated study, out of 75 tumor antigens, MUC1 was ranked the second most targetable antigen. Furthermore, the 72 amino acid cytoplasmic tail of MUC1 (MUC1 CT) is reported to aid in oncogenic signaling leading to tumor progression and metastasis by blocking apoptosis. A novel monoclonal antibody, TAB004, has been developed specifically against tMUC1. TAB004 detects tMUC1 with a high rate of specificity and sensitivity and spares recognition of normal epithelial MUC1. HYPOTHESIS: Treatment with TAB004 curbs PDAC cell survival by blocking MUC1 CT associated oncogenic signaling and renders the cells more susceptible to standard chemotherapy drugs. Methods: Several human PDAC cell lines based on their tMUC1 expression were grown in media with heat-inactivated serum to ensure that it is devoid of complement proteins. Cells were treated with various concentrations of TAB004 antibody, control IgG, 5-FU, paclitaxel, or gemcitabine and the IC50 was determined using the MTT or SRB cell survival assays. Once IC50 was determined, cells were treated with combinations of TAB004 and the chemotherapy drugs. To determine apoptosis, treated cells were stained with Annexin V-FITC and PI, and analyzed by flow cytometer. tMUC1 expression and localization was determined by staining cells with TAB004.Cy5.5 and imaged by confocal microscopy. Data analysis was performed using GraphPad Prism 7.0 and a p value of <0.05 was considered significant. Results: By itself, TAB004 treatment had minimal effect on most of the cell lines tested except for Capan2. The IC50 of TAB004 was ~400ugs/ml for Capan2. However, when combined with Gemcitabine, PTX, or 5-FU, TAB004 significantly increased anti-tumor efficacy of the drugs, especially 5-FU in Capan2, and 5-FU and gemcitabine, and PTX in BxPC3.MUC1. Annexin V-FITC and PI staining confirmed the MTT data in Capan2 cells. Fluorescent images showed that TAB004 internalizes in MUC1-positive PDAC cell lines but not in MUC1-negative cells. Conclusions: TAB004 has a dose-dependent, complement-independent growth-inhibitory effect on Capan2 cells (IC50 is ~400ug/ml). TAB 004 internalizes and activates apoptosis in Capan2 cells. The combination of TAB004 with chemotherapy drugs increases the efficacy of drug-induced killing of PDAC cells. Future studies will determine the mechanism of TAB004 induced apoptosis and sensitization of PDAC cells to drug-induced killing. Citation Format: Mukulika Bose, Pinku Mukherjee. A novel antibody blocks anti-apoptotic activity of MUC1 in pancreatic cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2052.

Abstract 1268: Externalized cytokeratin 8 : A relevant target at the crossroads of microenvironment and intracellular signaling

Abstract Accumulating evidence suggests the unusual presence of proteins at the external leaflet of plasma membrane of cancer cells while, in normal cells, these proteins are expressed in the intracellular compartments. They lack transmembrane domains and signal sequences that allow delivery to the plasma membrane and their role is not fully understood. As such, aberrantly externalized proteins represent a promising source of targets for cancer therapy. In order to find new targets for the treatment of metastatic colorectal cancer (CRC), we performed a comparative dynamic proteomic analysis of Isreco-1, a cell line characterized by a high invasiveness capacity. Among a set of proteins associated with invasion, we identified CK8, an intermediate filament protein of epithelial cells cytoskeleton involved in the maintenance of cell integrity of normal glandular epithelial cells. CK8 was shown to be expressed both intracellularly and associated at the plasma membrane of highly invasive Isreco-1 line (KRAS mutant) as well as in two other cell lines i.e. HCT116 (KRAS mutant) and HT29 (KRAS wild-type), by immunofluorescence. Using a biochemical approach, we demonstrated in all 3 cell lines, that plasma membrane-associated CK8 was strongly anchored to the plasma membrane, and subsequently referred as externalized CK8 (eCK8). eCK8 was co-localized with plasminogen and urokinase-type plasminogen activator (uPA) at the plasma membrane of Isreco-1 cells, consistent with its previously reported role as a plasminogen receptor. Among several murine monoclonal antibodies (MAbs) targeting specifically eCK8, one was able to reduce plasminogen induced invasion in vitro (Isreco-1) as well as tumor growth in vivo (Isreco-1 and HCT-116 subcutaneous xenograft models). It was specific of an extra-cellular portion of eCK8. Besides the inhibition of tumor growth, tumors were strikingly necrotic and characterized by apoptosis activation. No difference in terms of proliferation and microvascular density was observed. Altogether, our results suggest that in colorectal cancer, CK8 may be externalized and strongly anchored to the plasma membrane in highly invasive cells. eCK8 co-localize with plasminogen and uPA. Targeting a specific portion of eCK8 was associated with decreased tumor growth and apoptosis activation. We suggest that this particular domain of eCK8 is essential for bidirectional signaling, toward the tumor cell microenvironment by favouring the invasion process and the intracellular compartment by inhibiting apoptosis. Because the combination of cell invasion and inhibition of apoptosis are key mechanisms involved during metastasis, externalization of CK8 may represent a key event of the metastatic process and a relevant target in CRC, including in tumors with KRAS mutation. Citation Format: Marie Alexandra Albaret, Claudine Vermot-Desroches, Arnaud Paré, Jean-Xavier Roca-Martinez, Jad Essely, Laetitia Gerossier, Johan Brière, Nathalie Pion, Lucie Malet, Boris Vuillermoz, Carine Rousset, Hichem-Claude Mertani, Pierre Saintigny, Jean-Jacques Diaz. Externalized cytokeratin 8 : A relevant target at the crossroads of microenvironment and intracellular signaling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1268.

Specific MUC1 Splice Variants Are Correlated With Tumor Progression in Esophageal Cancer

Mucin 1 (MUC1) is a complex glycoprotein expressed on the apical surface of normal glandular epithelial cells. It plays a role in a number of biologic processes, and its overexpression is associated with various malignancies. A growing body of literature suggests that MUC1 is a potential diagnostic and therapeutic marker. Increasing numbers of variants are being identified for the MUC1 gene, but their role in carcinogenesis is unclear. Alternative splicing and a specific region on a variable number of tandem repeats are characteristic features of MUC1. However, the underlying mechanisms, overall prevalence, and the function of various MUC1 isoforms are not well characterized. In the present study, mRNA expression of nine variants of the MUC1 gene (A-D, X-Z, REP, SEC) was evaluated in normal and tumor tissues obtained from 50 patients with esophageal squamous cell carcinoma (ESCC). Associations between expression of various isoforms of MUC1 and important clinicopathologic factors were studied. Specific MUC1 splice variants (i.e., MUC1/C, D, and Z) are correlated with tumor progression in ESCC, whereas MUC1/B-previously suggested as a "normal" variant in some other cancers-has protective effects and is associated with more favorable tumor behavior and better prognosis. Specific isoforms of ESCC are associated with prognosis. Further characterization of different isoforms of MUC1 and their biologic effects is needed to explore their diagnostic and prognostic potential in clinical practice.

Lack of Functional TSLP Receptors Mitigates Th2 Polarization and the Establishment and Growth of 4T1 Primary Breast Tumours but has Different Effects on Tumour Quantities in the Lung and Brain

The 4T1 mammary carcinoma cell line produces TSLP. We had hypothesized that TSLP promotes the development of a permissive environment for the growth and metastasis of primary tumour and that this is associated with a Th2-polarized antitumour immune response. We found that, in Tslpr(-/-) mice, the mean tumour diameters were smaller from days 27 to 40, and relatively fewer tumour cells were present in the lung, compared with wild-type mice. Polarization of the Th2 cytokine profile was also diminished in Tslpr(-/-) mice. These findings confirmed those reported previously by others. Here, we further show that primary tumours are established less often in Tslpr(-/-) mice and that, unexpectedly, the relative number of tumour cells in the brain is greater in Tslpr(-/-) mice compared with wild-type mice. Findings from our cytotoxicity assays show that 4T1-directed lysis is undetectable in both WT and Tslpr(-/-) mice, ruling out the possibility that altered cytotoxic responses in Tslpr(-/-) mice are responsible for the differences we observed. In a human tissue microarray, positive staining for TSLP was seen in tumour cells from breast cancer tissue, but it was also seen in normal glandular epithelial cells from normal breast tissue, which has not been shown before. Thus, our findings provide new insight into the effects of TSLP in metastatic breast cancer.

Complete Response to Systemic Combination Therapy in a Gastric Cancer Patient in Inoperable Condition: A Case Report

Background: Gastric cancer is a leading cause of death worldwide and the average survival rate of patients within a year of diagnosis remains low. Currently, surgical resection is the major treatment strategy, and the effectiveness of chemotherapy remains debatable. Infections involving Helicobacter pylori (H. pylori) and the Epstein-Barr virus have been implicated in gastric cancer pathogenesis. In addition, extensive immunosuppression in patients caused by latent infections and chemotherapy limits their response to existing therapies. Case presentation: A 73-year-old man presented with stage IV gastric cancer (T2N0MII stage, cell group II). The tumor was inoperable due to concomitant multiorgan dysfunction and consequently, the patient was treated with a combinatorial therapy consisting of immunocorrective and antimicrobial therapy, as well as chemotherapy. The medications used for treatment include interferon-α, interleukin-2, statins, fenofibrate, macrolides, proton pumps, and antiviral drugs. Following treatment, tumor cells redifferentiated into normal glandular epithelial cells. A 2-year followup study of the patient after treatment revealed a complete remission from cancer. Conclusion: This case report demonstrates the possibility of using a systemic approach to treat inoperable gastric cancer. In addition, the observations made in this study should aid the design of novel treatment strategies for gastric cancer.

The expression of androgen‐responsive genes is Up‐Regulated in the epithelia of benign prostatic hyperplasia

Benign prostatic hyperplasia (BPH) is one of the most common diseases among aging men in the United States. In addition to aging, the presence of androgens is another major risk factor in BPH development. However, whether androgen signaling is altered in BPH remains unclear. To determine androgen signaling in BPH, we characterized the expression of four different androgen-responsive genes, Eaf2/U19, ELL2, FKBP5, and PSA, in BPH and adjacent normal glandular epithelial cells. A set of 17 BPH specimens were resected from patients over 60 years of age with clinical symptoms of BPH. Laser-capture microdissection (LCM) was used to isolate glandular epithelial cells from BPH areas and adjacent normal areas, separately. LCM isolated cells from individual specimens were lysed and RNA isolation, reverse transcription, and real-time PCR were performed using CellsDirect One-Step qRT-PCR Kit (Invitrogen, Carlsbad, CA). All of the assayed genes displayed increased expression, from approximately 2- to approximately 6-fold, in BPH as compared to the adjacent normal epithelial cells. We also generated a composite androgen response index based on the expression levels of the four genes, which provides a reliable readout for overall androgen action. Our study showed that the composite androgen response index in BPH is approximately 4-fold as compared to that in the adjacent normal tissues. Androgen signaling is significantly elevated in BPH relative to the adjacent normal prostate. Understanding the mechanisms causing elevated androgen signaling may lead to novel approaches for prevention and/or treatment of BPH.

Immunoglobulin mRNA and Protein Expression in Human Oral Epithelial Tumor Cells

In our previous work, we have reported the expression of immunoglobulin (Ig) molecules by numerous epithelial cancer cells and hyperplastic epithelial cells. In the present study, we extended our investigation to study the frequencies of expression of IgG and IgA in some types of oral epithelial tumor cells, and analyzed the oral tumor-derived V regions characteristic of Ig gamma chain gene transcripts by immunohistochemistry, in situ hybridization, laser capture microdissection-correlated reverse-transcription polymerase chain reaction, and sequencing. IgG and IgA immunoreactivity was prominent in the cytoplasmic or plasma membrane or secretion of malignant cells, pleomorphic adenoma tumor cells, and some normal glandular epithelial cells or squamous cells adjacent to tumors. More importantly, rearranged Ig gene transcripts were identified in these tumor cells, and in some normal glandular epithelial cells, the V-D-J region sequences revealed that IgG transcripts in 2 tested oral tumors were oligoclonal. These results support that the phenomenon of Ig could also be expressed in oral cavity epithelial tumor cells.

Expression Profile of Histone Deacetylase 1 in Gastric Cancer Tissues

Although histone deacetylases (HDACs) appear to play a crucial role in carcinogenesis, the expression status of HDACs in primary human cancer tissues has not yet been reported. In this study, we investigated the expression level of HDAC1 in 25 paired primary human gastric cancer (GC) tissues and corresponding normal tissues through semi‐quantitative RT‐PCR and immunoblot analysis. The HDAC1 expression pattern was also topologically examined through immunohisto‐chemistry. Overexpression of HDAC1 mRNA was detected in 68% of GC tissues (17 of 25), and the relative density of HDAC1 mRNA in GC tissue was increased 1.8‐fold versus the normal counterpart (P<0.01). Elevated expression of HDAC1 protein was also detected in 61% of GC samples (11 of 18), which also showed an increased mRNA level of HDAC. Immunohistochemically, overexpression of HDAC1 was predominantly localized in the nuclei of most neoplastic cells, including embolic tumor cells, whereas normal glandular epithelial cells revealed only weak HDAC1 expression that was focal in distribution. Thus, the present study clearly demonstrates that HDAC1 is overexpressed in GC and probably plays a significant role in gastric carcinogenesis.

Expression of B7.1 in a MUC1-expressing mouse mammary epithelial tumour cell line inhibits tumorigenicity but does not induce autoimmunity in MUC1 transgenic mice.

The MUC1 epithelial mucin, which is overexpressed and aberrantly glycosylated in breast and other carcinomas, is also expressed on the apical surface of most normal glandular epithelial cells. Since clinical trials evaluating the efficacy of MUC1-based vaccines have been initiated in breast cancer patients, it is important to address the question of whether an effective immune response to the cancer associated mucin can be generated without inducing autoimmunity. Since non-classic cytotoxic T lymphocyte (CTL) responses to MUC1 have been reported, it is also relevant to examine the role of costimulatory molecules in the effective presentation of MUC1 based antigens. We have therefore looked at the effect of expressing B7.1 on the tumorigenicity of a MUC1 expressing mammary epithelial cell line (410.4) in a transgenic mouse expressing MUC1 on its normal glandular epithelial tissues. Coexpression of B7.1 with MUC1 in 410. 4 cells resulted in a dramatic inhibition of tumour growth which depended on the activity of CD4+ and CD8+ T cells. The epithelial tissues in the transgenic mice able to reject the B7.1, MUC1-expressing tumours showed no evidence of degeneration and the mice survived their normal life span. The results demonstrate that an immune response to the MUC1 antigen can be induced in MUC1 transgenic mice and that presentation of the antigen, whether directly or by cross-priming, is markedly enhanced by coexpression of B7.1.

Heparin-binding EGF-like growth factor in the human prostate: synthesis predominantly by interstitial and vascular smooth muscle cells and action as a carcinoma cell mitogen.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is an activating ligand for the EGF receptor (HER1/ErbB1) and the high-affinity receptor for diphtheria toxin (DT) in its transmembrane form (proHB-EGF). HB-EGF was immunolocalized within human benign and malignant prostatic tissues, using monospecific antibodies directed against the mature protein and against the cytoplasmic domain of proHB-EGF. Prostate carcinoma cells, normal glandular epithelial cells, undifferentiated fibroblasts, and inflammatory cells were not decorated by the anti-HB-EGF antibodies; however, interstitial and vascular smooth muscle cells were highly reactive, indicating that the smooth muscle compartments are the major sites of synthesis and localization of HB-EGF within the prostate. In marked contrast to prostatic epithelium, proHB-EGF was immunolocalized to seminal vesicle epithelium, indicating differential regulation of HB-EGF synthesis within various epithelia of the reproductive tract. HB-EGF was not overexpressed in this series of cancer tissues, in comparison to the benign tissues. In experiments with LNCaP human prostate carcinoma cells, HB-EGF was similar in potency to epidermal growth factor (EGF) in stimulating cell growth. Exogenous HB-EGF and EGF each activated HER1 and HER3 receptor tyrosine kinases and induced tyrosine phosphorylation of cellular proteins to a similar extent. LNCaP cells expressed detectable but low levels of HB-EGF mRNA; however, proHB-EGF was detected at the cell surface indirectly by demonstration of specific sensitivity to DT. HB-EGF is the first HER1 ligand to be identified predominantly as a smooth muscle cell product in the human prostate. Further, the observation that HB-EGF is similar to EGF in mitogenic potency for human prostate carcinoma cells suggests that it may be one of the hypothesized stromal mediators of prostate cancer growth.

Monoclonal Antibodies Against Human Pancreatic Adenocarcinoma: Distribution of DU-PAN-2 Antigen on Glandular Epithelia and Adenocarcinomas&lt;xref ref-type="fn" rid="FN2"&gt;2&lt;/xref&gt;

This report describes the distribution of antigen DU-PAN-2, defined by a monoclonal antibody raised against human pancreatic carcinoma cells, on a variety of tumors and nonneoplastic tissues. With the use of an immunoperoxidase technique, the antigen was detected on 16 of 16 pancreatic carcinomas, on 5 of 5 gallbladder or bile duct carcinomas, on the great majority of stomach adenocarcinomas, and, less commonly, on adenocarcinomas of other primary sites. Substantial intratumor heterogeneity of antigen expression was noted. DU-PAN-2 antigen was present on many types of normal glandular epithelial cells but often was more weakly expressed than on the corresponding tumors. The immunomorphology of staining, coupled with biochemical information known about the antigen, supports the notion that the DU-PAN-2 antigen is a mucin-like substance. Its relative restriction of expression on different types of glandular epithelium suggests that DU-PAN-2 antibody might be a useful reagent for helping to determine the site of origin of adenocarcinomas.

癌細胞のコハク酸酸化酵素系

With a view to ascertain the common specific characteristics of the respiratory enzyme system of cancer cells and changes in the enzyme activities in consonant with their origin and grade of differentiation the authors carried out a series of histochemical and biochemical investigations by studying the succinoxidase system, cytochrome oxidase system, DPNH-dehydrogenase system, and endogenous dehydrogenase system in various tumorous tissues, homologous non-tumorous tissues, and precancerous tissues, all from human or animals, by using cell suspensions, tissue slices, tissue homogenates, and mitochondria fractions with tetrazolium salts (NT, Nitro-NT, and Nitro-BT); and arrived at the following conclusions:1. As for the activity of the succinoxidase system in normal glandular epithelial cells it is stronger in those matured and differentiated cells possessing vigorous metabolic function than in immature cells. However, in the cancer tissues, such as hepatoma and adenocarcinoma, the activity is generally lower than in the homologous normal tissues. In the cancer tissue the more highly differentiated cancer cells show stronger activity; and it is lowest in undifferentiated cancer cells.2. Normal squamous epithelial cells show generally a lower activity, and of them young basal cells present a higher activity. However, in the case of squamous cell cancer the activity is still stronger, and in contrast it is higher in poorly differentiated cancer cells.3. In normal non-epithelial cells, with a few exceptions (e. g. muscle cells), the activity of young cells is rather stronger than that of mature cells, and in the case of sarcomas (including leukemic cells) their activity is stronger than that of homologous tissue cells.4. The endogenous dehydrogenase system activity of both cancer cells and sarcoma cells (including leukemic cells) is stronger than that of homologous non-tumorous cells.5. The succinoxidase system activity of the rat liver tissue is elevated gradually at the precancerous stage by the DAB administration, and the activity is markedly diminished when the tissue became cancerous. On the other hand, the endogenous dehydrogenase system activity, rising gradually from the precancerous stage, does not fall even when cancerized.6. In cancer cells the shape and structures of the mitochondria and their respiratory enzyme activities are on the whole irregular.7. Cancer cells are apt to be easily destroyed in the course of the enzyme reaction, and moreover, it is liable to bring about the demolition and the decreased respiratory enzyme activity of mitochondria due to the destructive procedures as homogenization and or other treatments.