High MUC1 Research Articles

Autophagy is required during high MUC2 mucin biosynthesis in colonic goblet cells to contend metabolic stress.

Goblet cells are specialized for the production and secretion of MUC2 glycoproteins that forms a thick layer covering the mucosal epithelium as a protective barrier against noxious substances and invading microbes. High MUC2 mucin biosynthesis induces endoplasmic reticulum (ER) stress and apoptosis in goblet cells during inflammatory and infectious diseases. Autophagy is an intracellular degradation process required for maintenance of intestinal homeostasis. In this study, we hypothesized that autophagy was triggered during high MUC2 mucin biosynthesis from colonic goblet cells to cope with metabolic stress. To interrogate this, we analyzed the autophagy process in high MUC2-producing human HT29-H and a clone HT29-L silenced for MUC2 expression by lentivirus-mediated shRNA, and WT and CRISPR/Cas9 MUC2 KO LS174T cells. Autophagy was constitutively increased in high MUC2-producing cells characterized by elevated pULK1S555 expression and increased numbers of autophagosomes as compared with MUC2 silenced or gene edited cells. Similarly, colonoids from Muc2+/+ but not Muc2-/- littermates differentiated into goblet cells showed increased autophagy. IL-22 treatment corrected misfolded MUC2 protein and alleviated the autophagy process in LS174T cells. This study highlights that autophagy plays an essential role in goblet cells to survive during high mucin biosynthesis by regulating cellular homeostasis.NEW & NOTEWORTHY It is unclear how colonic goblet cells survive by producing high output MUC2 mucin that triggers endoplasmic stress by misfolded MUC2 proteins. To cope with metabolic stress, we interrogated if autophagy played an essential role in regulating cellular homeostasis. Indeed, high MUC2 mucin biosynthesis dysregulated autophagy processes that was regulated by IL-22 to maintain gut barrier innate host defenses.

Mucin-1 Protein Is a Prognostic Marker for Pancreatic Ductal Adenocarcinoma: Results From the CONKO-001 Study.

BackgroundThe Mucin-family protein, MUC1, impacts on carcinogenesis and tumor invasion. We evaluated the impact of MUC1 expression on outcome in a cohort of 158 patients with resected pancreatic ductal adenocarcinomas (PDAC) in the CONKO-001 study (adjuvant gemcitabine [gem] vs. observation [obs]).MethodsThe percentage of MUC1-positive tumor cells by immunohistochemistry (IHC) and the staining intensity were evaluated by two observers blinded to outcome. The numeric values of both parameters were multiplied, resulting in an immunoreactivity score (IRS) ranging from 0 to 12. The level of MUC1 expression was defined as follows: IRS 0–4 (low) vs IRS >4 (high). Outcomes in terms of disease-free (DFS) and overall survival (OS) were evaluated by Kaplan–Meier method, log-rank tests and Cox regressions.ResultsIn total, tumors of 158 study patients were eligible for immunohistochemistry of MUC1. High cytoplasmic MUC1 expression was associated with impaired DFS and OS in the overall study population (hazard ratio (HR) for DFS: 0.49, 95% CI 0.31 to 0.78, p = .003; HR for OS: 0.46, 95% CI 0.29 to 0.73, p = .001). In the study arms, prognostic effects of MUC1 were also evident in the observation group (HR for DFS: 0.55; 95% CI 0.29 to 1.04, p = .062; HR for OS: 0.34, 95% CI 0.17 to 0.67, p = .001) and trending in the gem group (HR for DFS: 0.48, 95% CI 0.24 to 0.95, p = .041; HR for OS: 0.56, 95% CI 0.28 to1.11, p = .093).ConclusionOur data suggest that MUC1 expression is a powerful prognostic marker in patients with PDAC after curatively intended resection.

Polymer stealthing and mucin-1 retargeting for enhanced pharmacokinetics of an oncolytic vaccinia virus

Vaccinia virus (VV) is a powerful tool for cancer treatment with the potential for tumor tropism, efficient cell-to-cell spread, rapid replication in cancer cells, and stimulation of anti-tumor immunity. It has a well-defined safety profile and is being assessed in late-stage clinical trials. However, VV clinical utility is limited by rapid bloodstream neutralization and poor penetration into tumors. These factors have often restricted its route of delivery to intratumoral or intrahepatic artery injection and may impede repeat dosing. Chemical stealthing improves the pharmacokinetics of non-enveloped viruses, but it has not yet been applied to enveloped viruses such as VV. In the present study, amphiphilic polymer was used to coat VV, leading to reduced binding of a neutralizing anti-VV antibody (81.8% of polymer-coated VV [PCVV] staining positive versus 97.1% of VV [p = 0.0038]). Attachment of anti-mucin-1 (aMUC1) targeting antibody, to give aMUC1-PCVV, enabled binding of the construct to MUC1. In high MUC1 expressing CAPAN-2 cells, infection with PCVV was reduced compared to VV, while infection was restored with aMUC1-PCVV. Pharmacokinetics of aMUC1-PCVV, PCVV, and VV were evaluated. After intravenous (i.v.) injection of 1 × 108 viral genomes (VG) or 5 × 108 VG, circulation time for PCVV and aMUC1-PCVV was increased, with ~5-fold higher circulating dose at 5 min versus VV.

Analysis of proliferation and apoptosis, MUC-1 and Cdx-2 expression in distal colonic polyps

Background. The universal molecular characteristics of malignancy are activation of cell proliferation and inactivation of apoptotic cell death. Besides genes-regulators of the processes other molecules are involved, including mucins and Cdx-2. Aim . To conduct comparative and correlation analysis of indexes of immunohistochemical expression of Ki-67, p53, caspase-3, as well as MUC-1 and Cdx-2 in distal colonic polyps. Methods. Pathohistological and immunohistochemical studies of biopsies of adenomas and hyperplastic polyps from 40 patients were carried out. Results. Distal colonic polyps are characterized by medium proliferation level: the median of Ki-67 expression for hyperplastic polyps is 42,20 (35,27; 52,38) %, while the median for adenomas is 65,39 (52,28; 76,23) % (р˂0,05). The polyps are characterized by low p53 expression levels: the median of the marker expression for hyperplastic polyps is 1,53 (1,17; 3,16) %, for adenomas – 9,15 (8,26; 12,39) % (р˂0,05). The studied polyps are characterized by low apoptosis levels: the median of caspase-3 expression for hyperplastic polyps is 33,78 (30,28; 42,34) CUOD, for adenomas – 22,19 (16,84; 40,51) CUOD (р˂0,05). Distal colonic polyps are characterized by medium MUC-1 and Cdx-2 expression levels: the medians of the markers expression for hyperplastic polyps are 62,13 (34,20; 71,85) CUOD and 71,81 (62,36; 77,77) CUOD, for adenomas – 80,25 (52,14; 95,21) CUOD and 95,14 (81,19; 112,17) CUOD, respectively (р˂0,05). The correlation analysis revealed several correlations: reverse medium strength correlation between p53 and caspase-3 expression levels (r = -0,57, р˂0,05), direct medium strength correlation between caspase-3 and MUC-1 expression levels (r = 0,52, p˂0,05) take place in hyperplastic polyps, whereas reverse strong correlation between caspase-3 and MUC-1 expression levels 1 (r = -0,88, p˂0,05) takes place in adenomas. Conclusions. Distal colonic adenomas differ by higher proliferation and p53 expression levels, comparing to hyperplastic polyps, and by lower apoptosis level that is associated with higher MUC-1 expression level as well. Hyperplastic polyps differ by higher apoptosis level that is associated with lower MUC-1 expression level. Cdx-2 expression increases on benign adenoma stage and does not correlate with proliferation and apoptosis indexes.

Abstract 1837: MUC1 confers sensitivity to STAT-3 inhibitor napabucasin in pancreatic ductal adenocarcinoma cells

Abstract Introduction: Pancreatic cancer is the third leading cause of cancer-related deaths in the US and 95% of pancreatic cancers are Pancreatic Ductal Adenocarcinomas (PDACs). Napabucasin is a small molecule inhibitor that targets the signal transducer and activator of transcription 3 (STAT-3) pathway in cancer stem cells. Napabucasin in combination with nab-paclitaxel and gemcitabine, was under phase III clinical trials for gastroenterological cancers including Pancreatic Cancer. The trials have been stopped recently due to futility. Therefore, it is important to find subpopulations of tumor cells that are likely to respond better to Napabucasin treatment alone or as a combination. Mucin1 or MUC1 is a transmembrane glycoprotein found in almost all glandular epithelial cells, and is overexpressed and aberrantly glycosylated in greater than 80% of PDACs. The tumor form of MUC1 is associated with poor prognosis. In PDAC cells, MUC1 expression is critical for tumor cell survival, oncogenic signaling, metastasis, and conferring stemness. Phosphorylated STAT-3 is known to regulate MUC1 expression. Thus, we hypothesize that 1) the STAT-3-MUC1 survival pathway is constitutively activated in MUC1-high PDAC cells and 2) Napabucasin will be more effective in MUC1-high compared to MUC1-low PDAC cells. Methods: Human PDAC cell lines with varying levels of MUC1 (CFPAC, Capan 2, Capan 1, HPAF II, HS766T, BxPc3.MUC1 and BxPC3.Neo) and a normal pancreatic ductal epithelial cell line (HPDE) were treated with increasing doses of Napabucasin (0.05 uM, 0.1uM, 0.2uM, 0.4uM, 0.8uM, 1.6uM, 3.2uM, 6.4uM, 12.8uM and 25.6uM) for 48 and 72 hours. Following treatment, MTT and colony formation assays were performed to determine survival and growth respectively. Results: The cells with high MUC1, CFPAC and Capan 1 showed increased susceptibility to Napabucasin at lower doses (IC50 - 2.088uM and 0.371uM respectively) compared to cells with low MUC1, Capan 2 and HS766T (IC50 - 6.885uM and 7.464uM respectively). MUC1-high HPAF II cells were resistant even at high doses. Data further confirmed that the IC50 of Napabucasin was significantly lower for Bx.Pc3.MUC1 (1.86uM) compared to Bx.Pc3.Neo cells (23.19uM). MUC1-high expressing cell lines lost their colony forming potential at lower doses compared to MUC1-low expressing cell lines. To test if oncogenic signaling linked to MUC1 dimerization confers sensitivity to Napabucasin, the PDA cells were pre-treated with GO-203, a small molecule that inhibits dimerization of MUC1 which renders signaling through the MUC1 cytoplasmic tail non-functional. Conclusion: MUC1 induces susceptibility to Napabucasin in PDACs. Napabucasin has shown anti-tumor activity in preclinical models and early clinical trials of gastroenterological cancers. However, lack of specificity led to increased toxicities and decreased efficacy. It is important to identify and validate biomarkers that better identify patients who have upregulated stemness pathways. MUC1 is one such specific biomarker that could be incorporated in future clinical trials to help identify patient subpopulations that may most likely benefit from combination therapies with stemness-inhibiting drugs. Further study of the crosstalk between stemness signaling pathways is necessary to understand mechanisms of synergistic effects and treatment resistance. Citation Format: Mukulika Bose, Aabha Vora, Taylor Colleton, Pinku Mukherjee. MUC1 confers sensitivity to STAT-3 inhibitor napabucasin in pancreatic ductal adenocarcinoma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1837.

Association between aberrant dynein cytoplasmic1 light intermediate chain1 expression levels, mucins and chemosensitivity in colorectal cancer.

Dynein transport along the cytoskeletal microtubules towards the minus end is essential for cell division, cell migration and other basic cellular functions. Dynein cytoplasmic 1 light intermediate chain 1 (DYNC1LI1) has been previously associated with pancreatic ductal adenocarcinoma, hepatocellular carcinoma and prostate cancer. Cytoskeletal structures are involved in the regulation of the mucosal barrier integrity. Thus, improving our understanding of the molecular mechanisms that regulate the mucosal barrier is critical for cancer management and treatment. The present study aimed to investigate DYNC1LI1 expression in colorectal cancer (CRC) tissues. The American Joint Committee on Cancer Stage II CRC cell line LS 174T was used to determine the association between the cellular expression levels of DYNC1LI1 and different types of mucin (MUC) by reverse transcription-quantitative PCR. The role of DYNC1LI1 in cell chemosensitivity and proliferation was also evaluated in the presence of the DNA analog 5-fluorouracil (5-FU) or the platinum-based drug, oxaliplatin by the MTT assay. LS 174T cells with decreased expression levels of DYNC1LI1 were discovered to be more sensitive to 5-FU compared with LS 174T cells with endogenous DYNC1LI1 expression levels. Moreover, LS 174T cells transfected with short hairpin RNA targeting DYNC1LI1 were associated with low MUC1 and high MUC2, MUC4 and MUC5AC expression levels. Notably, the CRC cells with low MUC1 expression levels and high expression levels of the other MUCs (MUC2, MU4 and MUC5AC) were shown to benefit from 5-FU treatment. In conclusion, the findings of the present study have suggested that DYNC1LI1 expression may be significantly associated with MUC expression levels and may be used to predict the chemotherapeutic efficiency. However, additional functional studies and clinical reports are required for an improved understanding of the significance of these molecular interactions in tumorigenesis.

MUC1, MUC2, MUC4, MUC5AC, Cdx2: immunohistochemical characteristics in polyps and adenocarcinoma of distal colon

Aim – to study MUC1, MUC2, MUC4, MUC5AC, Cdx2 immunohistochemical expression in polyps and adenocarcinoma of distal colon. Materials and methods. Pathomorphological and immunohistochemical studies of biopsies from 30 patients, which underwent colonoscopy with polypectomy, and surgical material from 30 patients, which underwent surgery for colorectal adenocarcinoma, were carried out. Results. Distal colonic polyps are different from unchanged mucosa by increased MUC-1 expression. Maximum expression levels of the marker are observed in serrated adenomas, which are characterized by high MUC-1 expression level. Median of MUC-1 expression in adenocarcinoma is greater than that in unchanged mucosa, however, less than in polyps. Comparative analysis of data of the study of MUC2 expression indicates that the maximum expression of the marker is characteristic of unchanged mucous membrane. Polyps are distinguished by lower MUC2 expression levels; among them, highest expression levels are observed in hyperplastic polyps, lesser – in serrated adenomas and adenomas. Distal colonic carcinoma has extremely low median of MUC2 expression. The highest levels of MUC4 expression are observed in hyperplastic polyps, somewhat lower – in unchanged mucosa. Serrated adenomas, adenomas and adenocarcinomas differs by lower MUC4 expression levels. The minimum level of the marker expression is observed in serrated adenomas. In polyps MUC5AC expression levels are greater than in adenocarcinoma. Expression of this marker is not detected in unchanged mucosa. The maximum median of MUC5AC expression is observed in serrated adenomas, which are characterized by high level of the marker expression, somewhat lower – in adenomas, and more lower in hyperplastic polyps. High Cdx-2 expression level characterizes serrated adenomas and adenocarcinoma. The average Cdx-2 expression level is observed in adenomatous polyps, hyperplastic polyps, and unchanged mucosa, with the medians of the marked expression decreasing in that sequence. Conclusions . Distal colon polyps are characterized by increased MUC1 and Cdx-2 expression levels, with parallel decrease of MUC2 expression level and aberrant MUC5AC expression. At the same time, serrated adenomas are distinguished by the greatest deviations of the studied markers expression levels. Adenocarcinoma of the distal colon, compared with polyps, is characterized by decrease of MUC1, MUC2 and MUC5AC expression levels.

Functional Expression of Mucin1 in Human Duodenal Adenocarcinoma

ObjectiveMucin1 (MUC1), a member of the mucin family, is a glycoprotein which is often expressed in malignant cells. However, the expression and function of MUC1 in human duodenal adenocarcinoma (DAC) has not yet been characterized because of its low frequency. Here, we examined the functional roles of core protein (MUC1-C) in DAC. Materials and methodsUsing a human duodenal cancer cell line, HuTu80, proliferation, migration, invasion, ALDH activity was assessed by cell counting kit–8, scratch wound healing, matrigel invasion, and ALDEFUOR assays, respectively. The function of MUC1 protein was evaluated with knockdown using specific siRNA as well as anti-MUC1-C peptide, GO203. MUC1 expression in human DAC was evaluated immunohistochemically in surgically resected tumors. ResultsThe positive expression of MUC1 in HuTu80 was confirmed by RT-PCR and flow cytometry. In vitro cell growth was inhibited by the addition of 50-100 μM GO203 as well as treatment with siRNA for MUC1-C. Silencing of MUC1-C also significantly reduced migration, invasion, ALDH activity. Local injection of GO-203 (14 mg/kg) significantly suppressed the growth of subcutaneous HuTu80 tumors in nude mice. Immunohistochemically, MUC1 was strongly detected in seven DAC cases, but not in 11 others. The outcome of patients with high MUC1 expression was significantly worse than those without MUC1 expression. ConclusionsThese results suggest that MUC1 is functionally associated with the malignant potential of DAC and could be a novel therapeutic target for this rare tumor.

P126 MULTIPLE SIGNALING PATHWAY DEFECTS IN THE INTESTINAL STEM CELL NICHE LIKELY TRIGGER CD-LIKE ILEITIS IN SAMP1/YITFC MICE

Intestinal epithelial barrier dysfunction is considered as a risk factor in the pathogenesis of Crohn’s disease (CD), and since there is no FDA approved therapy available, there is a need for identifying a relevant model to study and test therapies to correct this defect. Previously, we demonstrated that inflammatory environment of CD affects properties of intestinal stem cell (ISC) niche, ablating its ability to form enteroids (EnOs) in our mouse model for CD-like ileitis (SAMP/YitFc, SAMP) (doi.org/10.1016/S0016-5085(17)32213-8). However, EnOs from SAMP mice even prior to disease onset (5-wks, SAMP-5) harbor impaired features, such as smaller size (2.6 ± 0.4 vs 3.9 ± 0.2 fold to ctrl at day 1, P=0.003), number (0.6 ± 0.1 fold to ctrl, P=0.000), reduced viability (63.7%±7.5 vs ctrl P=0.003) and rudimental branching complexity [cyst-like structures (42.7%±2.3 vs 11%±1.2)], compared to controls (ctrls). These EnOs produce exacerbated expression of genes involved in epithelial secretory cell functions when challenged with purified bacterial ligands, such as LPS [higher Muc2 (10.8 ± 3.2 vs 2.5 ± 1.8 P=0.00), Lyz (11.0 ± 5.2 vs 1.1 ± 0.4 P=0.00), Reg3γ (6.7 ± 9.9 vs 1.1 ± 0.5 P=0.04), Crp (7.4 ± 5.4 vs 1.3 ± 0.6 P=0.05), and Defa1 (4006.0 ± 1472.5 vs 2.1 ± 0.7 P=0.00) mRNA vs AKR EnOs]. To further investigate this phenotype, we generated the first high-resolution transcriptional profile of SAMP ISC niche by laser capture microdissection and RNA sequencing. We found that signaling pathways involved in the cellular energy machinery were dramatically reduced in inflamed SAMP (25-wks, SAMP-25) ISC niche compared to that of inflammation-free mice [mitochondrial energy metabolism (P ≤ 0.00, Padj ≤ 0.002, normalized enrichment score NES ≤ -2.4); oxidative phosphorylation (P ≤ 0.00 Padj ≤ 0.001 NES ≤ -2.5)]. Fibrosis (P ≤ 0.04 Padj≤ 0.09 NES ≤ 1.3) and UPR (P ≤ 0.01 Padj ≤ 0.03 NES ≤ 1.5) pathways were enriched in SAMP-25 ISC niche compared to ctrls, but not to that of SAMP-5 mice. Bile acid metabolism (P ≤ 0.00, Padj ≤ 0.002 NES ≤ -1.8), ROS (P ≤ 0.001 Padj ≤ 0.003 NES ≤ -1.8), Wnt3a (P ≤ 0.001 Padj ≤ 0.01 NES ≤ -1.8), and Notch1 (P ≤ 0.001 Padj ≤ 0.02 NES ≤ -1.8) pathways were decrease in SAMP-25 ISC niche compared to ctrls, but not to that of SAMP-5 mice. Collectively, these data suggest that multiple signaling pathways contribute to ISC niche defect in SAMP mice, and that many key biological functions are already altered prior to ileitis onset and may be responsible for disease initiation. Finally, the evidence that altered gene expression is maintained in ex vivo expanded EnOs indicates that these cultures recapitulate reactive epithelial changes specific of experimental CD. This aspect is of paramount importance since it emphasizes the physiologically relevant value of EnOs generated from SAMP mice, rather than those originated from healthy individuals, to mimic CD-like ileitis.

P126 MULTIPLE SIGNALING PATHWAY DEFECTS IN THE INTESTINAL STEM CELL NICHE LIKELY TRIGGER CD-LIKE ILEITIS IN SAMP1/YITFC MICE

Intestinal epithelial barrier dysfunction is considered as a risk factor in the pathogenesis of Crohn’s disease (CD), and since there is no FDA approved therapy available, there is a need for identifying a relevant model to study and test therapies to correct this defect. Previously, we demonstrated that inflammatory environment of CD affects properties of intestinal stem cell (ISC) niche, ablating its ability to form enteroids (EnOs) in our mouse model for CD-like ileitis (SAMP/YitFc, SAMP) (doi.org/10.1016/S0016-5085(17)32213-8). However, EnOs from SAMP mice even prior to disease onset (5-wks, SAMP-5) harbor impaired features, such as smaller size (2.6 ± 0.4 vs 3.9 ± 0.2 fold to ctrl at day 1, P=0.003), number (0.6 ± 0.1 fold to ctrl, P=0.000), reduced viability (63.7%±7.5 vs ctrl P=0.003) and rudimental branching complexity [cyst-like structures (42.7%±2.3 vs 11%±1.2)], compared to controls (ctrls). These EnOs produce exacerbated expression of genes involved in epithelial secretory cell functions when challenged with purified bacterial ligands, such as LPS [higher Muc2 (10.8 ± 3.2 vs 2.5 ± 1.8 P=0.00), Lyz (11.0 ± 5.2 vs 1.1 ± 0.4 P=0.00), Reg3γ (6.7 ± 9.9 vs 1.1 ± 0.5 P=0.04), Crp (7.4 ± 5.4 vs 1.3 ± 0.6 P=0.05), and Defa1 (4006.0 ± 1472.5 vs 2.1 ± 0.7 P=0.00) mRNA vs AKR EnOs]. To further investigate this phenotype, we generated the first high-resolution transcriptional profile of SAMP ISC niche by laser capture microdissection and RNA sequencing. We found that signaling pathways involved in the cellular energy machinery were dramatically reduced in inflamed SAMP (25-wks, SAMP-25) ISC niche compared to that of inflammation-free mice [mitochondrial energy metabolism (P ≤ 0.00, Padj ≤ 0.002, normalized enrichment score NES ≤ -2.4); oxidative phosphorylation (P ≤ 0.00 Padj ≤ 0.001 NES ≤ -2.5)]. Fibrosis (P ≤ 0.04 Padj≤ 0.09 NES ≤ 1.3) and UPR (P ≤ 0.01 Padj ≤ 0.03 NES ≤ 1.5) pathways were enriched in SAMP-25 ISC niche compared to ctrls, but not to that of SAMP-5 mice. Bile acid metabolism (P ≤ 0.00, Padj ≤ 0.002 NES ≤ -1.8), ROS (P ≤ 0.001 Padj ≤ 0.003 NES ≤ -1.8), Wnt3a (P ≤ 0.001 Padj ≤ 0.01 NES ≤ -1.8), and Notch1 (P ≤ 0.001 Padj ≤ 0.02 NES ≤ -1.8) pathways were decrease in SAMP-25 ISC niche compared to ctrls, but not to that of SAMP-5 mice. Collectively, these data suggest that multiple signaling pathways contribute to ISC niche defect in SAMP mice, and that many key biological functions are already altered prior to ileitis onset and may be responsible for disease initiation. Finally, the evidence that altered gene expression is maintained in ex vivo expanded EnOs indicates that these cultures recapitulate reactive epithelial changes specific of experimental CD. This aspect is of paramount importance since it emphasizes the physiologically relevant value of EnOs generated from SAMP mice, rather than those originated from healthy individuals, to mimic CD-like ileitis.

MUC1/KL-6 expression confers an aggressive phenotype upon myeloma cells

The sialic glycoprotein, MUC1, is known to be involved in the pathogenesis of various types of cancers. KL-6 is one of the surface antigens of MUC1 and also a marker of interstitial pneumonitis. A fraction of patients with myeloma (3.9%) have elevated serum KL-6 levels without any evidence of interstitial pneumonitis and their myeloma cells have high MUC1 expression. We established a myeloma cell line designated EMM1 from a patient with multiple myeloma accompanied with elevated serum KL-6. EMM1 cells expressed high levels of MUC1 compared with other myeloma cell lines. Knockdown of MUC1 in EMM1 cells induced cell cycle arrest during S phase and apoptosis, suggesting that the MUC1 expression is involved in accelerated growth of EMM1 cells. RNA-seq analysis suggests that MUC1 expression activates k-ras and TNFα-induced NFκB pathways in EMM1 cells. We injected EMM1 cells subcutaneously into Rag2−/−Jak3−/− Balb/c mice to establish a mouse xenograft model. These mice had aggressive tumor growth that was accompanied by high serum KL-6 levels. In addition, MUC1 knockdown in EMM1 cells led to inhibited tumor growth. These findings demonstrate that MUC1 serves as a potential target for developing drugs for treatment of patients with KL-6+ myeloma, and EMM1 cells and EMM1-engrafted mice are useful tools for the development of such novel agents.

Chemoradiotherapy-induced Changes in Mucinous Components in Rectal Cancer Tissue: Evaluation on High Iron Diamine-alcian Blue and Mucin 1 Staining.

The standard treatment for rectal cancer is neoadjuvant chemoradiotherapy (nCRT) followed by surgery. Mucinous carcinoma responds poorly to nCRT. In some patients, mucin lakes (MLs) are induced by nCRT. Identifying whether MLs are induced or originally present would be of great importance. We studied 20 patients with MLs (CRT-MC group) among 205 patients who received nCRT. Among 88 patients who did not receive nCRT, we studied 9 patients with mucinous carcinoma (non-CRT-MC group) and 18 patients with MLs in differentiated adenocarcinoma (non-CRT-AC group). Tumors were stained with high iron diamine-Alcian blue (HID-AB) and MUC1 staining. Rate of AB>HID staining of cancer cells was significantly higher in the CRT-MC group than in non-CRT-MC group (p=0.0004). Rate of MUC1 staining in MLs was significantly higher in the CRT-MC group (p=0.0254). nCRT can induce qualitative changes in mucinous components, however, other methods are required to distinguish induced components from originally existing components.

High MUC2 Mucin Biosynthesis in Goblet Cells Impedes Restitution and Wound Healing by Elevating Endoplasmic Reticulum Stress and Altered Production of Growth Factors

Intestinal epithelial cell wound healing involves cell migration, proliferation, and differentiation. Although numerous studies have analyzed the migration of absorptive epithelial cells during wound healing, it remains unclear how goblet cells restitute and how MUC2 mucin production affects this process. In this study, we examined the role of high MUC2 production in goblet cell migration during wound healing and demonstrated that during high MUC2 output, goblet cells migrated slower because of impaired production of wound healing factors and endoplasmic reticulum (ER) stress. Two goblet cell lines, HT29-H and HT29-L, that produced high and low MUC2 mucin, respectively, were used. HT29-L healed wounds faster than HT29-H cells by producing significantly higher amounts of fibroblast growth factor (FGF) 1, FGF2, vascular endothelial growth factor-C, and matrix metallopeptidase 1. Predictably, treatment of HT29-H cells with recombinant FGF2 significantly enhanced migration and wound healing. High MUC2 biosynthesis in HT29-H cells induced ER stress and delayed migration that was abrogated by inhibiting ER stress with tauroursodeoxycholic acid and IL-22. FGF2- and IL-22-induced wound repair was dependent on STAT1 and STAT3 signaling. During wound healing after dextran sulfate sodium-induced colitis, restitution of Math1M1GFP+ goblet cells occurred earlier in the proximal colon, followed by the middle and then distal colon, where ulceration was severe. We conclude that high MUC2 output during colitis impairs goblet cell migration and wound healing by reducing production of growth factorscritical in wound repair.

Clinical effect of MUC1 and its relevance to BRAF V600E mutation in papillary thyroid carcinoma: a case-control study.

AimTo investigate the clinical effects of MUC1 on papillary thyroid cancer (PTC) and explore the relationship between MUC1 expression and BRAF mutation.MethodsThe data of 69 patients subjected to fine-needle aspiration biopsy in our hospital and 486 patient data downloaded from The Cancer Genome Atlas (TCGA) database were used. Univariate and multivariate analyses were performed.ResultsThe results on the 486 patients recorded in the TCGA indicated that high MUC1 expression was independently related to BRAF mutation, lymph node metastasis (LNM), and unifocal type. In the 69 fine-needle aspiration biopsy patients with PTC, high MUC1 expression was significantly related to LNM and extrathyroid extension (ETE). The result of Pearson’s correlation coefficient showed that BRAF mutation and MUC1 expression were moderately correlated. Moreover, in the subgroup with low MUC1 expression, the patients with BRAF mutation had higher ETE frequency and LNM than those without BRAF mutation. In the subgroup with BRAF mutation, patients with high MUC1 expression exhibited higher ETE frequency than those with low MUC1 expression, and high MUC1 expression occurred in older patients. In the subgroup with BRAF wild-type mutation, patients with high MUC1 expression had a higher incidence of ETE and LNM than those with low expression.ConclusionWe demonstrated that the MUC1 is an important oncogene in PTC and may have great significance on therapeutic cancer vaccine development.

Treatment of pancreatic ductal adenocarcinoma with tumor antigen specific-targeted delivery of paclitaxel loaded PLGA nanoparticles

BackgroundPancreatic ductal adenocarcinoma (PDA) remains the most aggressive cancers with a 5-year survival below 10%. Systemic delivery of chemotherapy drugs has severe side effects in patients with PDA and does not significantly improve overall survival rate. It is highly desirable to advance the therapeutic efficacy of chemotherapeutic drugs by targeting their delivery and increasing accumulation at the tumor site. MUC1 is a membrane-tethered glycoprotein that is aberrantly overexpressed in > 80% of PDA thus making it an attractive antigenic target.MethodsPoly lactic-co-glycolic acid nanoparticles (PLGA NPs) conjugated to a tumor specific MUC1 antibody, TAB004, was used as a nanocarrier for targeted delivery into human PDA cell lines in vitro and in PDA tumors in vivo. The PLGA NPs were loaded with fluorescent imaging agents, fluorescein diacetate (FDA) and Nile Red (NR) or isocyanine green (ICG) for in vitro and in vivo imaging respectively or with a chemotherapeutic drug, paclitaxel (PTX) for in vitro cytotoxicity assays. Confocal microscopy was used to visualize internalization of the nanocarrier in vitro in PDA cells with high and low MUC1 expression. The in vivo imaging system (IVIS) was used to visualize in vivo tumor targeting of the nanocarrier. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay was used to determine in vitro cell survival of cells treated with PTX-loaded nanocarrier. One-sided t-test comparing treatment groups at each concentration and two-way ANOVAs comparing internalization of antibody and PLGA nanoparticles.ResultsIn vitro, TAB004-conjugated ICG-nanocarriers were significantly better at internalizing in PDA cells than its non-conjugated counterpart. Similarly, TAB004-conjugated PTX-nanocarriers were significantly more cytotoxic in vitro against PDA cells than its non-conjugated counterpart. In vivo, TAB004-conjugated ICG-nanocarriers showed increased accumulation in the PDA tumor compared to the non-conjugated nanocarrier while sparing normal organs.ConclusionsThe study provides promising data for future development of a novel MUC1-targeted nanocarrier for direct delivery of imaging agents or drugs into the tumor microenvironment.

PD15-01 CHARACTERIZATION OF THE GENOMIC LANDSCAPE OF A PROSPECTIVE MUSCLE-INVASIVE BLADDER CANCER COHORT WITH A FOCUS ON SECONDARY VARIANT HISTOLOGY

You have accessJournal of UrologyBladder Cancer: Invasive II1 Apr 2018PD15-01 CHARACTERIZATION OF THE GENOMIC LANDSCAPE OF A PROSPECTIVE MUSCLE-INVASIVE BLADDER CANCER COHORT WITH A FOCUS ON SECONDARY VARIANT HISTOLOGY Vikram Narayan, Ewan Gibb, Nicholas Erho, Andrea Cronican, Tyler Kolisnik, Elai Davicioni, and Badrinath Konety Vikram NarayanVikram Narayan More articles by this author , Ewan GibbEwan Gibb More articles by this author , Nicholas ErhoNicholas Erho More articles by this author , Andrea CronicanAndrea Cronican More articles by this author , Tyler KolisnikTyler Kolisnik More articles by this author , Elai DavicioniElai Davicioni More articles by this author , and Badrinath KonetyBadrinath Konety More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.800AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Muscle invasive bladder cancer (MIBC) is a highly aggressive disease, often presenting with heterogeneous clinical and pathological features. Though these tumors are commonly urothelial, secondary variant histology is frequently encountered. Recent transcriptome profiling studies have revealed MIBC can be divided into at least four distinct molecular subtypes, with the benefits of neoadjuvant chemotherapy (NAC) potentially varying between subtypes. The presence of secondary variant histology may alter this molecular profile and the treatment responses of these tumors. In this study, we report the genomic characterization of a large prospective MIBC cohort enriched with secondary variant histology. METHODS The cohort consisted of 173 patients diagnosed with urothelial MIBC who received the Decipher Bladder genomic subtyping classifier test (GenomeDx Biosciences Laboratory, San Diego, CA). Descriptions of secondary variant histology were abstracted from clinical pathology reports of submitted cases. TURBT tissue samples were profiled using a high-density genome-wide expression microarray and de-identified for GRID software analysis in R-studio (v3.3.1). RESULTS The cohort included 136 males and 32 females with a mean age of 72±9 at TURBT. A total of 49 cases (28%) had variant histology including non-urothelial (squamous [24], neuroendocrine [4], glandular [4]) and urothelial (micropapillary [6], sarcomatoid [5], plasmacytoid [2], nested [3], clear cell [1]) variants. Molecular subtyping revealed 77% of variant cases were either pure basal-like (22) or basal claudin-low (16), with fewer cases being luminal (7) or infiltrated luminal (4). Squamous cases had profiles consistent with basal tumors, while neuroendocrine cases had high MKI67 and neuronal marker expression. Micropapillary profiles had high CDH3 and MUC1 expression levels. Sarcomatoid tumors were driven by robust expression of EMT markers, including VIM, ZEB1 and ZEB2, with a corresponding loss of CDH1 expression. One plasmacytoid case expressed high levels of CDH1 RNA, but was negative for IHC, while the other case had low CDH1 RNA and lacked IHC data. CONCLUSIONS Secondary variant histology in MIBC contributes to the molecular heterogeneity of these tumors, with the majority of variant histology cases in this cohort being associated with basal MIBC subtypes. These findings may have implications for tailoring NAC by molecular subtype and may contribute to the varied clinical responses encountered in treating patients with variant histology. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e310 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Vikram Narayan More articles by this author Ewan Gibb More articles by this author Nicholas Erho More articles by this author Andrea Cronican More articles by this author Tyler Kolisnik More articles by this author Elai Davicioni More articles by this author Badrinath Konety More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

High MUC2 Mucin Expression and Misfolding Induce Cellular Stress, Reactive Oxygen Production, and Apoptosis in Goblet Cells

MUC2 mucin is a large glycoprotein produced by goblet cells that forms the protective mucus blanket overlying the intestinal epithelium as the first line of innate host defense. High MUC2 production in inflammatory bowel disease and infectious colitis depletes goblet cells and the mucus layer by an unknown mechanism. Herein, we analyzed the effect of high MUC2 biosynthesis on endoplasmic reticulum (ER) stress and apoptosis in goblet cells using a high MUC2-producing human goblet cell line (HT29-H) and an HT29-H clone (HT29-L) silenced for MUC2 expression by lentivirus-mediated shRNA. Goblet cell ER stress and apoptosis were quantified during early onset of dextran sulfate sodium-induced colitis in C57BL/6 and Math1M1GFP mice. Compared with HT29-L and MUC2 nonproducing Caco-2 cells, high MUC2-producing HT29-H cells had significantly increased ER stress and apoptosis after treatment with ER stress-inducing agents. Apoptosis was driven by increased misfolded MUC2 that triggered elevated levels of reactive oxygen species. Correcting MUC2 folding and inhibiting reactive oxygen species alleviated ER stress and rescued cells from apoptosis. During early-onset colitis, mucus hypersecretion caused severe ER stress and apoptosis of goblet cells that preceded absorptive epithelial cell damage. Thus, in gastrointestinal inflammation, high MUC2 biosynthesis and goblet cell apoptosis lead to a dysfunctional epithelial barrier. Enhancing MUC2 folding may help alleviate goblet cell depletion and maintain mucosal integrity.

Abstract P1-01-13: Mucin-2 (Muc-2) modulates the biology of breast cancer

Abstract Background: Metastasis and therapeutic resistance are major issues in treating patients with breast cancer. Breast tumors that secrete mucus are generally resistant to chemotherapy. One type of secreted mucin, Mucin-2 (Muc2), is expressed in some breast cancers, but is absent in non-neoplastic breast tissue, suggesting that it plays a role in modulating breast cancer biology. Whether Muc2 expression correlates with breast cancer patient survival is controversial. Thus, the effects of Muc2 on the biology of breast cancer are largely unknown. This study examined the role of Muc2 in modulating breast cancer cell proliferation in vitro and in vivo, response to chemotherapy, and metastasis. Methods: Two novel cell lines were developed from patient derived tumor xenografts. These two cell lines, BCK4 and PT12, both express high levels of Muc2. In order to modulate Muc2 levels in both cell lines, shRNA targeted to Muc2 (shMuc2) were compared to non-targeting control shRNA (shNT). Decreased expression of Muc2 was confirmed using immunoblotting. Proliferation in vitro was measured using the IncuCyte live cell imaging system and crystal violet staining. Response to chemotherapy was measured by examining apoptosis using cleaved-caspase 3 expression. BCK4 and PT12 cells with shNT or shMuc2 were grown as solid tumors in immunocompromised mice and tumor volume measured by caliper. BCK4 cells with high Muc2 (shNT) and low Muc2 (shMuc2) were labeled with luciferase and examined in an experimental metastasis model where total disease was monitored by IVIS imaging. Results: Decreased Muc2 expression decreased proliferation in BCK4 and PT12 cells versus non-targeting control cells both in vitro and in vivo. Treatment with the chemotherapeutic Docetaxel induced minimal apoptosis in BCK4 control cells with high Muc2 however, apoptosis was significantly increased in BCK4 cells with reduced Muc2. In an experimental metastasis model, mice injected with BCK4 cells containing low Muc2 had decreased disease burden versus those injected with control cells with high Muc2. Endogenous Muc2 expression in wild-type BCK4 cells increased with addition of EGF and this effect was abolished by addition of the EGF-receptor inhibitor, Erlotinib. Conclusions: Muc2 expression plays an important role in mediating proliferation, apoptosis and metastasis of breast cancer cells. These data suggest that Muc2 is important in controlling the biology of Muc2 positive breast tumors. In addition, Muc2 may be important in guiding treatment and predicting outcomes in breast cancer patients. Citation Format: Astashchanka A, Shroka T, Jacobsen BM. Mucin-2 (Muc-2) modulates the biology of breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-01-13.

Bilateral blockade of MEK- and PI3K-mediated pathways downstream of mutant KRAS as a treatment approach for peritoneal mucinous malignancies.

Mucinous colorectal adenocarcinomas (MCAs) are clinically and morphologically distinct from nonmucinous colorectal cancers (CRCs), show a distinct spectrum of genetic alterations (higher KRAS mutations, lower p53, high MUC2), exhibit more aggressive behavior (more prone to peritoneal dissemination and lymph node involvement) and are associated with poorer response to chemotherapy with limited treatment options. Here, we report the effectiveness of combinatorial targeting of two KRAS-mediated parallel pathways in reducing MUC2 production and mucinous tumor growth in vitro and in vivo. By knockdown of mutant KRAS we show that, mutant KRAS (a) is necessary for MUC2 production in vitro and (b) synergistically engages PI3K/AKT and MEK/ERK pathways to maintain MUC2 expression in MCA cells. These results define a novel and a previously undescribed role for oncogenic KRAS in mucinous cancers. MCA cells were sensitive to MEK inhibition suggesting cellular dependence (‘addiction’) of KRAS-mutant MCA cells on hyperactivation of the MEK-driven pathway. Interestingly, MCA cells, though initially sensitive, were later resistant to PI3K single agent inhibition. Our studies suggest that this resistance involves dynamic rewiring of signaling circuits mediated through relief of RTK inhibition and MEK-ERK rebound activation. This resistance however, could be overcome by co-targeting of PI3K and MEK. Our studies thus provide a rational basis for MEK- and PI3K-targeted combination therapy for not only KRAS mutant MCA but also for other related mucinous neoplasms that overproduce MUC2 and have a high rate of KRAS mutations such as pseudomyxoma peritonei.

TET1-mediated DNA hypomethylation regulates the expression of MUC4 in lung cancer.

Lung cancer remains a disease of high mortality, despite advanced diagnostic techniques. Mucins (MUC) play crucial roles in carcinogenesis and tumor invasion in lung neoplasms. Our immunohistochemistry (IHC) studies have shown that high MUC4 expression correlates with a poor outcome. We have also shown that the expression of several mucin genes in cancer cell lines is regulated by DNA methylation. We evaluated the expression level of MUC4, mRNA and several DNA hypomethylation factors in lung tissue samples from 33 patients with various lung lesions. The results indicated that the DNA methylation status of MUC4 matched the expression level of mRNA. In addition, the TET1 (Ten-Eleven Translocation) mRNA showed a significant correlation with the status of DNA methylation of MUC4. Furthermore, the treatment of a lung cancer cell line with TET1 siRNA caused a reduction in MUC4 mRNA expression. Thus, we suggest that TET1 mediated DNA hypomethylation plays a key role in the expression of MUC4. This is the first report that TET1 mediated DNA hypomethylation regulates the expression of MUC4 in lung cancer. The analysis of these epigenetic changes may be useful for diagnosing carcinogenic risk.